Name: thermal limits determination for zooplankton using a heat block
Uploaded: 2022-11-18T14:50:00
Description: Watch this Scientific Journal Video about Thermal Limits Determination for Zooplankton Using a Heat Block at JoVE.com

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。

1. 加热块的制造
<ol><li>将 120 V、100 W 带状加热器连接到变阻器（参见 材料表）。</li>
	<li>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...

<ol>
	<li>Dowd, W. W., King, F. A., Denny, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+variation,+thermal+extremes+and+the+physiological+performance+of+individuals.">Thermal variation, thermal extremes and the physiological performance of individuals.</a> Journal of Experimental Biology. 218 (12), 1956-1967 (2015).</li><li>Garc&#237;a, F. C., Bestion, E., Warfield, R., Yvon-Durocher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temperature+alter+the+relationship+between+biodiversity+and+ecosystem+functioning.">Changes in temperature alter the relationship between biodiversity and ecosystem functioning.</a> Proceedings of the National Academy of Sciences. 115 (43), 10989-10994 (2018).</li><li>Sinclair, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+predict+ectotherm+responses+to+climate+change+using+thermal+performance+curves+and+body+temperatures.">Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures.</a> Ecology Letters. 19 (11), 1372-1385 (2016).</li><li>Lutterschmidt, W. I., Hutchison, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+thermal+maximum:+history+and+critique.">The critical thermal maximum: history and critique.</a> Canadian Journal of Zoology. 75 (10), 1561-1574 (1997).</li><li>Bennett, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+critical+thermal+limits+of+life+on+Earth.">The evolution of critical thermal limits of life on Earth.</a> Nature Communications. 12 (1), 1198 (2021).</li><li>Sunday, J. M., Bates, A. E., Dulvy, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+tolerance+and+the+global+redistribution+of+animals.">Thermal tolerance and the global redistribution of animals.</a> Nature Climate Change. 2 (9), 686-690 (2012).</li><li>Deutsch, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+climate+warming+on+terrestrial+ectotherms+across+latitude.">Impacts of climate warming on terrestrial ectotherms across latitude.</a> Proceedings of the National Academy of Sciences. 105 (18), 6668-6672 (2008).</li><li>Collin, R., Chan, K. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+urchin+Lytechinus+variegatus+lives+close+to+the+upper+thermal+limit+for+early+development+in+a+tropical+lagoon.">The sea urchin Lytechinus variegatus lives close to the upper thermal limit for early development in a tropical lagoon.</a> Ecology and Evolution. 6 (16), 5623-5634 (2016).</li><li>Wang, W., Ding, M. -. w., Li, X. -. x., Wang, J., Dong, Y. -. w. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+thermal+sensitivities+among+different+life+stages+of+the+pulmonate+limpet+Siphonaria+japonica.">Divergent thermal sensitivities among different life stages of the pulmonate limpet Siphonaria japonica.</a> Marine Biology. 164 (6), 1-10 (2017).</li><li>Mak, K. K. -. Y., Chan, K. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+effects+of+temperature+and+salinity+on+early+life+stages+of+the+sea+urchin+Heliocidaris+crassispina.">Interactive effects of temperature and salinity on early life stages of the sea urchin Heliocidaris crassispina.</a> Marine Biology. 165 (3), 1-11 (2018).</li><li>Strathmann, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+larva+of+marine+invertebrates.">Culturing larva of marine invertebrates.</a> Developmental Biology of the Sea Urchin and Other Marine Invertebrates. , 1-25 (2014).</li><li>Stillman, J. H., Somero, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+the+upper+thermal+tolerance+limits+of+Eastern+Pacific+porcelain+crabs,+Genus+Petrolisthes:+Influences+of+latitude,+vertical+Zonation,+acclimation,+and+phylogeny.">A comparative analysis of the upper thermal tolerance limits of Eastern Pacific porcelain crabs, Genus Petrolisthes: Influences of latitude, vertical Zonation, acclimation, and phylogeny.</a> Physiological and Biochemical Zoology. 73 (2), 200-208 (2000).</li><li>Sasaki, M. C., Dam, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+patterns+of+thermal+tolerance+and+phenotypic+plasticity+with+population+genetics+to+improve+understanding+of+vulnerability+to+warming+in+a+widespread+copepod.">Integrating patterns of thermal tolerance and phenotypic plasticity with population genetics to improve understanding of vulnerability to warming in a widespread copepod.</a> Global Change Biology. 25 (12), 4147-4164 (2019).