Name: a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms
Uploaded: 2015-01-11T14:00:00.000+00:00
Duration: 16 min 3 s
Description: Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

We thank James Maeda and Aaron Cooke for assistance with the filming and editing of this video. Funding was provided by the National Science Foundation (OCE-0550468, 1029515).

NOTA: Passo 3 do presente protocolo envolve indivíduos animais vertebrados. O procedimento foi concebido de modo que os animais recebam o tratamento mais humano possível e foi aprovado pelo cuidado e uso Comitê Institucional Animal (IACUC) da Universidade de North Carolina Wilmington. 1) extracção de tecidos <ol><li> Use tecido que está em seu estado natural de hidratação e não apertou, seca-out ou excessivamente úmido, pois isso irá alterar a concentração volumétrica de metabólitos secundários. Cortar ou cortar tecido para peças ou fatias que podem ser inseridos dentro de um tubo de centrífuga de 50 ml. Nota: O tecido fresco pode ser utilizado em alguns casos, mas muitas vezes é melhor cortar ou cortar tecido congelado, que não está sujeita a apertar, quando o corte. </li><li> Adicionar porções de tecido a 30 ml de uma mistura 1: 1 de diclorometano (DCM) e metanol (MeOH) em um tubo de centrífuga graduado até um volume final de 40 ml foi atingido. Certifique-se de realizar todas as etapas que envolvem a transferência desolvente em um exaustor com ventilação adequada. </li><li> Tapar o tubo e invertê-lo várias vezes, em seguida, agitar repetidamente durante um período de 4 horas de extracção. Nota: Durante este período, a água combina com o MeOH e o MeOH resultante: fase aquosa separa-se a fase de DCM. O tecido é exposto alternadamente a DCM e MeOH: água como uma emulsão como os tubos são agitados. </li><li> Transferir o extracto de DCM para um balão de fundo redondo e evapora-se até à secura num evaporador rotativo usando-se o calor baixo (&lt;40 ° C). Usando solvente mínima, transferir o extracto seco a um frasco de cintilação de 20 ml. Montar o frasco com um adaptador evaporador rotativo e evapora-se novamente até à secura num evaporador rotativo usando-se o calor baixo (&lt;40 ° C). Nota: O próximo passo requer o uso de um instrumento de compressão caseiro que pode ser montado por aparafusamento os seguintes artigos em ordem sequencial na extremidade de uma haste roscada (1) da porca (2), louça, e (3) da porca de bolota. A máquina de lavar ou deve ser perfurado oumontado de modo que seja menor que o diâmetro interno de um tubo de centrífuga de 50 ml. </li><li> Voltando para o tubo de centrífuga graduado que contém o tecido e o MeOH: extracto de água, espremer o meio de extracção para fora do tecido por meio de compressão. Transferir o MeOH: extracto de água ao mesmo balão de fundo redondo e armazenar refrigeradas (&lt;10 ° C). </li><li> Adicionar MeOH para o tubo de centrífuga graduado até que o tecido já desidratado está submerso para uma segunda extracção de 2 a 6 horas de duração, em seguida, transferir o extrato MeOH novo para o balão de fundo redondo arrefecido contendo o MeOH: extracto de água. Se houver alguma preocupação de que o tecido não tem sido totalmente extraída, repetir a 2 a 6 horas de extracção de MeOH. </li><li> Seque o MeOH num evaporador rotativo usando-se o calor baixo (&lt;40 ° C). Transferir o extracto aquoso remanescente a partir do balão de fundo redondo para o frasco de cintilação contendo o extracto não polar seco, usando um volume mínimo de MeOH para lavar o frasco de fundo redondo. </li><li> Evaporate o extracto aquoso até à secura utilizando calor baixo (&lt;40 ° C) num concentrador de vácuo. O frasco de cintilação agora contém o extracto em bruto seco orgânico total de 10 ml de tecido. Evacuar o espaço superior do frasco com gás N2 para evitar a oxidação, feche bem e armazenar congelados (-20 ° C). </li></ol> 2) Preparação de Alimentos <ol><li> Prepare pó liofilizado manto squid. Nota: manto Squid fornece uma fonte de alimentação que é comparável à de outros invertebrados bentônicos, e irá ser utilizada como um ingrediente nos sub-passos de 2,2. <ol><li> Anéis congelados descongelamento do manto squid em deionizada quente (DI) de água, em seguida, purê-los em um misturador de alta velocidade. </li><li> Verter uma fina camada de manto puré lulas em uma assadeira rasa e congelamento (-20 ° C), em seguida, romper a folha de puré lulas congeladas em pequenos pedaços para ser liofilizada. </li><li> Liofiliza os congelados purê manto lula seguindo os procedimentos