This work was funded, in part, by the Great Lakes Fishery Commission and the Annis Water Resources Institute. Use of trade, product, or firm names does not imply endorsement by the U. S. Government. This article is Contribution 1867 of the U. S. Geological Survey Great Lakes Science Center.

1 Laboratório de Experiência <ol><li> Obter o peixe presa para ser alimentado para o peixe predador durante a experiência. De preferência, estes peixe presa deve ser observado no campo, congelado e armazenado a cerca de -30 ° C. Considere operações de pesca comercial como uma fonte potencial para os peixes presas. </li><li> Introduzir a peixes predadores para os tanques de laboratório a ser utilizado para a experiência. Até 15 predador peixe foram introduzidos em cada um dos tanques 870-L, e até 30 peixes predadores foram introduzidos em cada um dos tanques de 2380-L, em estudos anteriores 16,18. </li><li> Aclimatar os peixes predadores de uma dieta do peixe presa seleccionado. Uma vez aclimatados, o peixe predador deve permanecer nesta dieta por 1-3 meses antes do início do experimento. </li><li> Separe amostras de peixes presa por seleção aleatória de 10 a 20 amostras compostas a partir do lote de peixe presa. Número de peixe presa em uma amostra de compósito pode variar entre 3 e 100, dependendo do tamanho da presapeixes. Cada amostra deve ser composto de embalagem dupla, congelado e armazenado a cerca de -30 ° C. </li><li> Inicia-se a experiência com o sacrifício de 30 a 50% dos peixes em cada um dos tanques. <ol><li> Para sacrificar o peixe, misture 8 g de Finquel com 45 L de água em um grande recipiente de plástico e, em seguida, coloque o peixe no recipiente com a solução Finquel. </li><li> Depois de sacrificados para colocar todos os peixes de um tanque sacrificados em um saco, bolsa, em seguida, e armazenar a cerca de -30 ° C até à altura de processamento. </li><li> Pesar cada um dos restantes peixes em cada um dos tanques, e registra os pesos; um anestésico provavelmente serão necessárias para realizar a pesagem. </li><li> Para anestesiar o peixe, misture 4,6 g de Finquel com 45 L de água em um recipiente de plástico grande, e em seguida, coloque o peixe no recipiente com a solução Finquel. </li><li> Aguarde alguns minutos para que o anestésico para entrar em vigor antes da pesagem do peixe. </li></ol></li><li> Em cada dia da experiência, descongelar umaquantidade adequada de peixe presa, e cortar o peixe presa em pedaços pesando aproximadamente 1-5 g. Pesa-se a quantidade de peixe presa para ser colocado em cada um dos tanques, em seguida, deixar cair os pedaços de peixe presa em cada um dos tanques e permitir que o peixe predador cerca de 1 hora para se alimentar. Em seguida, retire todos os restos de comida, deixe a comida secar ao ar por cerca de 20 min, e em seguida, pesar a restos de comida para cada um dos tanques. Registar a quantidade de comida colocada no tanque e da quantidade de restos de comida para cada um dos tanques de cada dia. NOTA: Para o experimento representativo, truta do lago foram alimentados com comida tanto quanto eles iriam consumir durante um período de alimentação a cada dia 18. No entanto, o peixe predador pode também ser colocado em rações fixos 16,19. </li><li> Terminar a experiência sacrificando todos os peixes predadores restante em cada um dos tanques. Para sacrificar o peixe, misture 8 g de Finquel com 45 L de água em um grande recipiente de plástico e, em seguida, coloque o peixe no recipiente com a solução Finquel. Grave tele peso de cada um dos peixes sacrificados. Para obter resultados confiáveis, o experimento deve ser