海洋蛀木甲壳类动物对木材生物降解性能的快速测试
Summary
该协议提出了一种通过测量粪便颗粒产生来评估蛀木甲壳类动物 Limnoria的进食速率的方法。该方法设计用于非专业实验室，并有可能纳入标准测试协议，以评估海洋条件下增强的木材耐久性。
Abstract
蛀木无脊椎动物迅速破坏海洋木材和沿海木制基础设施，每年在全球范围内造成数十亿美元的损失。由于使用广谱杀菌剂处理木材，如杂酚油和铬化砷酸铜（CCA），现在受到立法限制用于海洋用途，因此需要天然耐用的木材种类和新颖的木材保存方法。这些方法经过测试，以满足监管标准，例如针对海洋蛀虫测试木材防腐剂的欧洲标准EN 275。通过实验室测试可以快速、廉价地对耐用木材物种或木材防腐剂处理进行初步研究，这比通常成本高昂的长期海洋野外试验具有许多优势。许多种类的 Limnoria （gribble）是海洋蛀木甲壳类动物。 Limnoria 非常适合用于海洋蛀木者对木材生物降解的实验室测试，因为将它们饲养在水族箱中的实用性以及易于测量它们在木材上的进食率。在这里，我们概述了一种可标准化的实验室测试，用于使用gribble评估木材的生物降解。
Introduction
蛀木虫可对海洋木结构造成广泛损害，如海防、码头和水产养殖结构;更换或修复，全世界每年花费数十亿美元1，2，3。为了保护这些结构，木材通常经过处理以减少生物降解。然而，由于澳大利亚、欧盟、英国和美国在海洋环境中限制使用广谱杀菌剂，因此人们追捧对蛀虫具有天然耐久性的新型改性技术和木材种类4，5，6，7。在海洋环境中保存木材的新技术需要彻底的测试，以满足监管标准并限制任何化学防腐剂浸出等危害对环境的影响。例如，欧洲标准EN 275是1992年的现行欧洲标准，用于评估木材防腐处理对海洋蛀木器的损害8，9。该标准与其他禁止使用杀生物化合物的立法（如CCA4，5，6，7 和杂酚油10）一起，需要可持续的，无毒的木材保护方法，并使用天然耐用的木材物种来取代杀菌处理11，12.海洋试验，例如EN 275中规定的试验，需要较长的暴露期，因此成本高昂且缓慢，无法产生有意义的结果。然而，实验室测试为保护木材产品免受海洋蛀木虫攻击的测试方法提供了一种更快的替代方案，可以快速评估对处理时间表的调整13。这种快速实验室实验的结果旨在为木材的新型改性过程提供信息，并确定具有天然耐久性的木材物种，以应对蛀虫的损害。低进料速率和活力可能表明潜在产品的抗性增加，然后可以将此信息反馈给行业合作伙伴，以使他们能够改进设计。我们的方法允许灵活和快速的响应，这在工业中是可取的，一旦确定了有前途的产品，就可以用海洋试验的结果来补充。
Gribbles（Limnoria）是Limnoriidae家族中的等足甲壳类动物属。全世界有超过60种Limnoria13，14，15，在英国发现了三种常见物种，Limnoria lignorum，Limnoria tripunctata和Limnoria quadripunctata16。他们在淹没在海水中的木材表面开挖隧道，经常造成经济上的重大损害。Gribbles在英国沿海水域非常丰富，并且在实验室条件下易于维护，使其成为研究海洋蛀木无脊椎动物木材生物降解的理想生物。评估不同木材种类和木材保存方法上的进料速率和活力可以确定其抗生物降解的功效。以下协议列出了一种测量磨料速率的标准方法，该方法由Borges及其同事12，17开发而成，此外还简化了图像分析的引入，使该过程可以在非专业实验室中操作。图像分析还用于减少手动计数大量样品的实际限制。根据英国标准EN350-1：1994，长期海洋测试的耐久性根据樟子松边材18进行分级。在这里介绍的短期实验室测试中，我们使用苏格兰松（Pinus sylvestris L）边材作为测试ekki（Lophira alata Banks ex C.F Gaertn），山毛榉（Fagus sylvatica L），甜栗（Castanea sativa Mill）和松节油（Syncarpia glomulifera（Sm.）Nied）的心材的对照。使用每种木材品种八次重复的平均粪便颗粒产量和活力作为耐久性的指标。我们提供从典型评估中收集的说明性数据，使用格里布物种Limnoria quadripunctata和一系列天然耐用的木材物种。由Menzies（1951）提供的钥匙确定的Limnoria quadripunctata被选为生物降解试验的最佳物种，因为它是该家族中研究最充分的成员，并且被公认为用于生物降解试验的模型物种。该协议也适用于测试不同处理的木材，尽管使用的对照应该是未经处理的同一物种的复制。
Protocol
1. 准备测试棒 完成任何处理过程后，将干燥的木材切割成尺寸为2 mm x 4 mm x 20 mm的测试棒（图1）。在实验室条件下，风干粘在恒定的重量上。使用至少5个被测试木材的复制品。 图1：用于短期实验室测试的测试棒…