</li><li>Sasaki, M. C., Dam, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+differentiation+underlies+seasonal+variation+in+thermal+tolerance,+body+size,+and+plasticity+in+a+short-lived+copepod.">Genetic differentiation underlies seasonal variation in thermal tolerance, body size, and plasticity in a short-lived copepod.</a> Ecology and Evolution. 10 (21), 12200-12210 (2020).</li><li>Kelly, M. W., Sanford, E., Grosberg, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+potential+for+adaptation+to+climate+change+in+a+broadly+distributed+marine+crustacean.">Limited potential for adaptation to climate change in a broadly distributed marine crustacean.</a> Proceedings of the Royal Society B: Biological Sciences. 279 (1727), 349-356 (2012).</li><li>Rivera, H. E., Chen, C. -. Y., Gibson, M. C., Tarrant, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+in+parental+effects+confers+rapid+larval+thermal+tolerance+in+the+estuarine+anemone+Nematostella+vectensis.">Plasticity in parental effects confers rapid larval thermal tolerance in the estuarine anemone Nematostella vectensis.</a> Journal of Experimental Biology. 224 (5), 236745 (2021).</li><li>Sewell, M. A., Young, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+limits+to+fertilization+and+early+development+in+the+tropical+sea+urchin+Echinometra+lucunter.">Temperature limits to fertilization and early development in the tropical sea urchin Echinometra lucunter.</a> Journal of Experimental Marine Biology and Ecology. 236 (2), 291-305 (1999).</li><li>Walther, K., Crickenberger, S. E., Marchant, S., Marko, P. B., Moran, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+tolerance+of+larvae+of+Pollicipes+elegans,+a+marine+species+with+an+antitropical+distribution.">Thermal tolerance of larvae of Pollicipes elegans, a marine species with an antitropical distribution.</a> Marine Biology. 160 (10), 2723-2732 (2013).</li><li>Byrne, M., Gall, M. L., Campbell, H., Lamare, M. D., Holmes, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staying+in+place+and+moving+in+space:+contrasting+larval+thermal+sensitivity+explains+distributional+changes+of+sympatric+sea+urchin+species+to+habitat+warming.">Staying in place and moving in space: contrasting larval thermal sensitivity explains distributional changes of sympatric sea urchin species to habitat warming.</a> Global Change Biology. 28 (9), 3040-3053 (2022).</li><li>Zippay, M. L., Hofmann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+tolerances+across+latitudes:+thermal+sensitivity+of+larval+marine+snails+(Nucella+spp).">Physiological tolerances across latitudes: thermal sensitivity of larval marine snails (Nucella spp).</a> Marine Biology. 157 (4), 707-714 (2010).</li><li>Collin, R., Rebolledo, A. P., Smith, E., Chan, K. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+tolerance+of+early+development+predicts+the+realized+thermal+niche+in+marine+ectotherms.">Thermal tolerance of early development predicts the realized thermal niche in marine ectotherms.</a> Functional Ecology. 35 (8), 1679-1692 (2021).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+language+and+environment+for+statistical+computing.">R: A language and environment for statistical computing.</a> R Foundation for Statistical Computing. , (2021).</li><li>Venables, W. N., Ripley, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern Applied Statistics with S-PLUS. Fourth edn. , (2002).</li><li>Fumo, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contextualizing+marine+heatwaves+in+the+southern+California+bight+under+anthropogenic+climate+change.">Contextualizing marine heatwaves in the southern California bight under anthropogenic climate change.</a> Journal of Geophysical Research: Oceans. 125 (5), (2020).</li><li>Wheeler, M. W., Park, R. M., Bailer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+median+lethal+concentration+values+using+confidence+interval+overlap+or+ratio+tests.">Comparing median lethal concentration values using confidence interval overlap or ratio tests.</a> Environmental Toxicology and Chemistry: An International Journal. 25 (5), 1441-1444 (2006).</li><li>Kingsolver, J. G., MacLean, H. J., Goddin, S. B., Augustine, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+upper+thermal+limits+to+acute+and+chronic+temperature+variation+in+Manduca+sexta+larvae.">Plasticity of upper thermal limits to acute and chronic temperature variation in Manduca sexta larvae.</a> Journal of Experimental Biology. 219 (9), 1290-1294 (2016).</li><li>Kuo, E. S. L., Sanford, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+in+the+upper+thermal+limits+of+an+intertidal+snail:+implications+for+climate+envelope+models.">Geographic variation in the upper thermal limits of an intertidal snail: implications for climate envelope models.</a> Marine Ecology Progress Series. 388, 137-146 (2009).