使用加热块确定浮游动物的热限值
Summary
本协议说明了使用市售组件来产生稳定和线性的热梯度。然后，这种梯度可用于确定浮游生物，特别是无脊椎动物幼虫的热上限。
Abstract
热极限和广度已被广泛用于预测物种分布。随着全球气温的持续上升，了解热极限如何随着驯化而变化以及它在生命阶段和种群之间如何变化对于确定物种对未来变暖的脆弱性至关重要。大多数海洋生物具有复杂的生命周期，包括早期浮游阶段。虽然量化这些小的早期发育阶段（数十到数百微米）的热极限有助于识别发育瓶颈，但由于目标生物体体积小、工作台空间要求大以及初始制造成本高，这一过程可能具有挑战性。这里介绍了一种面向小体积（mL至数十mL）的设置。该设置结合了市售组件，以产生稳定和线性的热梯度。还介绍了该装置的生产规格，以及引入和枚举活体与死体以及计算致死温度的程序。
Introduction
耐热性是生物生存和功能的关键1，2。随着人为碳排放导致地球继续变暖，热极限的确定和应用越来越受到关注3.各种终点，如死亡率、发育迟缓和活动能力丧失，已被用于确定热上限和下限4。这些热极限通常被认为是生物体热生态位的代表。这些信息反过来用于识别更容易受到全球变暖影响的物种，以及预测未来的物种分布和由此产生的物种相互作用3，5，6，7。然而，确定热极限，特别是对于小型浮游生物，可能具有挑战性。
对于浮游生物，特别是海洋无脊椎动物的幼虫阶段，可以通过长期暴露来确定热极限。慢性暴露是通过在数天至数周内在几个温度下饲养幼虫并确定幼虫存活率和/或发育率降低的温度来实现的8，9，10。然而，这种方法相当耗时，需要大型孵化器和幼虫饲养经验（有关培养海洋无脊椎动物幼虫的良好介绍，请参见参考文献11）。
或者，急性暴露于热应力可用于确定热极限。通常，这种测定方法涉及将带有幼虫的小瓶放入温控干浴12，13，14中，利用PCR热循环仪15，16中的热梯度功能，或将玻璃小瓶/微量离心管沿着施加加热和冷却产生的热梯度放置在大铝块的末端，这些铝块带有小瓶紧贴的孔17， 18，19.典型的干浴产生单一温度;因此，必须同时运行多个单元，以评估不同温度范围内的性能。热循环仪产生梯度，但只能容纳少量样品（120 μL），并且需要仔细操作。与热循环仪类似，大型铝块可产生线性且稳定的温度梯度。这两种方法都可以与逻辑或概率回归相结合，以计算50%人口（LT50）的致死温度12，20，21。但是，使用的铝块长~100厘米;这种尺寸需要较大的实验室空间，并使用专门的计算机数控铣床来钻孔。再加上使用两个研究级水浴来保持目标温度，组装装置的财务成本很高。
因此，这项工作旨在开发一种替代方法，以使用市售部件生成稳定的线性温度梯度。这种产品必须具有较小的占地面积，并且应该能够轻松用于浮游生物的急性热应力暴露实验。该协议是用大小为<1 mm的浮游动物作为目标生物开发的，因此，它针对使用1.5或2 mL微量离心管进行了优化。较大的研究生物体将需要大于所用 1.5 mL 微量离心管的容器和铝块上的扩大孔。
除了使实验装置更易于访问之外，这项工作还旨在简化数据处理管道。虽然商业统计软件提供了使用逻辑或概率回归计算LT50 的例程，但许可成本并非微不足道。因此，依赖于开源统计程序R22 的易于使用的脚本将使数据分析更易于访问。
该协议展示了如何使用市售部件制造紧凑的加热块，并应用于将浮游动物（沙美元 Dendraster excentricus的幼虫）暴露于急性热应激以确定其热上限。
Protocol
1. 加热块的制造 将 120 V、100 W 带状加热器连接到变阻器（参见 材料表）。 通过在 6 x 10 网格中钻 60 个孔来准备 20.3 厘米 x 15.2 厘米 x 5 厘米（8 英寸 x 5 英寸 x 2 英寸）铝块（请参阅 材料表）。确保孔在两个方向上从中心到中心间隔 2 厘米。每个直径应为1.1厘米，深4.2厘米（图1）。注意： 使用高速钢钻头在铣床或钻床…
Representative Results
该协议的目标是确定浮游动物的热上限。为此，需要稳定和线性的热梯度。通过将水浴温度设置为8°C，将加热器设置为39°C，所提出的设置能够产生14°C至40°C的热梯度（图2A）。温度梯度可以通过改变端点值来缩小和移动。通过将加热器设置为37°C，将水浴设置为15°C，还产生了范围较窄（19°C至37°C）的热梯度。 块中的温度在设置后45分钟至1小时内稳定（图2…
Discussion
该协议提供了一种可访问和可定制的方法，通过急性热暴露来确定小型浮游生物的热极限。10孔设计和灵活的温度端点，由下端的水浴和上端的加热器控制，使人们能够精确地确定LT50。使用这种方法，可以检测到<1°C的热极限差异（图3）。这种方法可以快速确定各种物种的热极限（以小时为单位），并且结果值已应用于多种物种分布模型2，</sup…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了斯沃斯莫尔学院[KC]的教师研究基金以及BJ的Robert Reynolds和Lucinda Lewis’70暑期研究奖学金的支持。
Materials
| 0.45 µm membrane filter | VWR | 74300-042 | |
| ½” Acrylic sheet | McMaster-Carr | 8560K266 | Used to construct a ridged case with sufficient insulation. |
| 1 mL syringe | VWR | 76290-420 | |
| 2 Channel 7 Thermocouple Types Datalogger | Omega Engineering | HH506A | Can be replaced with any thermometer that will fit inside a microcentrifuge tube |
| Automatic pipette | Ranin | ||
| Bolt- and Clamp-Mount Strip Heater with 430 Stainless Steel Sheath, 120V AC, 1-1/2" Wide, 100W |
McMaster-Carr | 3619K32 | |