executado por pelo menos 100 dias, de preferência, pelo menos, 130 dias. Coloque todos os peixes de um tanque em um saco, então bag duplo, e armazenar a cerca de -30 ° C até o momento do processamento. </li></ol> Homogeneização 2. Peixe <ol><li> Selecione um conjunto de peixes predadores e / ou compósitos de peixe presa para descongelamento. Permitir que os compósitos para descongelar parcialmente. Cada composto pode exigir de 0,5 a 1 hora para homogeneizar. </li><li> Usando os liquidificadores de tamanho adequado, homogeneizar cada um dos compostos. Para cada composto, colocar uma amostra (de 50 a 100 g) do homogeneizado em um frasco limpo, acetona-lavado, e etiquetado. Em seguida, o frasco de tampa e armazenar o frasco a cerca de -30 ° C até o tempo de processamento. </li><li> Lave todos os equipamentos utilizados para homogeneizar o peixe, e depois enxaguar bem com água destilada e metanol, entre as amostras. </li></ol> 3. Extraction <ol><li> Pesar 20,0 gdescongeladas de tecido homogeneizado peixe num copo de 200 ml. </li><li> Adicionar cerca de 40 g de sulfato de sódio e misturar bem com uma espátula. </li><li> Adicionar solução de pico de substituição contendo os congéneres 30, 61, 161, e 166 a uma concentração pico que dá uma concentração final de 20 ng / ml no extracto. </li><li> Deixar a amostra secar à temperatura ambiente, enquanto se mistura a cada 20 min. </li><li> Deixar a amostra para alcançar uma consistência de areia seca, altura em que a amostra está pronta para a extracção. </li><li> Configure o aparelho de extração Soxhlet com um frasco de 500 ml contendo chips de Teflon fervura, Soxhlet, e do condensador. </li><li> Adicione a mistura de peixe seco para um dedal de vidro com um disco de fundo fritted-grossa ou dedal papel. </li><li> Adicionar 150 ml de 50% de hexano e 50% de diclorometano para o copo utilizado para a amostra e agitar durante a raspagem das paredes do balão com uma espátula. </li><li> Transferir o solvente para o topo da Soxhlet com o balão ligado e permitir que o ciclo através de Soxhlet e empara o balão. </li><li> Repetir uma segunda vez com 150 ml mais uma vez. </li><li> Coloque o Soxhlet com o balão anexado para o elemento de aquecimento e anexar o condensador. </li><li> Ligar o elemento de aquecimento e trazer o solvente a uma fervura suave, em seguida o extracto durante um período mínimo de 16 horas para garantir que a água fria é fornecida para os condensadores. </li><li> Depois de se permitir que o solvente de cool, verifica para ver se qualquer um dos frascos das amostras conter água. Para os frascos com água, adicionar sulfato de sódio e homogeneizar até que a água é absorvida pelo sulfato de sódio. </li><li> Concentra-se a amostra usando um concentrador de amostra de azoto ou uma configuração de artigos de vidro Kaderna dinamarquesa (KD) com um banho de água quente. </li><li> Deixar a amostra evaporar até um volume de menos de 2 ml, e, em seguida, levar a um volume final de 5 ml, utilizando pequenas lavagens de hexano para transferir a amostra do material de vidro utilizado para um balão volumétrico de 5 ml. </li><li> Transfira para um frasco de 10 ml e etiqueta com informações de amostra. </li></ol><ol><li> Prepare gel de sílica acidificado pela adição de 44 g de ácido sulfúrico concentrado e 100 g de gel de sílica activado. </li><li> Adicionar 10 g de gel de sílica acidificado numa pequena coluna de cromatografia contendo um pequeno tampão de lã de vidro no fundo. </li><li> Adicionar 1 ml de extracto da amostra à coluna após a pré-limpeza da coluna com 10 ml de hexano. </li><li> Eluir a coluna com 20 ml de hexano e