</li><li>Hammond, L. M., Hofmann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+tolerance+of+Strongylocentrotus+purpuratus+early+life+history+stages:+mortality,+stress-induced+gene+expression+and+biogeographic+patterns.">Thermal tolerance of Strongylocentrotus purpuratus early life history stages: mortality, stress-induced gene expression and biogeographic patterns.</a> Marine biology. 157 (12), 2677-2687 (2010).</li><li>Sasaki, M., Dam, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+patterns+in+copepod+thermal+tolerance.">Global patterns in copepod thermal tolerance.</a> Journal of Plankton Research. 43 (4), 598-609 (2021).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#181;m membrane filter</td>
			<td>VWR</td>
			<td>74300-042</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K266</td>
			<td>Used to construct a ridged case with sufficient insulation.</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>VWR</td>
			<td>76290-420</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Channel 7 Thermocouple Types Datalogger</td>
			<td>Omega Engineering</td>
			<td>HH506A</td>
			<td>Can be replaced with any thermometer that will fit inside a microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Automatic pipette&#160;</td>
			<td>Ranin&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt- and Clamp-Mount Strip Heater 
			with 430 Stainless Steel Sheath, 120V AC, 1-1/2&#34; Wide, 100W</td>
			<td>McMaster-Carr</td>
			<td>3619K32</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Sea Bioassay Mix</td>
			<td>Pentair</td>
			<td>CM2B</td>
			<td>Use to make aritifical seawater&#160;</td>
		</tr>
		<tr>
			<td>Denraster excentricus</td>
			<td>M-Rep&#160;</td>
			<td></td>
			<td>Sand dollars from California&#160;</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Nikon&#160;</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>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)</td>
			<td>Amazon</td>
			<td></td>
			<td>Connects to water bath and used to cool one end of the block.</td>
		</tr>
		<tr>
			<td>Easy-to-Machine MIC6 Cast Aluminum Sheet 2&#34; thick 8&#34; x 8&#34;&#160;</td>
			<td>McMaster-Carr</td>
			<td>86825K953</td>
			<td>Machined to 2&#34; x 6&#34; x 8&#34; with 60 equally spaced holes (11 mm dia., 42 mm depth) with two addition holes drilled in one side for thermostat probes.</td>
		</tr>
		<tr>
			<td>Economical Flexible Polyethylene Foam Pipe Insulation</td>
			<td>McMaster-Carr</td>
			<td>4530K121</td>
			<td>Covers the plastic tubing between chiller and block to reduce heat loss. Can be omitted if temperature range is close to room temperature&#160;</td>
		</tr>
		<tr>
			<td>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</td>
			<td>Amazon</td>
			<td></td>
			<td>Can be used in place of the lab-grade water bath&#160;</td>
		</tr>
		<tr>
			<td>Example with larval sand dollar&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GENNEL 100 g Silver Silicone Thermal Conductive Compound Grease Paste For GPU CPU IC LED Ovens Cooling</td>
			<td>Amazon</td>
			<td></td>
			<td>Improves the thermal conductance between the block and the heating and cooling elements.</td>
		</tr>
		<tr>
			<td>Inkbird WiFi Reptile Thermostat Temperature Controller with 2 Probes and 2 Outlets, IPT-2CH Reptiles Heat Mat Thermostat (Max 250 W per Outlet)</td>
			<td>Amazon</td>
			<td></td>
			<td>Monitors hot and cold ends. Maintains hot end in range</td>
		</tr>
		<tr>
			<td>Lauda Ecoline Silver Air-Cooled Refrigerated Circulators</td>
			<td>VWR</td>
			<td>89202-386</td>
			<td>Can be replaced with an aquarium chiller&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>VWR</td>
			<td>76019-014</td>
			<td>If larger animals are used, scanilation vials (VWR 66022-004) is a good alternative&#160;</td>
		</tr>
		<tr>
			<td>Nitex mesh filter</td>
			<td>&#160;Self made</td>
			<td></td>
			<td>Used hot glue to attached Nitex mesh to 1/2&#34; PVC tubing&#160;</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (0.35 M)&#160;</td>
			<td>Millpore-Sigma</td>
			<td>P3911-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>R statistical software.&#160;</td>
			<td>The R Project for Statistical Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>VWR</td>
			<td>89219-346</td>
			<td>Depending on size of target organism gague 14 and 16 can be used</td>
		</tr>
		<tr>
			<td>Tygon Tubing&#160;</td>
			<td>McMaster-Carr</td>
			<td>5233K65</td>
			<td>Adjust to match the chiller and block used&#160;</td>
		</tr>
		<tr>
			<td>Zoo Med Repti Temp Rheostat</td>
			<td>Chewy.com</td>
			<td></td>
			<td>Rated to 150 W and rewired to feed directly into the heating element. Used to control rate of heat output</td>
		</tr>
	</tbody>
</table>

