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=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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>D37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A41220</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C614-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryocool Heat Transfer Fluid</td>
			<td>Fisher Scientific</td>
			<td>20-548-146</td>
			<td>For vacuum concentrator</td>
		</tr>
		<tr>
			<td>Alginic Acid Sodium Salt High Viscosity</td>
			<td>MP Biomedicals</td>
			<td>154723</td>
			<td></td>
		</tr>
		<tr>
			<td>Squid mantle rings</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be purchased at grocery store</td>
		</tr>
		<tr>
			<td>Denatonium benzoate</td>
			<td>Aldrich</td>
			<td>D5765</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml graduated centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml scintillation vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bulbs for Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>03-448-24</td>
			<td></td>
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			<td>Red bulbs for pellet delivery</td>
			<td>Fisher Scientific</td>
			<td>03-448-27</td>
			<td></td>
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			<td>250 ml round-bottom flask</td>
			<td>Fisher Scientific</td>
			<td>10-067E</td>
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			<td>Scintillation vial adapter for rotavap</td>
			<td>Fisher Scientific</td>
			<td>K747130-1324</td>
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			<td>Weightboats</td>
			<td>Fisher Scientific</td>
			<td>02-202B</td>
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			<td>Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-53</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-54</td>
			<td></td>
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		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
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	</tbody>
</table>

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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9.3K 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

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Desvendando os jogadores invisíveis no Oceano - Um Guia de Campo para Química da Água e Microbiologia Marinha

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

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A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

A Volume Bioassay pequeno para avaliar Bacteriana / fitoplâncton Co-cultura utilizando água-Pulse-Amplitude Modulada (água-PAM) Fluorometria

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

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 ...

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 ...

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 ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extração de Pesticidas Organoclorados de Pelotização de Plástico e Análise de Tipo de Plástico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

Desenvolvimento de novos métodos para quantificar a densidade de peixes usando ferramentas de estéreo-vídeo subaquáticas

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

A craca Balanus improvisus como modelo Marine - cultivo e expressão gênica

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Acompanhamento a longo prazo de vídeo de animais aquáticos, Cohoused: um estudo de caso da atividade locomotora diária do lagostim (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

Microfocus X-Ray CT (microCT) imagem latente de Actinia equina da (Cnidaria), Harmothoe SP. (Annelida), e Xenoturbella japonica (xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...