recolha afilada em tubo de vidro de 20 ml. </li><li> Colocar o tubo de vidro sobre o aparelho de evaporador de azoto (N-Vap) sob uma corrente de azoto e imerso em água quente. </li><li> Evapora-se a menos de 1 ml mas não até à secura. </li><li> Retire do aparelho N-Vap e transferir para um frasco volumétrico de 1 ml com pequenas lavagens de hexano. </li><li> Transferir para um frasco de autoamostragem de 1,8 ml marcado com a informação da amostra. </li><li> Pico de 4 mL de padrão interno para dentro do frasco. A amostra está pronto para análise. </li></ol> 5. Analysis por Cromatografia Gasosa - Espectrometria de Massa Usando Negativa de Ionização Química <ol><li> Use padrões para calibrar o instrumento: As normas estão disponíveis em misturas que consistem em grupos de congêneres bem separados. Misturas 1-5 consistem em quase todos os compostos afins encontrados em Arochlors 1016, 1221, 1232, 1242, 1248, 1254, 1260 e uma mistura é utilizada como uma mistura de calibração multi-nível, e a linearidade do sistema é confirmada através da preparação de pelo menos cinco níveis de calibragem em concentrações entre 2 e 100 ng / ml. Mixes 2-5 são usados ​​como calibrações de um único ponto para cada congênere. </li><li> Defina-se a cromatografia em - sistema de espectrometria de massa no modo de ionização química negativa com hidrogénio como gás de arraste (1 ml / min) e metano como gás reagente. </li><li> Use uma sílica fundida, a coluna capilar (60 m x 0,25 milímetros de diâmetro interno) revestida com DB-XLB a espessura da película de 0,25 mm para a separação. Programa de temperatura do forno 60-212 ° C a 25 ° C / min, em seguida a 260 ° C a 1 ° C / min, e em seguida a 280 ° C a 4 ° C / min, com um tempo de retenção final de 4 min. Injector e linha de transferência temperaturas deve ser fixado em 280 ° C. Injectar 1 a 2 mL da amostra utilizando o modo de splitless injecção. </li><li> Analisar todos os padrões e as amostras pelo método de padrão interno usando 13 marcado com C decachlorobiphenyl. </li><li> Execute uma verificação da calibração inicial, executando um segundo padrão de fonte e Aroclors 1242 e 1260, e depois comparar os valores previstos para os congéneres Aroclor com os valores observados deste procedimento de verificação. </li><li> Uma vez que o procedimento de calibração inicial foi realizada com sucesso, uma análise completa de todas as amostras. Execute uma calibração verificar a cada dez amostras, utilizando qualquer uma das misturas de calibração a partir da calibração inicial. </li></ol> 6 Cálculo da Net Trófico Transferência Eficiência <ol><li> Calcular a eficiência da transferência líquida trófico, γ, para cada combinação of tanque e PCB congênere com a seguinte equação: <img alt="Equação 1" fo:content-width="2in" src="/files/ftp_upload/51496/51496eq1.jpg" /> , Onde [PCB f] é a concentração média de PCB congénere do peixe predador no tanque, no final da experiência, W f é o peso médio dos peixes predadores no tanque, no final da experiência, [PCB i] é a concentração média de PCB congénere do peixe predador no reservatório no inicio da experiência, W i é o peso médio dos peixes predadores no início da experiência, e a quantidade de PCB congénere ingerido refere-se ao peso do congéneres de PCB ingerida, em média, por cada truta do lago no tanque durante o curso do experimento. </li><li> Calcular o denominador na equação acima, multiplicando a concentração média dos congéneres de PCB nos compósitos de peixe presa pela quantidade média (peso) do peixe presa comido por peixes predadores no tanque durcontendo todo o curso da experiência. </li></ol>