operacionais da freeze de cabelo. </li><li> Pulverizar as peças liofilizadas de lula purê manto em um liquidificador de alta velocidade para formar um pó. </li><li> Em um exaustor, despeje o manto squid em pó em uma peneira rotativa e peneire a farinha para separar grandes pedaços de tecido a partir do pó fino. </li><li> Transfira a multa manto squid em pó para um recipiente lacrado. Evacuar o espaço de cabeça do recipiente com gás N2 para evitar a oxidação e armazenar congelada (-20 ° C). </li></ol></li><li> Preparar a mistura de alimentos. Nota: Quando executando vários ensaios consecutivos, é prático para preparar ~ 100 ml de mistura de alimentos, no entanto esta receita pode ser dimensionada para volumes menores, se necessário. <ol><li> Combinar uma mistura de 3 g de ácido algínico e 5 g de pó liofilizado manto lulas com 100 ml de água desionizada numa proveta de 150 ml. Agita-se vigorosamente com um microspatula durante alguns minutos até que o pó é completamente hidratada e a mistura é homogénea. NOTA: Se desejar, corante alimentar pode ser adicionado neste step: ele é mais fácil de adicionar à mistura de corante alimentar que vai gerar as duas misturas de controlo e tratados (mascarar o pigmento natural do extracto na mistura tratados com extracto) em vez de tentar fazer corresponder a cor da mistura tratada por adição de extracto tingir para a mistura de controlo. Uma cor esverdeada ou acastanhada alimentos é muitas vezes desejável para mascarar quaisquer pigmentos no extracto em bruto. </li><li> Coloque exactamente 10 ml da mistura de alimentos para uma seringa graduada. Tome cuidado para evitar a inclusão de bolhas de ar durante este processo. </li><li> Retirar o frasco de cintilação de 20 ml com extracto orgânico bruto seco do congelador. Adicionar uma ou duas gotas de MeOH, em seguida, misture o extrato em uma mistura homogênea com um microspatula. </li><li> Ejectar o carregado seringa de 10 ml de matriz de alimentos para o frasco de cintilação de 20 ml e agita-se com uma microspatula para homogeneizar a mistura de alimentos tratados com extracto. Nota: Pode ajudar a ejetar a seringa em incrementos menores (ou seja, ejetar 2 ml e homogeneizar, em seguida, r.epita até que todos os 10 ml foram homogeneizados). </li></ol></li><li> Prepare os grânulos de ensaio. <ol><li> Carregar um volume muito pequeno da mistura de extracto (~ 1 ml) com uma seringa, e submergir a ponta da seringa em uma solução de 0,25 M de CaCl2. Injectar o conteúdo da seringa para formar uma longa, cadeia espaguete semelhante. </li><li> Depois de alguns minutos, retire o fio endurecido, cortá-la em 4 mm de comprimento pelotas em uma placa de corte de vidro com uma lâmina de barbear, e depois enxaguar em água do mar. </li><li> Repita os passos 2.3.1 e 2.3.2 sem incluindo o extrato de tecido para fazer pelotas de controle. Certifique-se para tratar a pellets de controlo com um volume equivalente de solvente (ver adição de MeOH a mistura tratada no passo 2.2.3) para controlar a adição de solvente. Se um controlo negativo é desejado para confirmar que os peixes ensaio pode ser impedido de alimentação, adicionar benzoato de denatónio, a uma concentração de 2 mg ml-1 para a mistura de alimentos crus 27. </li></ol></li></ol> 3) A palatabilidadeBioensaios <ol><li> Realizar ensaios de alimentação com amarelo-fase Wrasse bluehead selvagens capturados, Thalassoma bifasciatum, mantidos em grupos de três em compartimentos opaco face de aquários laboratório. </li><li> Entregar pelotas de alimentos a partir de um copo de água do mar com uma pipeta de vidro com um bulbo de borracha. Nota: Pode demorar alguns dias para treinar peixes para receber o alimento desta forma. Um estímulo condicionado (por exemplo, alguns toques da pipeta no vidro do aquário) que precede a entrega de alimentos pode ser útil para treinar o peixe que esperar a adição de grãos de ração. </li><li> Pontuação peletes. Considere-se uma pelete aceite se prontamente consumida pelos peixes. Considere um pellet rejeitado se não comido depois de um mínimo de três tentativas por parte de um ou mais peixes para levá-la em sua cavidade bucal, ou se o pellet é abordado e ignorado após uma tentativa nesse sentido. </li><li> Amostras de pontuação. Nota: O procedimento de ensaio é descrito como um fluxograma na Figura 1, grupos de peixe que se recusam a comer.pelotas de controle em qualquer etapa do protocolo não são considerados ainda mais. Há dois resultados potenciais de uma única execução do ensaio: a amostra é aceitos ou