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: Fase 3 del presente protocollo coinvolge soggetti animali vertebrati. La procedura è stata progettata in modo che gli animali ricevano il trattamento più umano possibile ed è stato approvato dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) presso la University of North Carolina Wilmington. 1) Estrazione del tessuto <ol><li> Utilizzare tessuto che è nel suo stato naturale di idratazione e non spremuto, secchi-out o eccessivamente umido come questo altera la concentrazione volumetrica di metaboliti secondari. Tagliare o tagliare il tessuto in pezzi o fette che possono essere inseriti in un tubo da centrifuga da 50 ml. Nota: tessuto fresco può essere utilizzato in alcuni casi, ma spesso è meglio tagliare o tritare tessuti congelati, che non è soggetta a spremitura al taglio. </li><li> Aggiungere pezzi di tessuto a 30 ml di una miscela 1: 1 di diclorometano (DCM) e metanolo (MeOH) in una provetta da centrifuga graduata fino ad un volume finale di 40 ml è raggiunto. Assicurarsi di eseguire tutti i passaggi che comportano il trasferimento disolvente in una cappa aspirante con ventilazione adeguata. </li><li> Tappare la provetta e capovolgerla più volte, poi agitare più volte nel corso di un periodo di estrazione 4 ore. Nota: Durante questo periodo, l&#39;acqua si combina con il MeOH e il risultante MeOH: fase acquosa si separa dalla fase DCM. Il tessuto è alternativamente esposto al DCM e MeOH: acqua come emulsione come i tubi sono agitati. </li><li> Trasferire l&#39;estratto DCM in un pallone a fondo rotondo ed evaporare a secchezza su un evaporatore rotante utilizzando basse temperature (&lt;40 ° C). Utilizzando solventi minimal, trasferire l&#39;estratto secco di 20 ml di scintillazione flacone. Montare la fiala con un adattatore evaporatore rotante e di nuovo evaporare a secco in un evaporatore rotante con fuoco basso (&lt;40 ° C). Nota: Il passo successivo richiede l&#39;uso di uno strumento di compressione casalingo che possono essere assemblati avvitando i seguenti elementi in ordine sequenziale sull&#39;estremità di un&#39;asta filettata: (1) il dado (2) Rondella, e (3) dado cieco. La rondella deve o essere perforato omontata in modo che sia inferiore al diametro interno di un tubo da centrifuga da 50 ml. </li><li> Tornando al tubo da centrifuga graduato contenente tessuti e MeOH: estratto dall&#39;acqua, premere il mezzo di estrazione di tessuto attraverso la compressione. Trasferire il MeOH: estratto acquoso allo stesso pallone a fondo tondo e memorizzare refrigerati (&lt;10 ° C). </li><li> Aggiungere MeOH al tubo da centrifuga graduata finché il tessuto ora disidratato è sommerso per una seconda estrazione di 2 a 6 durata ore, poi trasferire la nuova MeOH estratto nel pallone a fondo tondo refrigerati contenente la MeOH: estratto acquoso. Se non vi è alcuna preoccupazione che il tessuto non è stato completamente estratto, ripetere l&#39;estrazione MeOH 2 a 6 ore. </li><li> Asciugare la MeOH su un evaporatore rotante con fuoco basso (&lt;40 ° C). Trasferire l&#39;estratto acquoso rimanente dal pallone a fondo tondo nella fiala di scintillazione contenente l&#39;estratto secco non polare, con un volume minimo di MeOH per risciacquare il pallone a fondo tondo. </li><li> Evaporate l&#39;estratto acquoso di essiccazione, a fuoco basso (&lt;40 ° C) su un concentratore a vuoto. La fiala di scintillazione ora contiene l&#39;estratto grezzo organico secco totale di 10 ml di tessuto. Evacuare spazio di testa del flaconcino con N 2 gas per prevenire l&#39;ossidazione, sigillare ermeticamente e conservare congelato (-20 ° C). </li></ol> 2) Preparazione alimentare <ol><li> Preparare calamari polvere manto liofilizzata. Nota: mantello Squid fornisce una fonte di alimentazione che è paragonabile a quella di altri invertebrati bentonici, e verrà utilizzato come ingrediente nelle sottofasi di 2.2. <ol><li> Anelli surgelati Scongelare del mantello calamari a deionizzata calda (DI) di acqua, poi purea in un frullatore ad alta velocità. </li><li> Versate un sottile strato di purè calamari mantello su una teglia bassa e congelamento (-20 ° C), quindi rompere il foglio di congelato purea calamari a pezzetti per essere liofilizzato. </li><li> Lyophilize la calamari congelati manto purea seguendo le modalità di funzionamento del freeze-essiccatore. </li><li> Polverizzare i pezzi liofilizzate di calamari mantello purea in un frullatore ad alta velocità per formare una polvere. </li><li> In una cappa aspirante, versare il mantello calamaro in polvere in una farina setaccio rotante e vagliare separare grossi pezzi di tessuto dalla polvere fine. </li><li> Trasferire la multa in polvere manto calamari in un contenitore sigillato. Evacuare spazio di testa del contenitore con N 2 gas per prevenire l&#39;ossidazione e memorizzare congelati (-20 ° C). </li></ol></li><li> Preparare l&#39;impasto alimentare. Nota: Durante l&#39;esecuzione di più test consecutivi, è pratico di preparare ~ 100 ml di miscela di cibo, ma questa ricetta può essere scalata a volumi più piccoli, se necessario. <ol><li> Combinare una miscela di 3 g di acido alginico e 5 g liofilizzata mantello polvere calamari con 100 ml di acqua deionizzata in un bicchiere da 150 ml. Mescolare energicamente con una microspatula per qualche