Name: A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae (Video) | JoVE Journal
Uploaded: 2018-06-25T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Learn a continuous thermal gradient assay to determine temperature preferences of Drosophila melanogaster larvae. This method enables reproducible analysis of somatosensory behavior across developmental stages.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

C.M. is supported by funding from the NEI (EY008117, EY010852), NIDCD (DC007864, DC016278) and the NIAID (1DP1AI124453).

<ol><li>Fowler, M. A., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+TRP+channels+and+animal+behavior%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">Drosophila TRP channels and animal behavior.</a> Life Sci. 92, 394-403 (2013).</li>
<li>Palkar, R., Lippoldt, E. K., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+and+cellular+basis+of+thermosensation+in+mammals%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">The molecular and cellular basis of thermosensation in mammals.</a> Curr Opin Neurobiol. 34, 14-19 (2015).</li>
<li>Vriens, J., Nilius, B., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+thermosensation+in+mammals%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Peripheral thermosensation in mammals.</a> Nat Rev Neurosci. 15 (9), 573-589 (2014).</li>
<li>Rosenzweig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li>
<li>Kwon, Y., Shim, H. S., Wang, X., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+thermotactic+behavior+via+coupling+of+a+TRP+channel+to+a+phospholipase+C+signaling+cascade%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade.</a> Nat Neurosci. 11, 871-873 (2008).</li>
<li>Kwon, Y., Shen, W. L., Shim, H. S., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fine+thermotactic+discrimination+between+the+optimal+and+slightly+cooler+temperatures+via+a+TRPV+channel+in+chordotonal+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons.</a> J Neurosci. 30 (31), 10465-10471 (2010).</li>
<li>Shen, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function+of+rhodopsin+in+temperature+discrimination+in+Drosophila%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Function of rhodopsin in temperature discrimination in Drosophila.</a> Science. 331 (6022), 1333-1336 (2011).</li>
<li>Sokabe, T., Chen, H. S., Luo, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+switch+in+thermal+preference+in+Drosophila+larvae+depends+on+multiple+rhodopsins%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins.</a> Cell Rep. 17, 336-344 (2016).</li>
<li>Ni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Ionotropic+Receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila.</a> Elife. 5, 13254(2016).</li>
<li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Navigational+decision+making+in+Drosophila+thermotaxis%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Navigational decision making in Drosophila thermotaxis.</a> J Neurosci. 30 (12), 4261-4272 (2010).</li>
<li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAshburner%2C+M+%22Drosophila%3A+a+laboratory+handbook%22">Drosophila: a laboratory handbook</a>. , Cold Spring Harbor Laboratory Press. (2005).</li>
</ol>

The authors have nothing to disclose.

To ensure the success of this protocol, it is important to take steps to obtain adequate numbers of larvae to perform the experiments. These include pre-feeding the flies in yeast paste-containing vials for 2-3 d to improve egg laying. The vials need to be placed in a tray containing water vials and enclosed in a clear plastic bag, which maintains the moisture of the food and promotes effective feeding by the larvae while permitting exposure to normal light-dark cycles. However, the yeast paste should not be so soft that...

