We thank Ellie McCabe for assistance with fly rearing. This work is supported by United States Department of Agriculture National Institute of Food and Agriculture Hatch Project grant 1010996 and National Science Foundation grant OIA-1826689 to N.M.T.

1. High-throughput CTmin assay
<ol>
	<li>Assembling the jacketed column (Figure 1A, Figure 2)
	<ol>
		<li>Cut the widest (7 cm x 6.35 cm x 0.3 cm) and narrowest (5.7 cm x 5.1 cm x 0.3 cm) acrylic tubes to equal lengths (31.5 cm) with a hacksaw (Figure 2A). These two tubes will be the outer and innermost walls of the jacketed column.</li>
		<li>Cut two rings (2 cm wide) from the midsized...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+rapid+measurement+of+thermal+tolerance+traits+in+studies+of+small+insects.">A method for the rapid measurement of thermal tolerance traits in studies of small insects.</a> Physiological Entomology. 33 (4), 389-394 (2008).</li><li>Andersen, J. L., 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=How+to+assess+Drosophila+cold+tolerance:+Chill+coma+temperature+and+lower+lethal+temperature+are+the+best+predictors+of+cold+distribution+limits.">How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits.</a> Functional Ecology. 29 (1), 55-65 (2015).</li><li>Hu, X. P., Appel, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+thermal+limits,+temperature+tolerance+and+water+balance+of+a+sub-Antarctic+kelp+fly,+Paractora+dreuxi+(Lepidoptera:+Tineidae).">Critical thermal limits, temperature tolerance and water balance of a sub-Antarctic kelp fly, Paractora dreuxi (Lepidoptera: Tineidae).</a> Journal of Insect Physiology. 43, 685-694 (1997).</li><li>Salachan, P. V., Burgaud, H., S&#248;rensen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+thermal+limits:+Non-linear+reaction+norms+drive+disparate+thermal+acclimation+responses+in+Drosophila+melanogaster.">Testing the thermal limits: Non-linear reaction norms drive disparate thermal acclimation responses in Drosophila melanogaster.</a> Journal of Insect Physiology. 118 (September), 103946 (2019).</li><li>Everatt, M. J., Bale, J. S., Convey, P., Worland, M. R., Hayward, S. 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=The+effect+of+acclimation+temperature+on+thermal+activity+thresholds+in+polar+terrestrial+invertebrates.">The effect of acclimation temperature on thermal activity thresholds in polar terrestrial invertebrates.</a> Journal of Insect Physiology. 59 (10), 1057-1064 (2013).</li><li>MacMillan, H. A., Sinclair, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+gut+in+insect+chilling+injury:+Cold-Induced+disruption+of+osmoregulation+in+the+fall+field+cricket,+Gryllus+pennsylvanicus.">The role of the gut in insect chilling injury: Cold-Induced disruption of osmoregulation in the fall field cricket, Gryllus pennsylvanicus.</a> Journal of Experimental Biology. 214 (5), 726-734 (2011).</li><li>Huey, R. B., Crill, W. D., Kingsolver, J. G., Weber, 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=A+method+for+rapid+measurement+of+heat+or+cold+resistance+of+small+insects.">A method for rapid measurement of heat or cold resistance of small insects.</a> British Ecological Society. 6 (4), 489-494 (1992).</li><li>Jenkins, N. L., Hoffmann, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+maternal+variation+for+heat+resistance+in+drosophila+from+the+field.">Genetic and maternal variation for heat resistance in drosophila from the field.</a> Genetics. 137 (3), 783-789 (1994).</li><li>Ransberry, V. E., MacMillan, H. A., Sinclair, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+chill-coma+onset+and+recovery+at+the+extremes+of+the+thermal+window+of+Drosophila+melanogaster.">The relationship between chill-coma onset and recovery at the extremes of the thermal window of Drosophila melanogaster.