Name: a temperature gradient assay to determine thermal preferences of drosophila larvae
Uploaded: 2018-06-25T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRP+channels+and+animal+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+thermosensation+in+mammals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+thermosensation+in+mammals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+thermotactic+behavior+via+coupling+of+a+TRP+channel+to+a+phospholipase+C+signaling+cascade.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+thermotactic+discrimination+between+the+optimal+and+slightly+cooler+temperatures+via+a+TRPV+channel+in+chordotonal+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+rhodopsin+in+temperature+discrimination+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+switch+in+thermal+preference+in+Drosophila+larvae+depends+on+multiple+rhodopsins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ionotropic+Receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigational+decision+making+in+Drosophila+thermotaxis.">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://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></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 the flies become trapped. The number of females per vial depends on the genotype. In the case of w1118, it is usually adequate to allow ~12 females and ~6 males to lay eggs for 3 h. Two vials typically provide enough larvae (100-200 larvae) to place on a plate for the single-directional assay. If the fly stock lays fewer eggs than wild-type or the proportion of larvae that hatch is reduced, add additional females (up to 30-35/vial) and males.
There are many causes of unintended variability when performing these assays. The developmental stage affects the temperature preference of the larvae. Therefore, it is imperative to carefully use larvae of a defined stage. To do so, collect larvae over a narrow time frame (such as 3 h). Since the rearing conditions (temperature, humidity, light-dark cycle and type of food) and some mutations affect the rate of development, do not rely strictly on the time after egg laying to analyze comparably aged larvae. It is also important to examine physical traits, such as the size of the mouth hooks and spiracles7 to determine whether larvae of different genotypes are at the same developmental stage. The times for 1st, 2nd and 3rd instar larvae to develop (Table 1) are based on using corn meal-based food and incubation at 25 &#176;C under 12 h light:12 h dark cycles. Larvae grow slower on molasses-based food. Proper hydration and food freshness also affects the growth of larvae. The amount of water in the food should leave the food neither too dry to peel back from the vial nor too loose due to excessive water. Ideally, the ~5 mm layer nearest the food surface is conditioned by the growing larvae and moves easily when the vial is tilted or tapped. This condition can be achieved using freshly made food with 50-100 larvae.
It is essential to establish a stable temperature gradient before initiating the assay. Therefore, turn on the water bath 1-2 h prior to initiating the assay, as the water baths produce heat and might change the ambient room temperature, which in turn have the potential to affect the gradient. After 1-2 h, the ambient temperature of most air-conditioned rooms equilibrates. However, this must be determined in each environment. In addition, when generating a gradient with a large temperature range (&#62;3.0 &#176;C/cm), it can be difficult to obtain a stable gradient. A tiny movement of the assay plate more significantly changes the temperature gradient when the range is large (e.g. &#62;3.0 &#176;C/cm). We found that a 1-2 &#176;C/cm gradient produces the most stable gradients.
There are several additional considerations that need to be controlled to limit variability in the thermotaxis assays. Washing the larvae is critical, as the presence of food particles or sucrose on the larvae can impact the assay. The washing steps must be performed thoroughly but quickly, because limiting their oxygen supply excessively while they are submerged in water may affect their health. Therefore, we recommend using the cell strainer (option 2) to clean the larvae. A strainer with a pore size of 300 &#181;m works well for larvae at the early-3rd instar stage (72 h AEL) or older. In addition, it is important to perform experiments at the same time of day, such as from Zeitgeber time (ZT) ZT4 to ZT8 (ZT=0 is when lights are turned on), to limit variability due to impacts of circadian rhythms on temperature selection. The level of moisture on the agarose plate can also cause variability in the results. While the surface of the plates needs to be moist, water droplets could trap the larvae, and therefore need to be avoided.
Limiting variability in the assays will make it possible to obtain robust results without performing large numbers of independent experiments. Typically, 3-8 independent experiments are sufficient to obtain a&#160;reliable outcome. Proper tabulation of the results is also critical for the success of the thermotaxis assays. Do not count larvae distributed in the regions within 0.5 cm from the aluminum walls, since the temperatures are not linear in these regions. Some larvae will be positioned at the border between two zones, at the time the assay is concluded. If more than 50% of the body length resides in one zone, then include the larva in the tabulation for that zone. If a larva is precisely 50% in each of two zones, then count the animal as 0.5 larvae in each zone.
While the gradient assay allows the larvae to discriminate a range of temperature differences, there are limitations to the lower and upper temperatures that can be effectively tested. It is not feasible to test temperatures &#60;10 &#176;C since the mobility of the larvae decreases greatly at lower temperatures. Although gradients with the upper temperature reaching 42 &#176;C can be established, almost no control larvae stay in any zone &#62;28 &#176;C. Therefore, continuous gradient assays cannot be used to discriminate temperature preferences between various zones &#62;28 &#176;C. However, gradient assays at these elevated temperatures could potentially be used to characterize mutants if they have highly shifted warm temperature preferences.
If a mutant has a locomotor defect, it may be necessary to conduct additional experiments to make sure that the apparent temperature preference is not inaccurate due to a movement impairment. To remedy this potential problem, it may be necessary to establish longer assay times to allow the animals additional time to move to their desired zones. The bidirectional gradient assay can also be employed to test whether the animals select the same preferred zone, regardless as to where they are placed initially on the plate.
In conclusion, the gradient assays described here have advantages over other thermotaxis assays. While two-way choice assays are useful, as they can yield robust results, especially when the temperature differences between the two zones are large, they are not effective in revealing the ideal temperature preferred by the larvae in a single experiment. To do so requires performing many two-way choice combinations to determine the most preferred temperature5,6,7. In contrast, the gradient assay provides the animal an opportunity to select their preferred temperature zone in an environment with a single continuous temperature landscape. Thus, it is unnecessary to devise a large combination of two-way choices to determine the preferred thermal environment. Studying thermotaxis using a round Petri dish has been employed to assess the effects of temperature on the moving dynamics of a larva10. However, it is less useful in discriminating between preferences between different temperatures, since each zone is a different size. Finally, due to the simplicity of the assay, and its utility in discriminating small temperature preferences in a single assay, it can be employed to screen dozens of candidate genes that are potentially involved in larval temperature preference that might otherwise be overlooked using other assays