rejeitados. <ol><li> Comece com uma bolinha de controle para confirmar que o grupo de peixes é cooperativa. Oferecer um pellet tratada. Se os peixes aceitar o pellet tratada, marcar a amostra que foi aceite. Se os peixes rejeitar o sedimento tratado, oferecer uma pelete de controlo subsequente para determinar se os peixes tenham cessado de alimentação. Se os peixes aceitar o pellet controle posterior, marcar a amostra como rejeitada. </li></ol></li><li> Replication. Repita o procedimento de ensaio com dez grupos independentes de peixe para cada extrato. </li></ol> 4) Importância Avaliando <ol><li> Avaliar a significância das diferenças no consumo de controle vs pelotas tratados com uma versão modificada do teste exato de Fisher 26. Modificar o teste para que os totais marginais para o controle e pelotas tratados são fixos, Tratando-os como duas amostras aleatórias. Nota: Este fornece p = 0,057 quando 7 pelotas são consumidos; Assim, qualquer extracto é considerada impedimento se 6 ou menos pelotas são comidos, e saboroso se 7 ou mais pelotas são comidos. </li><li> Para comparar a palatabilidade relativa entre os grupos de extractos, calcular um número significativo de peletes consumidos dentro de cada grupo. Manter o limiar a 6 peletes assim um grupo de extractos replicados são considerados dissuasor se o número médio de peletes consumidos + erro padrão (SE) ≤6. Nota: Nos resultados representativos, o trabalho de grupo é espécie, assim replicar extratos provenientes de indivíduos distintos e a palatabilidade relativa pode ser comparada entre as espécies. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+the+Caribbean+reef+sponge+Axinella+corrugata+against+predatory+fishes.">Chemical defense of the Caribbean reef sponge Axinella corrugata against predatory fishes.</a> Journal of Chemical Ecology. 25 (12), 2811-2823 (1999).</li><li>Chanas, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+predatory+reef+fish+II.+Spicules,+tissue+toughness,+and+nutritional+quality.">Defenses of Caribbean sponges against predatory reef fish II. Spicules, tissue toughness, and nutritional quality.</a> Marine Ecology Progress Series. 127 (1), 195-211 (1995).</li><li>Albrizio, S., Ciminiello, P., Fattorusso, E., Magno, S., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphitoxin,+a+new+high+molecular+weight+antifeedant+pyridinium+salt+from+the+Caribbean+sponge+Amphimedon+compressa.">Amphitoxin, a new high molecular weight antifeedant pyridinium salt from the Caribbean sponge Amphimedon compressa.</a> Journal of Natural Products. 58 (5), 647-652 (1995).</li><li>Assmann, M., Lichte, E., Pawlik, J. R., K&#246;ck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Marine Ecology Progress Series. 207, 255-262 (2000).</li><li>Kubanek, J., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antifeedant+triterpene+glycosides+from+the+Caribbean+sponge+Erylus+Formosus.">New antifeedant triterpene glycosides from the Caribbean sponge Erylus Formosus.</a> Natural Product Letters. 15 (4), 275-285 (2001).</li><li>Pawlik, J. R., McFall, G., Zea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+odor+from+sponges+of+the+genus+Ircinia+protect+them+from+fish+predators.">Does the odor from sponges of the genus Ircinia protect them from fish predators.</a> Journal of Chemical Ecology. 28 (6), 1103-1115 (2002).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+invertebrate+predators.+I.+Assays+with+hermit+crabs.">Defenses of Caribbean sponges against invertebrate predators. I. Assays with hermit crabs.</a> Marine Ecology Progress Series. 195, 125-132 (2000).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+of+Caribbean+sponges+against+invertebrate+predators.+II.+Assays+with+sea+stars.">Defense of Caribbean sponges against invertebrate predators. II. Assays with sea stars.</a> Marine Ecology Progress Series. 195, 133-144 (2000).</li><li>Burns, E., Ifrach, I., Carmeli, S., Pawlik, J. R., Ilan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-predatory+defenses+of+Red+Sea+and+Caribbean+sponges.+I.+Chemical+defense.">Comparison of anti-predatory defenses of Red Sea and Caribbean sponges. I. Chemical defense.</a> Marine Ecology Progress Series. 252, 105-114 (2003).</li><li>Jones, A. C., Blum, J. E., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+for+defensive+synergy+in+Caribbean+sponges:+Bad+taste+or+glass+spicules.">Testing for defensive synergy in Caribbean sponges: Bad taste or glass spicules.</a> Journal of Experimental Marine Biology and Ecology. 322 (1), 67 (2005).</li></ol>

