En fisk-fodring Laboratory bioassay i vurdering af den Antipredatory aktivitet af sekundære metabolitter fra væv af marine organismer
Summary
Dette bioassay anvender en model rovfisk til at vurdere forekomsten af fodring-afskrækkende metabolitter fra økologiske ekstrakter af væv af marine organismer ved naturlige koncentrationer ved hjælp af en ernæringsmæssigt sammenlignelig fødevare matrix.
Abstract
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.
Introduction
Kemisk økologi udviklet gennem et samarbejde med kemikere og økologer. Mens underdisciplin af jordbaseret kemisk økologi har eksisteret i nogen tid, at af havets kemiske økologi er kun et par årtier gammel, men har givet vigtig indsigt i den evolutionære økologi og samfund struktur af marine organismer 1-8. Ved at udnytte de emergente teknologier af dykning og NMR-spektroskopi, økologiske kemikere hurtigt genereret et stort antal publikationer, der beskriver nye metabolitter fra bundlevende marine hvirvelløse dyr og alger i 1970'erne og 1980'erne 9. Antages det, at sekundære metabolitter skal tjene et formål, mange af disse publikationer tilskrives økologisk vigtige egenskaber til nye forbindelser uden empiriske beviser. På nogenlunde samme tid, blev økologer også drage fordel af fremkomsten af dykning og beskrive fordelingerne og mængderne af bunddyr og planter tidligere kendte from relativt ineffektive prøvetagningsmetoder såsom uddybning. Antagelsen af disse forskere var, at noget siddende og blød og rørige skal kemisk forsvares at undgå forbrug af rovdyr 10. I et forsøg på at indføre empiri til, hvad der var ellers beskrivende arbejde arter mængderne, nogle økologer begyndte ekstrapolere kemiske forsvar fra toksicitet assays 11. De fleste toksicitet assays involverede eksponeringen af hele fisk eller andre organismer til vandige suspensioner af rå organiske ekstrakter af hvirvelløse væv, med efterfølgende bestemmelse af tørvægt koncentrationer af ekstrakter, der er ansvarlige for at dræbe halvdelen af assay organismer. Men toksicitet analyser ikke efterligne den måde, hvorpå potentielle rovdyr opfatter bytte under naturlige forhold, og efterfølgende undersøgelser har fundet nogen sammenhæng mellem toksicitet og velsmag 12-13. Det er overraskende, at publikationer i prestigefyldte tidsskrifter brugte teknikker, der har lidt eller ingen Ecological relevans 14-15, og at disse undersøgelser stadig er almindeligt citeret i dag. Det er endnu mere alarmerende at bemærke, at undersøgelser baseret på toksicitetsdata fortsat blive offentliggjort 16-18. Den heri beskrevne bioassay metode blev udviklet i slutningen af 1980'erne for at give et økologisk relevant tilgang til marine kemiske økologer at vurdere antipredatory kemiske forsvar. Metoden kræver en model rovdyr at prøve en rå organisk ekstrakt fra målorganismen på en naturlig koncentration i et ernæringsmæssigt sammenlignelig fødevare matrix, der giver smagen data, der er mere økologisk meningsfulde end toksicitetsdata.
Den generelle tilgang til vurdering af antipredatory aktivitet af væv marine organismer indeholder fire vigtige kriterier: (1) en passende generalist rovdyr skal anvendes i foder assays, (2) organiske metabolitter af alle polariteter skal være udtømmende udvindes af væv i målrette organisme, (3) metabolitterne skal be blandet ind i en ernæringsmæssigt egnet eksperimentel mad på samme volumetriske koncentration som findes i organismen, hvorfra de blev udvundet, og (4) den eksperimentelle design og statistiske metode skal give en meningsfuld måling for at angive relative distastefulness.
Proceduren beskrevet nedenfor er designet specielt til at vurdere antipredatory kemiske forsvar i Caribien marine hvirvelløse dyr. Vi anvender den bluehead wrasse, Thalassoma bifasciatum, som model rovfisk, fordi denne art er almindelig på caribiske koralrev og er kendt for at prøve et bredt sortiment af bunddyr 19. Væv fra målet organismen først udvindes, derefter kombineret med en fødevare blanding, og endelig tilbydes til grupper af T. bifasciatum at observere, om de forkaster ekstrakt-behandlede fødevarer. Assay data ved hjælp af denne metode har givet vigtig indsigt i den defensive kemi af marine organismer 12,20-21, lIFE historie afvejninger 22-24 og samfund økologi 25-26.
Protocol
Representative Results
Discussion
Den heri beskrevne fremgangsmåde tilvejebringer en relativt enkel, økologisk relevant laboratorieprotokol for vurderingen antipredatory kemiske forsvar i marine organismer. Her gennemgår vi de vigtige kriterier, som er opfyldt med dette sæt af metoder:
(1) Passende rovdyr. Dette fodring assay anvender bluehead wrasse, Thalassoma bifasciatum, en af de mest udbredte fisk på koralrev i hele Caribien. Den bluehead er en generalist kødædende kendt for at prøve et bredt s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Dichloromethane | Fisher Scientific | D37-20 | |
| Methanol | Fisher Scientific | A41220 | |
| Anhydrous Calcium Chloride | Fisher Scientific | C614-500 | |
| Cryocool Heat Transfer Fluid | Fisher Scientific | 20-548-146 | For vacuum concentrator |
| Alginic Acid Sodium Salt High Viscosity | MP Biomedicals | 154723 | |
| Squid mantle rings | N/A | N/A | Can be purchased at grocery store |
| Denatonium benzoate | Aldrich | D5765 | |
| 50 ml graduated centrifuge tube | Fisher Scientific | 14-432-22 | |
| 20 ml scintillation vial | Fisher Scientific | 03-337-7 | |
| Disposable Pasteur pipets | Fisher Scientific | 13-678-20D | |
| Rubber bulbs for Pasteur pipets | Fisher Scientific | 03-448-24 | |
| Red bulbs for pellet delivery | Fisher Scientific | 03-448-27 | |
| 250 ml round-bottom flask | Fisher Scientific | 10-067E | |
| Scintillation vial adapter for rotavap | Fisher Scientific | K747130-1324 | |
| Weightboats | Fisher Scientific | 02-202B | |
| Microspatula | Fisher Scientific | 21-401-10 | |
| 5 ml graduated syringe | Fisher Scientific | 14-817-53 | |
| 10 ml graduated syringe | Fisher Scientific | 14-817-54 | |
| Razor blade | Fisher Scientific | S17302 |
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