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

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 &#176;C-28 &#176;C single-directional gradient, we set the temperatures of two water baths to 16.8 &#176;C and 31 &#176;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 + 16.19, R2 = 0.9961) (Figure 2E).
We assayed the temperature preferences of control (w1118) larvae at various ages. 1st (24 &#177; 1.5 h AEL), 2nd (48 &#177; 1.5 h AEL) and early-3rd larvae (72 &#177; 1.5 h) showed peaks in the 24 &#176;C zone (Figure 3A, B). The temperature preferences changed during 3rd instar larval development. The largest percentage of mid-3rd instar (96 &#177; 1.5 h AEL) accumulated in the 18 &#176;C zone (Figure 3A, B), and this bias increased with age. Among late-3rd instar larvae (pre-climbing; 120 &#177; 1.5 h AEL), ~50% clustered in the 18 &#176;C zone, and the selection of this temperature was ~4-fold higher than the adjacent 20 &#176;C zone (18 &#176;C zone, 50.2%; 20 &#176;C zone, 15.1%; Figure 3A, B). The proclivity to accumulate in the 18 &#176;C zone in w1118 was not due to an edge effect since the late-3rd instar larvae still accumulated in the 18 &#176;C zone, using a bidirectional temperature gradient (Figure 3E, F).
We also tested late-3rd instar larvae (120 &#177; 1.5 h AEL) with mutations in genes required for discriminating temperature differences in the comfortable range. These include trpA1, which is required for normal temperature selection in the 18 &#176;C-24 &#176;C range5,7,8. Larvae with a null mutation in trpA1 (trpA11) distribute equally over the entire 18 &#176;C&#8212;28 &#176;C gradient (Figure 3C). Larvae missing just the A and B isoforms (trpA1-ABG4), or the C and D isoforms (trpA1-CDG4) also show severe impairments (Figure 3C). Flies encode two isoforms of phospholipase C&#946; (PLC21C and NORPA), and mutations affecting norpA (norpAP24) but not plc21c (plc21cP319) also disrupt accumulation in the 18 &#176;C range (Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57963/57963fig1.jpg" /> 
Figure 1. Apparatus for performing the single-directional temperature gradient assay. (A) An aluminum test plate used for assaying larval thermotaxis behavior. The 13 black lines at the top and bottom demarcate 12 zones (10 mm each). The bottom of the plate is anodized with black paint so that it is easier to visualize the larvae. (B) Dimensions of the aluminum plate (indicated in mm). The outer size of the aluminum plate is 140 x 100 x 9 mm. The inner size of the aluminum plate is 130 x 90 x 8 mm. The demarcations are separated by 10 mm. The first and last demarcation is 5 mm from the edges of the inner area of the plate. (C) Top and side views of one of the aluminum blocks used to control the temperature of the gradient. The block has two connectors used to attach to silicon tubes, which connect to a water bath. (D) Dimensions of an aluminum block. The outer size of the aluminum block is 255 x 50 x 14 mm. The diameter of the inner water path is 7 mm. Two 30 mm connectors on the left connect with silicon tubing that extends to the water bath. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57963/57963fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57963/57963fig2.jpg" /> 
Figure 2. Single and bidirectional gradient assay setups. (A) Single-directional gradient setup with two aluminum assay plates on two aluminum blocks. The temperatures of the aluminum blocks are controlled by circulating water from two water baths. (B) The arrangement of the three aluminum blocks, water baths and an aluminum plate (250 x 220 mm) for a bidirectional gradient. The left and right blocks are connected to the same water bath and the middle block is connected to the other water bath. The aluminum assay plate is wrapped with tape to form a 10 mm wall to contain the 1% agarose. (C) Positions to check temperatures (indicated by dots) and to release larvae on the plate. Before initiating an experiment, check the temperature at two points within each zone to confirm that the desired linear temperature gradient is established. Larvae are released within the indicated area near the midline. The larvae are counted within each of the 2-cm zones. (D) Positions to check temperatures (indicated by dots) and the release zones for the larvae on a bidirectional gradient. An equal number of larvae are released along the midline of each half of the bidirectional gradient. The numbers of larvae are counted in each of the 10 (2 cm) zones. One typical set of temperatures (18 &#176;C-26 &#176;C) on the agarose surface is indicated. (E) Temperatures measured along the border lines and midlines of each zone in a sample single-directional gradient. Data represent mean temperatures &#177; SD. n=8 assays (150 &#177; 50 larvae/assay). Parts of this figure are reproduced from Sokabe et al.8 with slight modifications. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57963/57963fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57963/57963fig3.jpg" /> 
