Summary

了鱼喂食实验室生物测定,从海洋生物的组织评估次生代谢产物的Antipredatory活动

Published: January 11, 2015
doi:

Summary

这种生物测定采用模型肉食性鱼类,以评估喂食威慑代谢产物从海洋生物的天然浓度的组织有机提取物用营养可比食品基质的存在。

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

通过化学家和生态学家的合作化学生态学的发展。虽然陆地化学生态学的分支学科已经有一段时间了,那海洋化学生态学的只有几十年的历史,但已经提供了重要的见解海洋生物1-8的进化生态学和群落结构。以水肺潜水和核磁共振光谱的新兴技术的优势,有机化学迅速产生,描述上世纪70年代和80年代,从9底栖海洋无脊椎动物和藻类代谢产物的小说出版了大量。假设次级代谢产物必须为某种目的,许多这些出版物没有经验证据冲高重要生态价值属性的新化合物。大约在同一时间,生态学家还采取水肺潜水的来临优势和描述的分布和底栖动物和植物的前身来回的丰度米相对无效的抽样方法,如疏浚。这些研究人员的假设是,任何无柄和软体必须捍卫化学,以避免掠食者10消费。在努力引进经验什么是关于物种丰度,否则描述工作中,一些生态学家开始从毒性试验11推断的化学防御。大多数毒性测定法涉及的整鱼或其他生物体的脊椎动物组织的粗有机萃取水性悬浮液的曝光,随后确定负责杀死半数试验生物提取物的干重的浓度。然而,毒性试验不效仿其潜在的掠食者自然条件下感知猎物的方式,和随后的研究发现毒性和12-13适口性之间没有任何关系。令人惊讶的是,在著名期刊出版物使用具有很少或没有。生态技术升相关14-15,今天,这些研究仍然被广泛引用。而更令人震惊的要注意的是基于毒性数据研究继续出版16-18。这里所描述的生物测定方法的开发,在20世纪80年代后期,为海洋化工生态学家的生态相​​关的方法来评估antipredatory化学防御。该方法需要一个模型捕食样品从目标生物体的粗有机提取物在营养食品可比矩阵天然浓度,从而提供适口的数据,比毒性数据更加生态有意义。

一般的方法来评估海洋生物的组织的antipredatory活动包括四个重要的标准:(1)一个适当的通才捕食必须馈送测定法中使用,(2)所有极性有机代谢物必须详尽地从所述的组织中提取靶生物,(3)的代谢物必须BE在相同体积浓度混入营养合适的实验食品如在先前从其中提取,并且(4)的实验设计和统计方法必须提供一个有意义的度量,以指示相对于令人厌恶的生物体中发现。

下面的程序是专为评估加勒比海海洋无脊椎动物antipredatory化学防御。我们采用了双带锦鱼,Thalassoma bifasciatum,作为一种模式掠食性鱼类,因为这是种常见的对加勒比海的珊瑚礁,被称为采样底栖无脊椎动物19各式各样。从目标生物体组织被首先提取出来,然后与一种食物混合物,最后提供给T的组bifasciatum,观察他们是否拒绝提取物治疗食品。使用这种方法测定的数据提供了重要的见解海洋生物12,20-21,L的化学防御IFE历史权衡22-24和群落生态学25-26。

Protocol

注:本协议的第3步涉及脊椎动物科目。该程序已被设计成动物获得了最人性化的治疗可能和已经批准的机构动物护理和使用委员会(IACUC)在北卡罗莱纳州威尔明顿的大学。 1)组织提取使用组织是在其天然水,而不是挤压,干涸的或过于潮湿的状态,因为这将改变次生代谢产物的体积浓度。切剁的组织,以片或片可插入到50ml离心管中。注意:新鲜组织可以在某些情?…

Representative Results

在这里,我们报告该生物测定6种常见加勒比海绵( 图2)的结果。这些数据最初发表于1995年由Pawlik 等 12并证明了这种方法的力量调查共发生类群之间的化学防御策略的差异。结果被报告为标准误差(SE),针对每个物种食用+食物颗粒的平均数量。几乎没有颗粒被吃掉的测定与象耳海绵,Amphimedon牛鞭 ,并Aplysina cauliformis粗制有机提取物。与此相反,从Ca…

Discussion

这里所描述的程序提供了一个相对简单的,生态的相关实验室的协议,以评估海洋生物antipredatory化学防御。在这里,我们回顾了由这套方法满足重要的标准:

(1)适当的捕食者这种喂养法采用双带锦鱼,Thalassoma bifasciatum,珊瑚礁整个加勒比地区最丰富的鱼类之一。该bluehead是一个通才食肉动物称为采样底栖无脊椎动物19各式各样。通才天敌是最好的?…

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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Cite This Article
Marty, M. J., Pawlik, J. R. A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms. J. Vis. Exp. (95), e52429, doi:10.3791/52429 (2015).

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