The authors declare that they have no competing financial interests.

O procedimento aqui descrito fornece um protocolo laboratorial relativamente simples, ecologicamente relevantes para a avaliação defesas químicas anti-predação em organismos marinhos. Aqui vamos rever os critérios importantes que sejam satisfeitas por este conjunto de métodos: (1) A condição de predador. Este ensaio alimentação emprega o bodião bluehead, Thalassoma bifasciatum, um dos peixes mais abundantes nos recifes de coral em todo o Caribe. O bluehead é um...

Ecologia química desenvolvido com a colaboração de químicos e ecologistas. Embora a especialidade da ecologia química terrestre tem sido em torno de algum tempo, o de ecologia química marinha é apenas algumas décadas de idade, mas tem fornecido importantes contribuições para a ecologia e comunidade estrutura evolutiva dos organismos marinhos 1-8. Aproveitando-se das tecnologias emergentes de mergulho e espectroscopia de RMN, químicos orgânicos rapidamente gerou um grande número de publicações que descrevem novos metabolitos de invertebrados marinhos bentônicos e algas na década de 1970 e 1980 9. Supondo-se que metabólitos secundários devem servir para alguma coisa, muitas dessas publicações atribuídas propriedades ecologicamente importantes para novos compostos sem evidência empírica. Mais ou menos ao mesmo tempo, os ecologistas também foram aproveitando o advento do mergulho e descreve as distribuições e abundâncias de animais bentônicos e plantas já conhecidas frosou métodos de amostragem relativamente ineficazes, como a dragagem. A hipótese dos pesquisadores foi que nada sésseis e de corpo mole deve ser quimicamente defendidas para evitar o consumo por predadores 10. Em um esforço para introduzir o empirismo ao que foi o trabalho de outra forma descritiva em abundância das espécies, alguns ecologistas começaram extrapolando defesas químicas a partir de ensaios de toxicidade 11. A maioria dos ensaios de toxicidade envolveu a exposição dos peixes inteiros ou em outros organismos para suspensões aquosas de extratos brutos da tecidos de invertebrados, com posterior determinação das concentrações de massa seca de extratos responsáveis ​​por matar metade dos organismos de ensaio. No entanto, os ensaios de toxicidade não imitar a maneira pela qual os predadores potenciais percebem presas em condições naturais, e estudos posteriores não encontraram nenhuma relação entre a toxicidade e palatabilidade 12-13. É surpreendente que as publicações em revistas de prestígio utilizadas técnicas tendo pouca ou nenhuma ecological relevância 14-15 e, hoje, que esses estudos ainda são amplamente citado. Ele é ainda mais alarmante notar que estudos baseados em dados de toxicidade continuam a ser publicados 16-18. O método de bioensaio aqui descrito foi desenvolvido no final de 1980 para fornecer uma abordagem ecologicamente relevantes para os ecologistas marinhos químicos para avaliar as defesas químicas anti-predação. O método requer um modelo de predador para provar um extrato bruto do organismo alvo em uma concentração natural em uma matriz alimentar comparável nutricionalmente, fornecendo dados palatabilidade que são ecologicamente mais significativo do que os dados de toxicidade. A abordagem geral para a avaliação da actividade anti-predação dos tecidos dos organismos marinhos inclui quatro critérios importantes: (1) um predador generalista apropriado devem ser usadas em ensaios de alimentação, (2) de todos os metabolitos orgânicos polaridades deve ser exaustivamente extraído do tecido do organismo alvo, (3) os metabolitos deve be misturados em um alimento nutricionalmente adequada experimental com a mesma concentração volumétrica como encontrado no organismo a partir do qual foram extraídos, e (4) o desenho experimental e abordagem estatística deve fornecer uma métrica significativa para indicar distastefulness relativa. O procedimento descrito a seguir é projetado especificamente para avaliar as defesas químicas anti-predação de invertebrados marinhos do Caribe. Nós empregamos o bodião bluehead, Thalassoma bifasciatum, como um peixe predatório do modelo, porque esta espécie é comum em recifes de corais do Caribe e é conhecido por experimentar uma grande variedade de invertebrados bentônicos 19. De tecido do organismo alvo é extraído em primeiro lugar, em seguida, combinada com uma mistura alimentar, e finalmente oferecido para grupos de T. bifasciatum observar se eles rejeitam os alimentos tratados com extrato. Dados de ensaio usando este método forneceram importantes insights sobre a química defensiva de organismos marinhos 12,20-21, lhistória ife trade-offs 22-24 e 25-26 comunidade ecologia.