Representative Results
L.2020年11月，使用五种不同的木材类型（苏格兰松（Pinus sylvestris L）边材和山毛榉（Fagus sylvatica L），ekki（Lophira alata Banks ex C. F Gaertn），甜栗子（Castanea sativa Mil）和松节油（Syncarpia glomulifera （Sm.） Neid））的心材（见材料表）进行了为期20天的四分鼎目。每个木种使用八根复制棒，每根木棍喂养一个四尖瓣木的标本。所有鲷鱼都?…
Discussion
在选择用于喂养实验的试样之前，应对个体进行筛选以评估其适用性。由于体型的差异，个体之间的进食速率可能存在一些差异，因此应仅选择完全生长的成年标本。Borges等人在1.5毫米和3毫米长之间的个体的进食速率之间没有显着差异，200917。雌性 Limnoria 孵化卵，在此期间喂养率降低。因此，在选择标本时，应检查并丢弃任何育雏雌性。同样，蜕皮个体的进食率也会降?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
感谢挪威研究委员会（奥斯陆区域基金，Alcofur rffofjor 269707）和朴茨茅斯大学（理学院博士研究助学金）为露西·马丁的研究提供资金。此外，Gervais S. Sawyer提供了用于产生代表性结果的木材。松节油由不列颠哥伦比亚大学的Philip Evans教授提供。
Materials
| 12-well cell culture plates | ThermoFisher Scientific | 150200 | |
| 50ml Falcon tubes | Fisher Scientific | 14-432-22 | |
| Adjustable volume pipette | Fisher Scientific | FBE10000 | 1-10 ml |
| Beech | G. Sawyer (consultant in timber technology) | Fagus sylvatica | Taxonomic authority: L |
| Ekki | G. Sawyer (consultant in timber technology) | Lophira alata | Taxonomic authority: Banks ex C. F. Gaertn. |
| Forceps | Fisher Scientific | 10098140 | |
| Incubator | LMS LTD | INC5009 | |
| Microporous specimen capsules | Electron Microscopy Sciences | 70187-20 | |
| Petri dish | Fisher Scientific | FB0875713 | |
| Scots Pine | G. Sawyer (consultant in timber technology) | Pinus sylvestris | Taxonomic authority: L. |
| Size 00000 paintbrush | Hobby Craft | 5674331001 | Size 000 or 0000 also acceptable |
| Sweet Chestnut | G. Sawyer (consultant in timber technology) | Castanea sativa | Taxonomic authority: Mill |
| Turpentine | P. Evans (Professor, Dept. Wood Science, University of British Columbia) | Syncarpia glomulifera | Taxonomic authority: (Sm.) Nied. |
| Vacuum desiccator | Fisher Scientific | 15544635 |
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Cite This Article
Martin, L. S., Shipway, J. R., Martin, M. A., Malyon, G. P., Akter, M., Cragg, S. M. Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans. J. Vis. Exp. (179), e62776, doi:10.3791/62776 (2022).