</a> Physiological and Biochemical Zoology. 84 (6), 553-559 (2011).</li><li>S&#248;rensen, M. H., 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=Rapid+induction+of+the+heat+hardening+response+in+an+Arctic+insect.">Rapid induction of the heat hardening response in an Arctic insect.</a> Biology Letters. 15 (10), (2019).</li><li>Shuman, D., Coffelt, J. A., Weaver, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-based+electronic+fall-through+probe+insect+counter+for+monitoring+infestation+in+stored+products.">A computer-based electronic fall-through probe insect counter for monitoring infestation in stored products.</a> Transactions of the American Society of Agricultural Engineers. 39 (5), 1773-1780 (1996).</li><li>MacKay, T. F. C., 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+Drosophila+melanogaster+Genetic+Reference+Panel.">The Drosophila melanogaster Genetic Reference Panel.</a> Nature. 482 (7384), 173-178 (2012).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. , (2005).</li><li>Garcia, M. J., Teets, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+stress+results+in+sustained+locomotor+and+behavioral+deficits+in+Drosophila+melanogaster.">Cold stress results in sustained locomotor and behavioral deficits in Drosophila melanogaster.</a> Journal of Experimental Zoology Part A: Ecological and Integrative Physiology. 331 (3), 192-200 (2019).</li><li>Teets, N. M., Hahn, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+shape+of+cold-survival+curves+in+a+single+fly+population+suggests+potential+for+selection+from+climate+variability.">Genetic variation in the shape of cold-survival curves in a single fly population suggests potential for selection from climate variability.</a> Journal of Evolutionary Biology. 31 (4), 543-555 (2018).</li><li>Kelty, J. D., Lee, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+rapid+cold+hardening+by+cooling+at+ecologically+relevant+rates+in+Drosophila+melanogaster.">Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster.</a> Journal of Insect Physiology. 45 (8), 719-726 (1999).</li><li>MacMillan, H. A., Sinclair, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+insect+chill-coma.">Mechanisms underlying insect chill-coma.</a> Journal of Insect Physiology. 57 (1), 12-20 (2011).</li><li>Salachan, P. V., S&#248;rensen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+thermal+limits+affected+differently+by+developmental+and+adult+thermal+fluctuations.">Critical thermal limits affected differently by developmental and adult thermal fluctuations.</a> Journal of Experimental Biology. 220 (23), 4471-4478 (2017).</li><li>Hoffmann, A. A., Hallas, R., Anderson, A. R., Telonis-Scott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+robust+sex-specific+trade-off+between+cold+resistance+and+starvation+resistance+in+Drosophila+melanogaster.">Evidence for a robust sex-specific trade-off between cold resistance and starvation resistance in Drosophila melanogaster.</a> Journal of Evolutionary Biology. 18 (4), 804-810 (2005).</li><li>Kelty, J. D., Lee, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+cold-hardening+of+Drosophila+melanogaster+(Diptera:+Drosophilidae)+during+ecologically+based+thermoperiodic+cycles.">Rapid cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles.</a> Journal of Experimental Biology. 204 (9), 1659-1666 (2001).</li><li>Sinclair, B. J., Vernon, P., Klok, C. J., Chown, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insects+at+low+temperatures:+An+ecological+perspective.">Insects at low temperatures: An ecological perspective.</a> Trends in Ecology and Evolution. 18 (5), 257-262 (2003).</li></ol>