minuto fino a quando la polvere è completamente idratata e la miscela è omogenea. Nota: Se si desidera, colorante alimentare può essere aggiunto a questo step: è facile aggiungere alla miscela colorante alimentare che genererà entrambe le miscele trattate e di controllo (mascherare il pigmento naturale dell&#39;estratto nella miscela estratto trattati) anziché cercare di abbinare il colore della miscela dell&#39;estratto trattati aggiungendo tingere alla miscela di controllo. Colorante alimentare verde o marrone è spesso desiderabile per mascherare eventuali pigmenti nell&#39;estratto greggio. </li><li> Caricare esattamente 10 ml di miscela di cibo in una siringa graduata. Fare attenzione ad evitare l&#39;inclusione di bolle d&#39;aria nel corso di questo processo. </li><li> Rimuovere l&#39;20 ml scintillazione flacone con estratto biologico greggio asciutto dal freezer. Aggiungere una o due gocce di MeOH, quindi mescolare l&#39;estratto in una miscela omogenea con un microspatula. </li><li> Espellere il caricata siringa da 10 ml di matrice alimentare in 20 ml di scintillazione fiala e mescolare con una microspatula per omogeneizzare la miscela alimentare estratto trattati. Nota: Può essere utile per espellere la siringa in incrementi più piccoli (cioè, espellere 2 ml e omogeneizzare, poi r.EPEAT fino tutti i 10 ml sono stati omogeneizzati). </li></ol></li><li> Preparare il pellet di analisi. <ol><li> Caricare un volume molto piccolo della miscela dell&#39;estratto (~ 1 ml) in una siringa, e immergere la punta della siringa in una soluzione di 0,25 M CaCl 2. Estrarre il contenuto della siringa per formare un lungo spaghetto-like. </li><li> Dopo pochi minuti, togliere il filo indurito, tritarlo in 4 mm di lunghezza pellet su un tagliere di vetro con una lama di rasoio, poi sciacquare in acqua di mare. </li><li> Ripetere i punti 2.3.1 e 2.3.2 senza includere l&#39;estratto di tessuto per fare pellet di controllo. Assicurarsi di trattamento pellets di controllo con un volume equivalente di solvente (vedi aggiunta di MeOH al composto trattata nel passaggio 2.2.3) per controllare per aggiunta di solventi. Se un controllo negativo è desiderato per confermare che il pesce saggio può essere indotta a rinunciare alimentazione, aggiungere denatonium benzoato ad una concentrazione di 2 mg ml -1 al cibo miscela grezza 27. </li></ol></li></ol> 3) AppetibilitàBioassays <ol><li> Eseguire test di alimentazione con il giallo-fase wrasse bluehead catturati, Thalassoma bifasciatum, tenuti in gruppi di tre in compartimenti opaco facciate di acquari di laboratorio. </li><li> Invia pellets alimentari da un bicchiere di acqua di mare con una pipetta di vetro con un bulbo di gomma. Nota: Potrebbero essere necessari alcuni giorni per addestrare i pesci per ricevere il cibo in questo modo. Uno stimolo condizionata (es pochi tocchi della pipetta sul vetro dell&#39;acquario) che precede la consegna di cibo può essere utile per addestrare il pesce aspettarsi l&#39;aggiunta di pellets alimentari. </li><li> Punteggio pellet. Si consideri un pellet accettato se prontamente consumato dai pesci. Si consideri un pellet respinta se non mangiato, dopo un minimo di tre tentativi da parte di uno o più pesci di prenderlo nella loro cavità orale, o se il pellet si avvicina e ignorato dopo un tentativo del genere. </li><li> Campioni di punteggio. Nota: La procedura di test è raffigurato come un diagramma di flusso in Figura 1 Gruppi di pesci che si rifiutano di mangiare.pellets di controllo in qualsiasi fase del protocollo non sono considerati ulteriormente. Ci sono due possibili risultati di una singola esecuzione del test: il campione viene accettato o respinto. <ol><li> Inizia con un pellet di controllo per confermare che il gruppo di pesci è cooperativa. Offrire un pellet trattata. Se i pesci accettano il pellet trattata, segnare il campione accettata. Se i pesci rifiutano il pellet trattato, offrire una successiva pellet di controllo per determinare se i pesci hanno cessato l&#39;alimentazione. Se i pesci accettano la successiva pellet di controllo, segnare il campione respinta. </li></ol></li><li> Replica. Ripetere la procedura di test con dieci gruppi indipendenti di pesce per ogni estratto. </li></ol> 4) Significato Valutazione <ol><li> Valutare la significatività delle differenze di consumo di controllo vs pellet trattate con una versione modificata del test esatto di Fisher 26. Modificare il test in modo che siano fissati i totali marginali per il controllo e pellet trattati, Trattandoli due campioni casuali. Nota: Questo fornisce p = 0,057 quando 7 pellet sono mangiati; quindi, ogni estratto è considerato deterrente se 6 o meno pellet sono mangiati, e appetibile se 7 o più pellet sono mangiati. </li><li> Per confrontare l&#39;appetibilità relativa tra i gruppi di estratti, calcolare un numero medio di pellet consumato all&#39;interno di ogni gruppo. Mantenere la soglia a 6 pellet così un gruppo di estratti repliche sono considerati deterrente se il numero medio di pellet consumato + errore standard (SE) ≤6. Nota: Nei risultati rappresentativi, l&#39;assegnazione del gruppo è di specie, così replicano estratti provengono da individui distinti e la relativa appetibilità possono essere confrontati tra le specie. </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.