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Para as estimativas mais precisas de γ, o experimentador deve ser capaz de controlar com precisão a quantidade de ambos os alimentos colocados em cada um dos tanques e a quantidade de restos de comida em cada um dos tanques durante o curso da experiência. Para realizar isto, o experimentador deve ser capaz de remover todo o alimento não consumido a partir dos tanques e determinar com precisão o seu peso. Além controle preciso do alimento comido efectivamente pelos peixes predadores, estimativa exacta de γ também...

Of all of the factors affecting the rate at which fish accumulate contaminants, the efficiency with which fish retain contaminants from the food that they eat is one of the most important1-3. Risk assessment models have been developed to predict future risks to both people and wildlife eating contaminated fish under various scenarios of environmental contamination, and the reliability of these predictions critically depends on the accuracy of the estimates of the efficiency at which fish retain contaminants from their food4.
The efficiency with which the contaminant in the food ingested by the predator is transported through the gut wall is known as gross trophic transfer efficiency5. A portion of the quantity of the contaminant transported through the gut wall of the predator may eventually be lost through depuration and/or metabolic transformation. The efficiency with which the contaminant in the food ingested by the predator is retained by the predator, including any losses due to elimination and metabolic transformation, is known as net trophic transfer efficiency6.
Gross trophic transfer efficiency of organic contaminants to fish from their prey appears to vary with the contaminant&#8217;s chemical properties, including lipid affiliation as measured by the octanol-water partition coefficient, Kow3,7. According to an empirical relationship developed by Thomann3, gross trophic transfer efficiency is relatively high when log Kow is equal to a value between 5 and 6. Gross trophic transfer efficiency declines exponentially at a rate of 50% per unit of log Kow as log Kow increases from 6 to 10, according to the Thomann3 relationship.
Nevertheless, the gross and net trophic transfer efficiencies of polychlorinated biphenyl (PCB) congeners to fish from their prey do not appear to follow the Thomann3 relationship in most cases. Although the trophic transfer efficiencies of PCB congeners to lake whitefish (Coregonus clupeaformis) from its food followed the relationship proposed by Thomann8, trophic transfer efficiencies of PCB congeners were either just weakly related or not related at all to log Kow for Atlantic salmon (Salmo salar)9, rainbow trout (Oncorhynchus mykiss)10, coho salmon (Oncorhynchus kisutch)11, and northern pike (Esox lucius)11.
The overall goal of this study was to present a laboratory technique for estimating the net trophic transfer efficiencies of PCB congeners to a piscivorous fish from its prey. Lake trout (Salvelinus namaycush) was chosen as the piscivorous fish for our experiment because lake trout are relatively easy to maintain in laboratory tanks. Bloater (Coregonus hoyi) was selected as the prey fish to be fed to the lake trout because bloater is eaten by lake trout in its natural setting12. In addition, we determined whether the net trophic transfer efficiencies for lake trout estimated from our laboratory experiment followed the Thomann3 relationship. We also determined whether the degree of activity by the lake trout had a significant effect on net trophic transfer efficiency (&#947;) of the PCB congeners. Activity by lake trout in the Laurentian Great Lakes is believed to have recently increased because changes in the food webs have caused lake trout to allocate more energy toward searching for food13. Lake trout were forced to exercise in one set of tanks by subjecting these lake trout to relatively high flow rates, whereas the other lake trout were permitted to remain relatively inactive by subjecting them to relatively low flow rates. Finally, the specific details of our laboratory procedure that need to be carefully followed to ensure the highest degree of accuracy in the &#947; estimates and to make the laboratory results applicable to the field are discussed, as well as future directions for research building on our laboratory technique. Net trophic transfer efficiency can be estimated both in the laboratory and in the field, and advantages and disadvantages are associated with both approaches. Accuracy in the estimate of &#947; depends on the accuracy of the estimate of food consumption. The amount of food eaten by fish in the laboratory can be accurately determined when proper protocols are followed, whereas the amount of food eaten by fish in the field is typically estimated via bioenergetics modeling. Use of bioenergetics modeling to derive the amount of food eaten has the potential to introduce a substantial amount of uncertainty into the estimates of food consumption. Fish bioenergetics models have been shown to estimate food consumption with no detectable bias for the case of lake trout14,15, but considerable bias in bioenergetics model estimates of food consumption has been detected for the case of lake whitefish15,16. On the other hand, estimates of net trophic transfer efficiency estimated in the laboratory may not be applicable to the field due to a difference in feeding rates between the laboratory and the field17. Evidence from both the laboratory and the field suggest that feeding rate can influence &#947;14,17.
The methodology used in the present study for estimating &#947; in the laboratory is applicable to situations where the predator fish is fed prey fish, and the amount of prey fish eaten by the predator can be accurately tracked. To accomplish this, the experimenter must weigh all of the food before placement in the tank; and the experimenter must be able to remove all of the uneaten food from the tank, and then weigh the uneaten food. In addition, an adequate suite of mixers and blenders should be available to obtain a sufficient degree of homogenization of the samples of both predator and prey fish. Finally, the gas chromatography &#8211; mass spectrometry instrumentation used to determine the PCB congener concentrations must be capable of detecting and quantifying individual PCB congeners at relatively low concentrations.