The two methods detailed above generate high-throughput data of ecologically relevant metrics for upper and lower thermal limits. These protocols build upon previously established methodologies common to research on insect thermal limits (summarized in Sinclair et al.)6. Both protocols can be completed in a short amount of time (~2 h each), produce data sets with large sample sizes, do not sacrifice repeatability or accuracy, and minimize experimenter error by eliminating manual data record...

Thermal limits of insects 
Variation in environmental conditions, including temperature, is a major factor influencing the performance, fitness, survival, and geographic distribution of organisms1,2. Upper and lower thermal limits determine the theoretical range of environments an organism can tolerate, and, therefore, these limits are important predictors of plant and animal distributions, especially in the face of climate change3,4. Thus, protocols to accurately measure thermal limits are important tools for ecologists, physiologists, evolutionary biologists, and conservation biologists, among others.
As the most abundant and diverse terrestrial animals, insects are frequently used for measurements of thermal limits.&#160;Critical thermal maxima (CTmax) and critical thermal minima (CTmin) are commonly used to assess intra- and interspecific variation in thermal tolerance5,6,7. While CTmax and CTmin can be measured for multiple phenotypes, including growth, reproductive output, and behavior, they are most commonly applied to locomotor function5,6,7. Thus, CTmax (also called heat knockdown temperature) and CTmin are often defined as the high and low temperatures at which insects lose motor function and are unable to remain upright5,6,7,8,9,10,11. CTmin coincides with the onset of chill coma, a reversible paralysis brought on by cold temperatures6. While paralysis at the thermal limits is often reversible, continued exposure to these temperatures leads to ecological death5.
Common methods for measuring thermal limits 
A variety of apparatuses have been used to measure thermal limits (summarized in Sinclair et al.)6. Briefly, insects are heated or cooled in incubators12,13, containers submerged in fluid baths11,14,15,16, aluminum blocks10,17, or jacketed containers18, and monitored until locomotion ceases. To monitor insects during the assay, the most common method is direct observation, in which individuals are continuously monitored in real time or retrospectively with recorded video6,9,10,11,15,17. While direct observation methods have minimal equipment requirements, they are labor-intensive and limit throughput. Alternatively, insects can be observed indirectly by collecting individuals at discrete times as they fall from perches6,19,20,21 or using activity monitors13.
Indirect methods for measuring thermal limits are generally higher-throughput and potentially less error prone than direct observation methods. The most common method for indirect monitoring uses a jacketed temperature-controlled column6,8,19,20,21. Insects are placed inside a column with perches, and the temperature of the inner chamber is controlled by pumping fluid from a temperature-controlled fluid bath through the jacketed lining of the column. Individuals that reach their thermal limit fall from their perch and are collected at discrete temperatures or time intervals. While this method works well for CTmin, it has been found unsuitable for CTmax, because flies voluntarily walk out of the bottom of the column when the temperature increases. The new method described here circumvents this issue by individually containing flies during automated measurements.
In addition to the method of observation, two types of temperature regimes are commonly used to assess upper thermal limits. Dynamic assays consist of gradually increasing temperature until motor function is lost; that temperature is the dynamic CTmax7,8,9,13. In contrast, static assays consist of a constant stressful temperature until motor function is lost; that time point is the heat knockdown time (heat KDT), also called the static CTmax (sCTmax) in a recent paper by J&#248;rgensen et al.7,8,9,16,22. Although CTmax and heat knockdown assays (heat KD assays) produce metrics with different units, mathematical modeling of the two traits indicates they give comparable information on heat tolerance and are both ecologically relevant8,9. Dynamic assays yield a temperature that can be compared to environmental conditions, and they are preferable when there are large differences in heat tolerance, such as comparisons between species with widely different thermal niches. However, due to the high Q10 for heat injury accumulation, a static assay may be preferable for detecting small effect sizes, such as intraspecific variation in heat tolerance9. Also, practically speaking, a static assay requires less sophisticated equipment than a dynamic assay.
Objective 
The objective of this paper is to formalize methods for CTmin and heat KD assays that can be used in future research to assess the thermal limits of motile insects. The protocols are adapted from previously established methodologies and are designed to be high-throughput, automated, and cost-effective. Both assays can be completed in a short amount of time (~2 h), which means that multiple experiments can be conducted in a single day, producing large amounts of data without sacrificing repeatability or accuracy. With this setup, the heat tolerance of 96 flies can be measured simultaneously, while the column for CTmin can hold more than 100 flies, provided there is adequate surface area for perching.
The high-throughput method for observing CTmin modifies the common jacketed column methodology with the addition of an infrared sensor to automatically count flies. The use of an infrared sensor for counting was first proposed by Shuman et al. in 199623 but has not been widely adopted. The addition of the infrared sensor allows for the generation of continuous data rather than collecting data at discrete intervals. This protocol also minimizes experimenter error by eliminating manual data entry and the need to manually switch collection tubes below the jacked column at discrete time points.