La procedura qui descritta prevede un protocollo di laboratorio relativamente semplice, ecologicamente rilevanti per valutare le difese chimiche antipredatory negli organismi marini. Qui passiamo in rassegna i criteri importanti che sono soddisfatte da questo insieme di metodi: (1) appropriata predatore. Questa analisi alimentare impiega il wrasse bluehead, Thalassoma bifasciatum, uno dei pesci più abbondanti sulle barriere coralline in tutti i Caraibi. Il bluehead è un c...

Ecologia chimica sviluppata attraverso la collaborazione di chimici ed ecologisti. Mentre la sotto-disciplina di terrestre ecologia chimica è stato intorno per qualche tempo, quello di ecologia chimica marina è solo pochi decenni vecchio, ma ha fornito importanti conoscenze sulla ecologia e comunità la struttura evolutiva degli organismi marini 1-8. Sfruttando le tecnologie emergenti di immersioni subacquee e di spettroscopia NMR, i chimici organici rapidamente generato un gran numero di pubblicazioni che descrivono nuovi metaboliti bentoniche invertebrati e alghe marine negli anni 1970 e 1980 9. Supponendo che metaboliti secondari devono servire a qualcosa, molte di queste pubblicazioni ascritti ecologicamente importanti proprietà a nuovi composti senza prove empiriche. Nello stesso tempo, gli ecologisti sono stati anche approfittando dell&#39;avvento di immersioni subacquee e descrivere le distribuzioni e abbondanze di animali bentonici e piante precedentemente noti from metodi di campionamento relativamente inefficaci come il dragaggio. L&#39;assunzione di questi ricercatori è che qualsiasi cosa sessili e corpo molle devono essere difesi chimicamente per evitare il consumo da parte di predatori 10. Nel tentativo di introdurre l&#39;empirismo a quello che era il lavoro altrimenti descrittivo sulle specie abbondanze, alcuni ecologisti hanno cominciato estrapolando difese chimiche da saggi di tossicità 11. La maggior parte dei test di tossicità l&#39;esposizione di pesci interi o di altri organismi a sospensioni acquose di estratti organici grezzi di tessuti invertebrati, con successiva determinazione delle concentrazioni in massa a secco di estratti responsabili dell&#39;uccisione metà gli organismi di analisi. Tuttavia, i saggi di tossicità non emulare il modo in cui i potenziali predatori percepiscono preda in condizioni naturali, e gli studi successivi hanno trovato alcuna relazione tra la tossicità e l&#39;appetibilità 12-13. E &#39;sorprendente che le pubblicazioni su riviste prestigiose utilizzate tecniche minimo o nullo ecological rilevanza 14-15 e oggi che questi studi sono ancora ampiamente citati. E &#39;ancora più allarmante notare che gli studi basati su dati di tossicità continuano ad essere pubblicati 16-18. Il metodo di analisi biologico descritto è stato sviluppato alla fine del 1980 per fornire un approccio ecologicamente rilevante per gli ecologi marini chimiche per valutare le difese chimiche antipredatory. Il metodo richiede un modello predatore di assaggiare un estratto organico greggio da parte dell&#39;organismo bersaglio ad una concentrazione naturale in un punto di vista nutrizionale analoga matrice alimentare, fornendo dati appetibilità che sono più ecologicamente significativo di dati sulla tossicità. L&#39;approccio generale per valutare l&#39;attività antipredatory dei tessuti di organismi marini comprende quattro criteri importanti: (1) un appropriato predatore generalista deve essere utilizzato in saggi di alimentazione, (2) metaboliti organici di tutte le polarità devono essere esaustivo estratte dal tessuto del bersaglio organismo, (3) i metaboliti devono be mescolato in un alimento nutrizionalmente sperimentale appropriata alla stessa concentrazione volumetrica come trovato nell&#39;organismo da cui sono stati estratti, e (4) il disegno sperimentale e approccio statistico devono fornire una metrica significativa per indicare relativa distastefulness. La procedura descritta di seguito è stato progettato specificamente per valutare le difese chimiche antipredatory in Caraibi invertebrati marini. Impieghiamo il wrasse bluehead, Thalassoma bifasciatum, come un modello di pesce predatore, perché questa specie è comune in barriere coralline dei Caraibi ed è noto per degustare un vasto assortimento di invertebrati bentonici 19. Tissue da parte dell&#39;organismo bersaglio viene dapprima estratta, poi combinata con una miscela di cibo, e infine offerto ai gruppi di T. bifasciatum osservare se rifiutano i cibi estratto trattati. Dati del dosaggio con questo metodo hanno fornito importanti conoscenze sulla chimica di difesa degli organismi marini 12,20-21, lstoria ife compromessi 22-24, e la comunità ecologia 25-26.