Thermotaxis is employed by mobile animals to select an environment with the most favorable conditions1,2,3. If the climate is excessively hot or cold, this behavior is vital for the survival. In addition, many animals are sensitive to very small differences in temperature in the comfortable range and seek out surroundings with an ideal temperature. This is of particular importance for poikilothermic organisms such as fruit flies, which equilibrate their body temperature with the environment. Assays to monitor larval thermotaxis have been instrumental in identifying and clarifying the roles of molecular sensors such as Drosophila Transient Receptor Potential (TRP) channels4,5,6, rhodopsins7,8, and ionotropic receptor receptors (IRs)9, which endow these animals with temperature sensitivities over different temperature ranges.
A two-way choice test provides one approach to study thermal preferences in larvae6,7. The assay entails establishing two distinct temperature zones and allows&#160;the animals to select one side over the other. The results from two-way choice tests can be robust, especially if the temperature differences between the two options are&#160;large. In addition, since each assay involves tabulating only two groups, the data can be expressed as a simple preference index. The ease and simplicity of two-way choice assays are also amenable to genetic screens. However, a major limitation is that many experiments are required to establish the preferred temperature of the wild-type or mutant animals.
A gradient assay offers the opportunity to establish the preferred temperature in a single assay8. Moreover, unlike the two-way choice test, it permits the evaluation of the distribution of a group of animals, when confronted with a continuous range of temperatures. One gradient assay uses a Petri dish and single animals and is well-suited for characterizing the detailed behavior of individual animals10. However, since Petri dishes are round, the sizes of the temperature zones vary and are progressively smaller depending on the distance from the center. Therefore, this setup is not ideal for monitoring the temperature selections of populations of animals.
A continuous thermal gradient apparatus that is well-suited to assess the temperature preferences of groups of larvae employs a rectangular arena and is described here. The apparatus is simple to construct and assemble. In addition, the gradient is linear, and is flexible, as it can be used to assess thermotaxis over large temperature ranges from 10 &#176;C to 42 &#176;C. The assay is rapid and straightforward to perform and yields reproducible data. In addition to reporting the favored temperature of larvae, it reveals the preferences of the population of animals over an entire linear range in a single experiment. Due to these advantages, it is an excellent choice for identifying genes required for thermotaxis.

<table><tbody><tr><td>&lt;strong&gt;Gradient assay apparatus&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PolyScience 9106, Refrigerated/Heated 6L Circulating Bath</td><td>Thomas Scientific</td><td>9106</td><td>This model is discontinued. Updated replacement models include: 1186R00 and 1197U04 for 120 V, 60 Hz, or 1184L08 and 1197U04 for 240 V, 50 Hz.</td></tr><tr><td>Aluminum assay plate (for single directional gradient)</td><td></td><td></td><td>Outer size: 14 x 10.1 x 0.9 cm, inner size: 12.9 x 8.7 x 0.8 cm, black anodized.</td></tr><tr><td>Aluminum plate (for bidirectional gradient)</td><td></td><td></td><td>25 x 22 x 0.2 cm, black anodized.</td></tr><tr><td>Aluminum block</td><td></td><td></td><td>Outer size: 25.5 x 5 x 1.4 cm, parameters of inner channels are shown in Figure 1D.</td></tr><tr><td>Connector for aluminum blocks and tubing</td><td>McMaster-Carr</td><td>91355K82</td><td></td></tr><tr><td>Tygon Sanitary Silicone Tubing</td><td>Tygon</td><td>57296</td><td>1/4&quot; ID x 3/8&quot; OD x 1/16&quot; wall</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Items and reagents for assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pestle</td><td>USA Scientific</td><td>17361</td><td>Pestle for 1.5 mL microcentrifuge tubes</td></tr><tr><td>Thermometer</td><td>Fluke</td><td>51II</td><td></td></tr><tr><td>Thermocouple</td><td>Fluke</td><td>K type</td><td></td></tr><tr><td>Universal microplate lid</td><td>Corning</td><td>6980A77</td><td></td></tr><tr><td>35 mm dish</td><td>Corning</td><td>9380D40</td><td></td></tr><tr><td>Labeling tape (for bidirectional gradient)</td><td>Fisher Scientific</td><td>15-951</td><td>Fisherbrand labeling tape 2 in x 14 yds</td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 1% solution</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389-5KG</td><td>Prepare 18% solution right before starting assay</td></tr><tr><td>Paint brush</td><td>Fisher Scientific</td><td>11860</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Denville</td><td>C1062-P</td><td></td></tr><tr><td>Scoopula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>500 mL round wide-mouth bottle</td><td>Pyrex</td><td>1395-500</td><td></td></tr><tr><td>Cell strainer (300 mm pore)</td><td>PluriSelect</td><td>43-50300</td><td>Optional item for larvae washing</td></tr><tr><td>Cardboard box (vial tray)</td><td>Genesee Scientific</td><td>FS32-124</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Drosophila&lt;/em&gt;&lt;/strong&gt;&lt;strong&gt; food&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td>22,400 mL</td></tr><tr><td>Cornmeal, yellow (extra fine mesh,flocked) 20 kg</td><td>LabScientific Inc.</td><td>NC0535320</td><td>1,609 g</td></tr><tr><td>Brewers yeast 100 lbs</td><td>MP Biomedicals</td><td>ICN90331280</td><td>379 g</td></tr><tr><td>NutriSoy&amp;reg; Soy Flour (10 kg/unit)</td><td>Genesee Scientific</td><td>62-115</td><td>221 g</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Agar, Type II (5 kg)</td><td>Genesee Scientific</td><td>66-103</td><td>190 g</td></tr><tr><td>Karo light corn syrup</td><td>Karo</td><td></td><td>1,700 mL</td></tr><tr><td>Methyl 4-hydroxybenzoate (suspend in 200 proof ethanol)</td><td>Sigma Aldrich</td><td>H5501-5KG</td><td>72 g/240 mL</td></tr><tr><td>Propionic acid puriss. p.a.,&gt;99.5% (GC)</td><td>Sigma Aldrich</td><td>81910-1 L</td><td>108 mL</td></tr><tr><td>Phosphoric acid&amp;nbsp;ACS reagent, &amp;ge;85 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>438081-500 mL</td><td>8.5 mL</td></tr></tbody></table>