The high-throughput method for recording heat KDT is modified from two previous studies of heat tolerance in insects10,12. Individual flies are stored in a 96 well plate in a temperature-controlled incubator and video is recorded. This protocol minimizes experimenter bias in determining heat KDT because experiments can be reviewed and verified by playing back the recording. This protocol also provides a set of custom Python scripts that can be used to speed up video analysis. The use of individual wells eliminates interference that can occur when other individuals move around or fall over, which can be a problem when groups of individuals are observed in the same arena10,17. Furthermore, the temperature-controlled incubator provides a stable temperature across all 96 wells, unlike the temperature gradient sometimes observed across a temperature-controlled aluminum block10. Also note that the 96 well recording method can be adapted to measure dynamic CTmax and potentially CTmin (see Discussion).
To demonstrate each protocol, the thermal limits of adult Drosophila melanogaster females from select lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) were compared24. These lines were selected because preliminary experiments indicated significant differences in thermal tolerance. These assays proved to be robust methods for discriminating differences in thermal tolerance. The following two protocols, high-throughput CTmin assay (section 1) and high-throughput heat KD assay (section 2), describe the necessary actions to produce CTmin and heat KDT data for any motile insect life stage capable of fitting in the apparatuses, such as adult Drosophila. For CTmin it is also essential that the insect be able to perch. Here, each assay is demonstrated in adult Drosophila melanogaster. However, modifications may be required for other taxa or life stages6. Minor changes might include using perching material with larger openings to accommodate larger specimens in the CTmin assay or using a higher quality camera to discern the subtle KDT of a slow moving insect or life stage in the heat KD assay. This protocol does not describe methods for preparing flies, but it is important to standardize rearing protocols to ensure repeatability25 (see Garcia and Teets26 and Teets and Hahn27). The protocols provided include information on how to build and set up the apparatuses, how to record measurements, and a brief description of data analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARCTIC A40 Refrigerated fluid circulator (Programable teperature ramps)</td>
			<td>Thermo Scientific; Waltham, MA</td>
			<td>153-5401</td>
			<td></td>
		</tr>
		<tr>
			<td>C922 Pro Stream Webcam</td>
			<td>Logitech; Newark, CA</td>
			<td>960-001087</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular adjustable steel clamp &#8211; 5.08 cm to 7.62 cm</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 5.7 cm x 5.1 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>44036</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 6.35 cm x 5.7 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>440515</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 7 cm x 6.35 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>44041</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear silicone sealant</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube (15 ml)</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless Drill</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Funnel Monitor (DFM)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>DFM</td>
			<td>Used to count the number of flies that fall through the funnel at a given time point</td>
		</tr>
		<tr>
			<td>DAM data collection software</td>
			<td>TriKinetics; Waltham, MA</td>
			<td></td>
			<td>Records data input from the DFM</td>
		</tr>
		<tr>
			<td>Fly Storage Lid</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-96LID-5PK</td>
			<td>Used to load flies into the storage plate for the sCTmax assay</td>
		</tr>
		<tr>
			<td>Fly Storage Plate</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-96PLATE-5PK</td>
			<td>Used to hold flies during in the sCTmax assay</td>
		</tr>
		<tr>
			<td>Fly Food Tray</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-TRAY-5PK</td>
			<td>Used to keep flies on food after loading into the 96-well plate until the sCTmax assay</td>
		</tr>
		<tr>
			<td>Glass funnel</td>
			<td>Kimax</td>
			<td>28950-75</td>
			<td>75mm</td>
		</tr>
		<tr>
			<td>Gutter guard</td>
			<td>Any</td>
			<td>Any</td>
			<td>~0.5 cm diameter openings</td>
		</tr>
		<tr>
			<td>Hacksaw</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Heratherm Thermo Scientific incubator</td>
			<td>Thermo Scientific; Waltham, MA</td>
			<td>OMS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hose nylon adapters (2) &#8211; &#188; MNPT x 3/8</td>
			<td>United States Plastic Corp., OH</td>
			<td>61135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot glue gun and glue</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnets</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>OMEGA TC-08 Recorder and TC-08 Player Software</td>
			<td>OMEGA; Norwalk, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OMEGA thermocouple (Type T)</td>
			<td>OMEGA; Norwalk, CT</td>
			<td>5LRTC-TT-K-20-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic funnel</td>
			<td>Any</td>
			<td>Any</td>
			<td>2&#34; diameter</td>
		</tr>
		<tr>
			<td>Plastic tubing - 0.6 cm diameter</td>
			<td>United States Plastic Corp., OH</td>
			<td>62852</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort ring</td>
			<td>Any</td>
			<td>Any</td>
			<td>2&#34; diameter</td>
		</tr>
		<tr>
			<td>Retort stand</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort three-prong clamp</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serial port connector (PSIU9)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>PSIU9</td>
			<td>Intermediate connection between the DFM and computer, allows for multiple DFM connections</td>
		</tr>
		<tr>
			<td>Styrofoam (2&#34; thick)</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Uninterrupted Power Supply (PS9-1)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>PS9-1</td>
			<td>Power supply for the DFM and PSIU9</td>
		</tr>
		<tr>
			<td>Weld-on #4 Acrylic Cement</td>
			<td>United States Plastic Corp., OH</td>
			<td>45737</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput assays of critical thermal limits in insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