Figure 3. Representative results using single-directional and bidirectional gradient assays. (A, B) Mean percentages of larvae in 6 zones on the single-directional gradient. Data include 3rd instar larvae at the indicated hours after egg laying (AEL). n=6-7. The error bars in A represent &#177;SEM. (C) Thermal distributions of the pre-climbing late-3rd instar larvae of control (w1118) and trpA1 mutants on the single-directional gradient. n=3-4. The error bars represent &#177;SEM. (D) Thermal distributions of the late-3rd instar larvae of control (w1118) and PLC&#946; mutants on the single-directional gradient. n=4-6. The error bars represent &#177;SEM. (E) Representative distribution of pre-climbing, late-3rd instar larvae (w1118) on the bidirectional gradient. The left and right assay zones are indicated by dotted lines and separated by the no-count zone in the center (shaded region). (F) Percentage of pre-climbing late-3rd instar larvae (w1118) in each zone along the thermal gradient. Larvae were placed in the left and the right release zones. The assay zones are separated by a 3-cm no-count zone in the center and the distributions are calculated independently. The error bars represent SEMs. n=3 assays (200-400 larvae/assay). Parts of this figure are reproduced from Sokabe et al.8 with slight modifications. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57963/57963fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hours AEL</td>
			<td colspan="2">Larval stage</td>
		</tr>
		<tr>
			<td>24</td>
			<td colspan="2">1st instar</td>
		</tr>
		<tr>
			<td>48</td>
			<td colspan="2">2nd instar</td>
		</tr>
		<tr>
			<td>72</td>
			<td colspan="2">Early-3rd instar</td>
		</tr>
		<tr>
			<td>96</td>
			<td colspan="2">Mid-3rd instar</td>
		</tr>
		<tr>
			<td>120</td>
			<td colspan="2">Late-3rd instar, just before climbing stage</td>
		</tr>
	</tbody>
</table>
Table 1. The relationship between the hours after egg laying (AEL) and larval stages.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Temperature gradient on agarose plate (slope)</td>
			<td>Temperatures of water baths</td>
			<td>Temperatures of aluminum blocks</td>
		</tr>
		<tr>
			<td>10.0-25.0&#176;C (1.5&#176;C/cm)</td>
			<td>~6.5-7&#176;C/~28.5&#176;C</td>
			<td>&#160;~8.5&#176;C/~26.8&#176;C</td>
		</tr>
		<tr>
			<td>18.0-28.0&#176;C (1&#176;C/cm)</td>
			<td>~16.8&#176;C/~31.0&#176;C</td>
			<td>~17.8&#176;C/~29.7&#176;C</td>
		</tr>
		<tr>
			<td>14.0-34.0&#176;C (2&#176;C/cm)</td>
			<td>~10.0&#176;C/~40.0&#176;C</td>
			<td>~11.8&#176;C/~36.8&#176;C</td>
		</tr>
		<tr>
			<td>12.5-42.0&#176;C (2.95&#176;C/cm)</td>
			<td>&#160;~7.0&#176;C/~55.0&#176;C</td>
			<td>&#160;~9.4&#176;C/~49.4&#176;C</td>
		</tr>
	</tbody>
</table>
Table 2. Typical temperature gradients and the corresponding temperatures of the water baths and aluminum blocks for single directional gradients.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Temperature gradient on agarose plate</td>
			<td>Temperatures of water baths</td>
			<td>Temperatures of aluminum blocks</td>
		</tr>
		<tr>
			<td>22-10-22&#176;C (1.5&#176;C/cm)</td>
			<td>&#160;~5.0&#176;C /~25.0&#176;C</td>
			<td>&#160;~7.5&#176;C/~24.0&#176;C</td>
		</tr>
		<tr>
			<td>26-18-26&#176;C (1&#176;C/cm)</td>
			<td>~15.8&#176;C /~30.6&#176;C</td>
			<td>~16.9&#176;C/~28.4&#176;C</td>
		</tr>
		<tr>
			<td>30-14-30&#176;C (2&#176;C/cm)</td>
			<td>&#160;~8.5&#176;C /~36.4&#176;C</td>
			<td>~10.9&#176;C/~32.8&#176;C</td>
		</tr>
		<tr>
			<td>36-12.5-36&#176;C (2.95&#176;C/cm)</td>
			<td>&#160;~5.0&#176;C /~47.2&#176;C</td>
			<td>&#160;~7.9&#176;C/~40.9&#176;C</td>
		</tr>
	</tbody>
</table>
Table 3. Typical temperature gradients and the corresponding temperatures of the water baths and aluminum blocks for bidirectional gradients.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Larval age (AEL)</td>
			<td>Assay time (single directional)</td>
			<td>Assay time (bidirectional)</td>
		</tr>
		<tr>
			<td>&#160; 24 h</td>
			<td>30 min</td>
			<td>35 min</td>
		</tr>
		<tr>
			<td>&#160; 48 h</td>
			<td>22 min</td>
			<td>27 min</td>
		</tr>
		<tr>
			<td>&#160; 72 h</td>
			<td>16 min</td>
			<td>21 min</td>
		</tr>
		<tr>
			<td>&#160; 96 h</td>
			<td>13 min</td>
			<td>18 min</td>
		</tr>
		<tr>
			<td>120 h</td>
			<td>10 min</td>
			<td>15 min</td>
		</tr>
	</tbody>
</table>
Table 4. Different larval ages (AEL) and the corresponding assay times.

Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

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

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.5K Views.  University of California, Santa Barbara. Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Video: A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

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

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

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

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.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Behavior

High-Throughput Heat Tolerance Measurement in Drosophila Using DAM2 Systems

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

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to assessing heat tolerance being to monitor the time to knockdown (TKD) after exposure to an elevated temperature. Classically, flies are housed in individual vials and placed inside a heated water bath. TKD is then monitored manually by researchers. While very well-established, there remain problems of subjectivity and consistent application of a tangible definition of cessation of all movement, including muscular spasms, when implementing these manual assays. We have developed a high-throughput method for automating heat tolerance assays using the TriKinetics Drosophila Activity Monitors (DAM2). To accompany the DAM2 system, we have written a program and created an easy-use executable to automatically read the last time of movement from the activity data generated. This script then writes to a .csv file the time to heat paralysis (TKD) for each fly. Our data show that this automated DAM2 method is consistent and reliable. Meanwhile, activity profiles created from the activity count data are of interest. These activity profiles can be compiled and have the potential to expand heat tolerance assays to include the relatively unstudied behavioral components of heat tolerance. This protocol will describe in detail how to use the DAM2 system and the HoTDAM! software to estimate heat tolerance in D. melanogaster.

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.

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

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.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

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

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

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

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&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Biology

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

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.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

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.

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 ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

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.

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

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

Source: Chattopadhyay, A., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;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.

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Larva

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

Source: Liu, J., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57963/" target="_blank">A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae</a>. J. Vis. Exp. (2018).
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.

Source: Liu, J., et al. A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae. J. Vis. Exp. (2018).
Drosophila fruit flies ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Summary

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Chapters in this video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

Measurement of Larval Activity in the Drosophila Activity Monitor

High-Throughput Assays of Critical Thermal Limits in Insects

Determining Temperature Preference of Mosquitoes and Other Ectotherms

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

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