a temperature gradient assay to determine thermal preferences of drosophila larvae

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

To establish an 18 °C-28 °C single-directional gradient, we set the temperatures of two water baths to 16.8 °C and 31 °C. We obtain the temperatures at 13 points by measuring the temperature at 26 positions within the upper and lower portions of all 6 zones, the border lines between the zones, and at the extreme ends of the agarose gel surface (Figure 2C, 2E). The temperature distribution along the gradient was nearly linear (Y = 0.9672*X + 1...

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

a-temperature-gradient-assay-to-determine-thermal-preferences

Research

JoVE Journal

Neuroscience

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

This video describes the taste preference assay, a behavioral method used to measure attraction or avoidance towards colored solutions that taste differently by assessing the fly's abdominal coloration after ingestion of the preferred substance. The featured protocol demonstrates the procedure used to measure flies' preference towards solutions of varying sucrose concentrations.

Taste Preference Assay: A Method for Measuring Feeding Behavior in Drosophila

Drosophila larvae have dedicated sensory neurons–nociceptors–whose response to potentially harmful thermal stimuli triggers aversive–or so-called "nocifensive"–behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Drosophila fruit flies can distinguish small changes in temperature and therefore select environments with favorable thermal conditions. This video describes a behavioral assay that tests the temperature preference of Drosophila larvae on a linear thermal gradient.

Temperature Gradient Assay: A Method to Test Temperature Preference in Drosophila Larvae

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here we describe the design and analysis of TPR in Drosophila.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Measurement of Larval Activity in the Drosophila Activity Monitor

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

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.

High-Throughput Assays of Critical Thermal Limits in Insects

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Determining Temperature Preference of Mosquitoes and Other Ectotherms

This report describes a method for measuring adult Drosophila melanogaster time to knockdown using a Drosophila Activity Monitor (DAM2) in response to an air conduction heat stressor within an incubator chamber. The DAM2 measures activity by recording individual fly movements as they cross an infrared beam. Data analysis is facilitated by a novel executable file created by the authors.

Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

Learn a continuous thermal gradient assay to determine temperature preferences of Drosophila melanogaster larvae. This method enables reproducible analysis of somatosensory behavior across developmental stages.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Explore More Videos