| Crystal Sea Bioassay Mix | Pentair | CM2B | Use to make aritifical seawater |
| Denraster excentricus | M-Rep | Sand dollars from California | |
| Dissecting microscope | Nikon | SMZ645 | |
| DIYhz Aluminum Water Cooling Block, Liquid Water Cooler Heat Sink System for PC Computer CPU Graphics Radiator Heatsink Endothermic Head Silver(40 mm x 120 mm x 12 mm) | Amazon | Connects to water bath and used to cool one end of the block. | |
| Easy-to-Machine MIC6 Cast Aluminum Sheet 2" thick 8" x 8" | McMaster-Carr | 86825K953 | Machined to 2" x 6" x 8" with 60 equally spaced holes (11 mm dia., 42 mm depth) with two addition holes drilled in one side for thermostat probes. |
| Economical Flexible Polyethylene Foam Pipe Insulation | McMaster-Carr | 4530K121 | Covers the plastic tubing between chiller and block to reduce heat loss. Can be omitted if temperature range is close to room temperature |
| EVERSECU 72w 110-240v Aquarium Water Chiller Warmer/Cooler Temperature Controller for Fish Shrimp Tank Marine Coral Reef Tank Below 20 L/30 L Aquarium Chiller | Amazon | Can be used in place of the lab-grade water bath | |
| Example with larval sand dollar | |||
| GENNEL 100 g Silver Silicone Thermal Conductive Compound Grease Paste For GPU CPU IC LED Ovens Cooling | Amazon | Improves the thermal conductance between the block and the heating and cooling elements. | |
| Inkbird WiFi Reptile Thermostat Temperature Controller with 2 Probes and 2 Outlets, IPT-2CH Reptiles Heat Mat Thermostat (Max 250 W per Outlet) | Amazon | Monitors hot and cold ends. Maintains hot end in range | |
| Lauda Ecoline Silver Air-Cooled Refrigerated Circulators | VWR | 89202-386 | Can be replaced with an aquarium chiller |
| Microcentrifuge Tubes | VWR | 76019-014 | If larger animals are used, scanilation vials (VWR 66022-004) is a good alternative |
| Nitex mesh filter | Self made | Used hot glue to attached Nitex mesh to 1/2" PVC tubing | |
| Pasteur pipette | VWR | 14673-010 | |
| Potassium Chloride (0.35 M) | Millpore-Sigma | P3911-500G | |
| R statistical software. | The R Project for Statistical Computing | ||
| Syringe needle | VWR | 89219-346 | Depending on size of target organism gague 14 and 16 can be used |
| Tygon Tubing | McMaster-Carr | 5233K65 | Adjust to match the chiller and block used |
| Zoo Med Repti Temp Rheostat | Chewy.com | Rated to 150 W and rewired to feed directly into the heating element. Used to control rate of heat output |
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Citer Cet Article
Chan, K. Y. K., Jorgensen, B. K., Scoma, S. Thermal Limits Determination for Zooplankton Using a Heat Block. J. Vis. Exp. (189), e64762, doi:10.3791/64762 (2022).