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			<td height="21" style="height:21px;width:259px;">870-L fiberglass tanks</td>
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			<td style="width:167px;">Eugenol could also be used as an anesthetic.</td>
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			<td height="35" style="height:35px;width:259px;">Soxhlet extraction glassware (500 mL)</td>
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			<td style="width:119px;">&#160;LG-6900-104</td>
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			<td height="35" style="height:35px;width:259px;">Hexane</td>
			<td style="width:87px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:119px;">Cat. 211-4</td>
			<td></td>
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			<td height="35" style="height:35px;width:259px;">Dichloromethane</td>
			<td style="width:87px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:119px;">Cat. 300-4</td>
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			<td height="21" style="height:21px;width:259px;">Silica gel</td>
			<td style="width:87px;">BDH</td>
			<td style="width:119px;">Cat. BDH9004-1KG</td>
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			<td height="35" style="height:35px;width:259px;">Labl Line 5000 mult-unit extraction heater</td>
			<td style="width:87px;">Lab Line Instruments</td>
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			<td height="35" style="height:35px;width:259px;">Agilent 5973 GC/MS with chemical ionization</td>
			<td style="width:87px;">Agilent</td>
			<td style="width:119px;">5973N</td>
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			<td height="52" style="height:52px;width:259px;">Internal standard solution&#160;</td>
			<td style="width:87px;">Cambridge Isotope Laboratories</td>
			<td style="width:119px;">EC-1410-1.2</td>
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			<td height="21" style="height:21px;width:259px;">PCB congener calibration standards</td>
			<td style="width:87px;">Accustandard</td>
			<td style="width:119px;">C-CSQ-SET</td>
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			<td height="21" style="height:21px;width:259px;">DB-XLB column (60m x 0.25mm, 0.25 micron)</td>
			<td style="width:87px;">Agilent/ J&#38;W</td>
			<td style="width:119px;">122-1262</td>
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laboratory estimation of net trophic transfer efficiencies of pcb congeners to lake trout (salvelinus namaycush) from its prey