thermal limits determination for zooplankton using a heat block

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。

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...

Watch this Scientific Journal Video about Thermal Limits Determination for Zooplankton Using a Heat Block at JoVE.com

Thermal Limits Determination for Zooplankton Using a Heat Block

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。

使用加热块确定浮游动物的热限值

Thermal limits and breadth have been widely used to predict species distribution. As the global temperature continues to rise, understanding how thermal ...

随着全球气温持续上升，量化热极限以及与驯化和个体发生的关系对于确定物种对未来变暖的脆弱性至关重要。对于具有复杂生活史的海洋生物，确定热极限在逻辑上可能具有挑战性。该协议引入了一种占地面积小，易于构建的方法，以估计小型浮游生物的临界温度。虽然该方法是为小于一毫米的小型浮游生物开发的，但它可以用于适合约50毫升体积的闪烁瓶的大型海洋生物。首先将带状加热器连接到变阻器。在 6 x 10 尺寸的网格中钻 60 个孔，以准备尺寸为 20.3 x 15.2 x 5 厘米的铝块，并确保孔在两个方向上从中心到中心间隔 2 厘米。在第一列和第二列以及第九列和第十列之间钻两个额外的孔，与温度控制器探头的尺寸相匹配。为了将元件固定到位并对完成的加热块进行绝缘，请用透明丙烯酸板构建一个外壳，确保在加热元件的背面应用两层。在最终装配中，涂抹导热膏以最大限度地提高从加热元件到块以及从块到冷却元件的热传导。用Tygon管连接水浴，并将恒温器探头插入铝块侧面的孔中。在所有铣削的孔中，放置1.5毫升的微量离心管，装满自来水。打开温度控制器，将探头一的停止加热温度设置为35至37摄氏度，探头二设置为21.5至22.5摄氏度。旋转变阻器以打开加热元件并将其设置为中等。打开水浴并将冷却器温度设置为 15 摄氏度。使用带有K型电极的热电偶，之后每10分钟检查一次每个微量离心管内的温度。根据需要通过更改温度控制器和水浴的设置来调整端点的值。打开循环水浴和加热器，分别设置为15摄氏度和37摄氏度，以产生从19.5摄氏度到37摄氏度的温度梯度。一旦将微量离心管放置在铣削孔中并达到加热块的温度，请使用带有K型电极的热电偶检查每个微量离心管内的温度。并记下这些温度。用海水填充1.5毫升微量离心管，通过0.45微米网过滤。通过反向过滤浓缩研究生物体的培养物，使生物体保持在烧杯底部。用过滤的海水冲洗浓缩的培养物，并再次重复反向过滤以浓缩样品。在解剖显微镜下计数小型浮游生物，并使用玻璃巴斯德移液管将已知数量的生物转移到半填充的微量离心管中。现在加入过滤的海水，直到这些管中的最终体积达到一毫升。现在将这些管成对放入加热块中，从冷端开始，让生物体逐渐升温到所需的实验温度。等待 10 分钟，然后将成对的微量离心管移动到温度较高的相邻钻孔中。在冷端的每一排中放置额外的微量离心管，并继续成对将它们移向较温暖的一端。填充整个块后，在指定温度下孵育两个小时。在孵育期结束时，测量每个试管中的温度并记下。然后将所有试管转移到预先标记的支架中，并在预定温度下孵育一小时以使其恢复。为了计数活生物体部分，使用玻璃移液管将单个微量离心管的内容物转移到35毫米培养皿中。在解剖显微镜下，数活生物体和死生物体并记下数字。观察到的生物数量应与最初采集的生物体相匹配。如果没有，请检查微量离心管和培养皿的侧面，生成CSV格式的数据表，其中标题对感兴趣的变量，管的温度（以摄氏度为单位），活着的个体数和死亡的个体数。要使用逻辑回归拟合数据，请使用具有二项分布的广义线性模型。若要运行模型，请键入 source，并使用 R 文件 modelloop.r。计算 50% 个体存活的预测变量值，以确定中位数热上限。使用该协议，在受精后两天，四天和六天测量幼虫沙美元的存活率，该存活率在19至37摄氏度的温度范围内。随着幼虫砂美元的发展，热上限从受精后两天的28.6摄氏度增加到受精后四天的28.8摄氏度，以及受精后六天的约29摄氏度。孵化和恢复时间因物种而异。进行初步测试以确保所选时间产生可靠的活与死估计非常重要。

Research

JoVE Journal

Biology

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

在蚊子（埃及斑蚊）感染引起的表型测定为登革热感染病例的议定书“

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

科研

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Rapid PCR Thermocycling using Microscale Thermal Convection

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

使用串行块面和聚焦的Ion光束扫描电子显微镜进行有针对性的研究

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

使用热像仪测量通过鸟羽毛大衣的热损失

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...