<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>
		</tr>
		<tr>
			<td>Red bulbs for pellet delivery</td>
			<td>Fisher Scientific</td>
			<td>03-448-27</td>
			<td></td>
		</tr>
		<tr>
			<td>250 ml round-bottom flask</td>
			<td>Fisher Scientific</td>
			<td>10-067E</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation vial adapter for rotavap</td>
			<td>Fisher Scientific</td>
			<td>K747130-1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Weightboats</td>
			<td>Fisher Scientific</td>
			<td>02-202B</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
	</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.

Qui riportiamo i risultati di questo saggio biologico per sei specie di spugne caraibiche comuni (Figura 2). Questi dati sono stati inizialmente pubblicati nel 1995 da Pawlik et al. 12 e dimostrano la potenza di questo approccio per esaminare le differenze nelle strategie di difesa chimiche tra taxa concomitanti. I risultati sono stati riportati come un numero medio di pellet cibo mangiato + errore standard (SE) per ogni specie. Quasi nessun pellet sono stati consumati in test con es...

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

Questo saggio biologico si avvale di un modello di pesce predatore per valutare la presenza di metaboliti di alimentazione-deterrenti da estratti organici dei tessuti degli organismi marini a concentrazioni naturali utilizzando un punto di vista nutrizionale analoga matrice alimentare.

Un pesce-alimentare Laboratorio Bioassay per valutare l&#39;attività di antipredatory Secondary metaboliti dai tessuti di organismi marini

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 Views.  University of North Carolina Wilmington.  Questo saggio biologico si avvale di un modello di pesce predatore per valutare la presenza di metaboliti di alimentazione-deterrenti da estratti organici dei tessuti degli organismi marini a concentrazioni naturali utilizzando un punto di vista nutrizionale analoga matrice alimentare. 

Video: 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.

Svelare i giocatori invisibili del Mare - Una guida del campo di Acqua Chimica e microbiologia marina

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.

Un piccolo volume Bioassay per valutare batterica / fitoplancton Co-cultura Utilizzando WATER-Pulse-modulata in ampiezza (WATER-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 Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

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.

Simulazione Laboratorio di ferro (II) ricchi di Precambriano Marine Upwelling sistema per esplorare la crescita di batteri fotosintetici

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.

Metodo ecotossicologico con i batteri marini<em&gt; Vibrio anguillarum</em&gt; Per valutare la tossicità acuta dei contaminanti ambientali

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.

Estrazione di pesticidi di organochlorato da pellicole di plastica e analisi di tipo plastico

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.

Sviluppo di nuovi metodi per quantificare la densità di pesce utilizzando strumenti di Stereo-video subacquei

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.

Il Barnacle Balanus BalanUs come modello Marine - coltura e l'espressione genica

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.

A lungo termine dei Video rilevamento di Cohoused animali acquatici: un caso di studio dell'attività locomotrice quotidiana di scampi (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) Imaging di Actinia equina (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. ...