The thermal limits (i.e., CTmin and heat KDT) of females from the Drosophila melanogaster Genetic Reference Panel (DGRP) were measured to demonstrate the high-throughput data generated from the two described protocols. CTmin was assayed using the DGRP lines 714 (n = 37) and 913 (n = 45). Heat KDT was assayed and compared with the DGRP lines 189 (n = 42) and 461 (n = 42), and video files were manually analyzed. The total time of the experiments, including wat...

Watch this Scientific Journal Video about High-Throughput Assays of Critical Thermal Limits in Insects at JoVE.com

High-Throughput Assays of Critical Thermal Limits in Insects

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Thermal tolerance is a critical component of species distributions and responses to climate change. Here we provide methods for rapidly assessing heat and cold tolerance in small insects. These methods for measuring cold tolerance, via the critical thermal minimum and heat tolerance, via heat knocked down time are high throughput, semi-automated and require minimal equipment.Demonstrating the procedure will be David Awde, a postdoc from my laboratory. To set up a critical thermal minimum assay, power on a temperature controlled fluid bath and press the play button to run a program, raising and maintaining the temperature of the bath to 25 degrees Celsius. Wait five to 10 minutes to allow the fluid bathroom column to reach 25 degrees Celsius, before replacing the plug at the top of the column with a 5.08 centimeter diameter filter.Tap approximately 40 flies from their food vial into the column through the funnel and quickly replace the funnel with the plug, taking care not to let any flies escape. Then allow the flies to settle. Occasionally tapping the bottom plug to encourage the flies to climb.After five minutes, press the start button on the fluid bath gain, to begin the critical thermal minima ramping program. Click to open the thermal couple recording software on the computer and click record to begin recording the temperature inside the column, every second for the duration of the assay. Ensure that each temperature recording includes the timestamp specific to the second, so that temperature data can later be merged with data from the Drosophila funnel monitor.Add five milliliters of 90%ethanol to a 15 milliliter conical centrifuge tube and place the tube in a rack below the column. Occasionally tap the bottom plug of the column to entice any flies on the bottom to climb. Most of the flies will be on a perch or near the top of the column by 15 degrees Celsius.At 15 degrees Celsius, place a 75 millimeter outer diameter glass funnel into the Drosophila funnel monitor. Remove the bottom plug and collect any flies still on the plug in the ethanol. Adjust the retort ring, funnel monitor and funnel, so that they are under the column.Make sure that the lip of the funnel completely seals the bottom of the column and insert the bottom of the funnel into the 15 milliliter collection tube. Open the Drosophila funnel monitor software. The software will immediately start recording the time and date at which the flies reach their critical thermal minima.Flies that reach their critical thermal minima, will lose their neuromuscular function and fall from their perches, through the Drosophila funnel monitor. To monitor whether all of the flies have reached their critical thermal minima, as the temperature decreases, check the top plug and perches to see if any flies are still perched. When all the flies, have reached their critical thermal minima, move the Drosophila funnel monitor and funnel away from the column opening.On rare occasions, flies may reach their critical thermal minima, but remain stuck in the column. Open the top plug to remove these flies. To set up a high throughput heat knocked down assay, use an aspirator and septum lid, to load individual carbon dioxide sedated flies, into each well of a modified 96 well no bottom plate and seal the plate with a clear tight-fitting lid.Then place the plate over a supply of food and allow the flies to acclimatize to the 96 well plate for at least 48 hours. On the day of the assay, set the incubator to 37.5 degrees Celsius, after 30 minutes place the fly culture plate into the incubator with the bottom of the plate against the white paper on the bottom of the tray. Note the orientation of the well column and row names on the tray and in the frame of the webcam and place colored tape along the sides and edges of the plate to help verify the orientation.When the plate is in position, close the incubator door and click record in the video recording software. After two hours, check the recording to see whether all of the flies, have reached their final resting spot and stopped moving. When all of the flies have stopped moving, click stop and dispose of the flies.Open the video file and record the knockdown time of each fly in each well. The most consistent measure of the heat knocked down downtime between the trials and observations is the time at which a fly reaches its final resting spot. Then track the video in reverse, focusing on a single well and noting the time at which the fly first moves off its final resting spot.Determining the final resting time of each fly can be difficult. To ensure an accurate analysis, it is important to take your time and to observe each well individually. In this representative analysis, females from the Drosophila genetic reference panel line, had significantly lower mean thermal minima temperatures than females from the DGRP line 714.In addition, the heat knocked down time at 37.5 degrees Celsius, differ significantly between females from the 73 and 461 DGRP lines. With minor modifications, this method for measuring the heat knocked down time, could be used to measure other thermal tolerance traits, such as chill-coma recovery time or critical thermal maximum. By significantly reducing the hands-on time for investigators, these methods have allowed the exploration of genetic variation and thermal tolerance traits at a scale that was not previously possible.

high-throughput-assays-of-critical-thermal-limits-in-insects

Research

JoVE Journal

Biology

5.0K Aufrufe.  University of Kentucky. Thermische Toleranz ist ein entscheidender Bestandteil der Artenverteilungen und Reaktionen auf den Klimawandel. Hier bieten wir Methoden zur schnellen Beurteilung der Wärme- und Kältetoleranz bei kleinen Insekten. Diese Methoden zur Messung der Kältetoleranz über die kritische thermische Mindest- und Wärmetoleranz über die abgeschlagene Wärmezeit sind durchdiesam, halbautomatisch und erfordern minimale Ausrüstung.Demonstriert wird das Verfahren von David Awde, einem Postdoc au...