Uma técnica para estimativa de laboratório da rede trófica eficiência de transferência (γ) de bifenil policlorados (PCB) congêneres de peixes piscívoros de suas presas é aqui descrito. Durante um experimento de laboratório de 135 dias, nos alimentamos arenque defumado (Coregonus hoyi), que tinha sido capturado no lago Michigan para a truta do lago (Salvelinus namaycush) mantidos em oito tanques de laboratório. Bloater é uma presa natural para a truta do lago. Em quatro dos tanques, uma taxa de fluxo relativamente elevada foi usado para assegurar a actividade relativamente elevada da truta do lago, ao passo que uma baixa taxa de fluxo foi utilizado nas outras quatro tanques, permitindo a baixa actividade truta do lago. Em uma base de tanque-a-tanque, a quantidade de alimentos ingeridos pela truta do lago, em cada dia do experimento foi gravado. Cada truta do lago foi pesado no início e no fim da experiência. De quatro a nove truta do lago de cada um dos oito tanques foram sacrificados no início da experiência, e todas as 10 a truta do lago restante em cada um dos tanques foram euthanzado no final da experiência. Foram determinadas as concentrações de 75 congéneres de PCB na truta do lago, no início da experiência, na truta do lago, no final da experiência, e em bloaters alimentada à truta do lago, durante a experiência. Com base nestas medições, γ foi calculada para cada um dos 75 congéneres de PCB em cada um dos oito tanques. A média de γ foi calculada para cada um dos 75 congéneres de PCB, tanto para ativos e inativos truta do lago. Como o experimento foi repetido em oito tanques, o erro padrão sobre significar γ pode ser estimada. Os resultados deste tipo de experiência são úteis em modelos de avaliação de risco para prever risco futuro para os seres humanos e os animais selvagens que comem peixes contaminados em vários cenários de contaminação ambiental.

Truta do lago mostrou uma quantidade substancial de crescimento durante o experimento, como a truta do lago iniciais significa pesos variaram 694-907 g, enquanto as trutas finais lago significa peso variou de 853 a 1.566 g (Tabela 1). A quantidade média de comida consumida por uma truta do lago, durante o decurso da experiência de 135 dias variou de 641 a 2.649 g. As concentrações de PCB de congéneres significa na truta do lago aumentado durante o ensaio, em termos de concentração média de cong?...

Watch this Scientific Journal Video about Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey at JoVE.com

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

Uma técnica para estimativa de laboratório da rede trófica eficiência de transferência de bifenil policlorados (PCB) congêneres de peixes piscívoros de suas presas é apresentado. Para maximizar a aplicabilidade dos resultados do laboratório para o campo, o piscívoro deve ser alimentado presa peixe que são tipicamente consumidos no campo.

Estimativa Laboratório de Líquido tróficas eficiências de transferência de PCB para Lake Trout (<em&gt; Salvelinus namaycush</em&gt;) De sua rapina

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

laboratory-estimation-net-trophic-transfer-efficiencies-pcb-congeners

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JoVE Journal

Environment

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

10.9K visualizações.  U. S. Geological Survey.  Uma técnica para estimativa de laboratório da rede trófica eficiência de transferência de bifenil policlorados (PCB) congêneres de peixes piscívoros de suas presas é apresentado. Para maximizar a aplicabilidade dos resultados do laboratório para o campo, o piscívoro deve ser alimentado presa peixe que são tipicamente consumidos no campo. 

Vídeo: Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Determinados pelo Laboratório Fósforo Flux do Lago Sedimentos como medida de Fósforo Interno Carregando

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Ambiente

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

A Laboratory Bioassay alimentando-Fish para avaliar a actividade anti-predação de metabolitos secundários a partir de tecidos de organismos marinhos

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Automated Gel seleção de tamanho para melhorar a qualidade das bibliotecas de sequenciação de próxima geração Preparado a partir de amostras de água do ambiente

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

A remoção do Trace Elements por Óxido de Cupric nanopartículas de urânio<em&gt; In Situ</em&gt; Recuperação sangro água e seu efeito na viabilidade celular

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

Laboratório de Simulação de um ferro (II) rico em Precambrian Marinha Ressurgência do sistema para explorar o crescimento de bactérias fotossintéticas

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

Desenvolvimento de<em&gt; Metarhizium anisopliae</em&gt; Como um Mycoinsecticide: do isolamento ao desempenho do campo

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Procedimentos de laboratório de fumigação para controle de pragas com gás de óxido nítrico

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

Aderência de bactérias para plantar superfícies medidas em laboratório

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Protocolos de fabricação (EcoFAB) ecossistema para a construção de ecossistemas de laboratório projetado para estudar interações planta-micróbio

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Laboratório e campo protocolo para estimar as taxas de erosão folha de Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...