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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.

Aqui nós relatamos resultados deste ensaio biológico para seis espécies de esponjas do Caribe comuns (Figura 2). Estes dados foram inicialmente publicado em 1995 por Pawlik et al. 12 e demonstrar o poder dessa abordagem para examinar as diferenças de estratégias de defesa química entre taxa co-ocorrência. Os resultados foram relatados como uma média do número de peletes de alimento comido + erro padrão (SE) para cada espécie. Quase não há sedimentos foram consumidos em e...

Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

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

Este bioassay emprega um modelo de peixes predadores para avaliar a presença de metabólitos alimentação-de dissuasão a partir de extratos orgânicos de tecidos de organismos marinhos em concentrações naturais utilizando uma matriz alimentar comparável nutricionalmente.

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 ...

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9.4K visualizações.  University of North Carolina Wilmington.  Este bioassay emprega um modelo de peixes predadores para avaliar a presença de metabólitos alimentação-de dissuasão a partir de extratos orgânicos de tecidos de organismos marinhos em concentrações naturais utilizando uma matriz alimentar comparável nutricionalmente. 

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

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

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

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

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

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 ...

Ambiente

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

Ensaiar comportamentos alimentares predadores em<em&gt; Pristionchus</em&gt; E outros nematóides

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Método Ecotoxicológico com Bactérias Marinhas<em&gt; Vibrio anguillarum</em&gt; Avaliar a Toxicidade Aguda dos Contaminantes Ambientais

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

A identificação das feromonas de lampreia do mar usando guiada por bioensaio fracionamento

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Bioquímica

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Protocolo experimental pela análise de perfis de resposta comportamental em peixes Larval: aplicação para a Neuro-estimulantes cafeína

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Identificação da Atividade Hemolítica e Fosfolipase em Extratos Brutos de Anêmonas do Mar por Bioensaões Simples

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Bioensaio de Letalidade Utilizando Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Química

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Biologia

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidez baseado Triagem Interação Extracelular (AVEXIS) para a detecção escalável de baixa afinidade Extracelular receptor-ligante Interações

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Bateria de Testes Comportamentais para Avaliação de Efeitos Neurotóxicos em Peixe-Zebra Adulto

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Autor em destaque: Avaliação de mudanças comportamentais ambientais e induzidas por drogas em peixes-zebra adultos 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

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

Resumo

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