This work was supported by NIH P20GM103643 to I. Meng.

1. Preparation of Larvae
<ol>
	<li>To analyze a desired larva for locomotion or position preference, grow larvae under standard conditions to the desired age to assay10 using standard fly food11.</li>
	<li>Make a mesh filter by stretching silk-screen grade nylon mesh over a funnel. Secure the mesh at the funnel neck with rubber band. Place the funnel in a beaker.</li>
	<li>To collect larvae for analysis, scoop a spatula-full of food containing foraging larvae from the culture bottle and wash with RT tap water over the mesh filter, collecting individual larvae directly from the mesh with a paintbrush10.</li>
</ol>
2. Preparation of Assay Tubes
<ol>
	<li>To prepare to plug the assay tubes, carefully boil a 4% agar gel and pour into a petri dish to a depth of 1.5 cm. This will provide a plug for either side of the tube when animal is enclosed. The 4% solution is sufficiently dense to prevent larvae from penetrating the plugs.</li>
	<li>Ensure that tubes are clean and clear prior to insertion of larva. If they are not, this may block movements from being recorded.</li>
	<li>To ensure larvae have sufficient moisture for movement, prepare a squeeze bottle containing a small amount of hot water. Invert the bottle, carefully orient the outlet toward a sink and squeeze gently to expel water from the pickup tube.</li>
	<li>Insert the bottle outlet into the assay tube. Squeeze the bottle to deliver water vapor into the assay tube until a thin layer of condensation appears on the wall of the tube. Video 1 shows a larva moving in a tube with an appropriate amount of moisture.</li>
	<li>To seal the larva in the tube, remove the 1.5 cm thick agar gel from the petri dish and place over a mesh surface to allow airflow beneath the agar so that an agar plug can be inserted into the tube. Press one end of the assay tube into the gel twice to insert two plugs of gel into one end.</li>
	<li>With a paintbrush, place one larva into the tube approximately 1.5 inches deep and seal the tube by pressing the end nearest the larva into the agar gel. The resulting pressure will force out the second plug of agar gel from the opposite end and seal the animal within the tube.</li>
	<li>Place tubes in the MB5 Multi-Beam Drosophila Activity Monitor(DAM) device, and adjust the tubes&#8217; positions so that the animals cannot move beyond the range of the sensors.</li>
	<li>To ensure that assay tubes do not slide out of the device&#8217;s infrared reading frame during transport, hold the tube in place by fastening a ring of putty around each tube where it contacts the recording device.</li>
</ol>
3. Measuring Activity
<ol>
	<li>Record activity in an incubator to prevent inaccurate readings that may occur due to shadows, fluorescent lights or to temperature variations in the lab. See Figure 5 for a suggested arrangement of the system.</li>
	<li>Set an incubator to 20 &#176;C (see Figure 1). To avoid false recordings due to interference from incandescent light sources during recording, turn off fluorescent incubator lights and use a separate LED light source while recording in incubator. Perform a trial without any animals to ensure that light conditions do not aberrantly trigger sensors. There should be no recorded movement data after this test.</li>
	<li>Allow larvae to acclimate to the 20&#176;C incubator settings for 5 min prior to start of assay.</li>
	<li>To set the DAM System to desired recording intervals (e.g., 1 min), ensure DAM system recording software is downloaded to host computer12 and open the DAM System file prior to connecting the recording chamber to the PSIU interface.</li>
	<li>To set the desired recording frequency at which data will be saved, select preferences and then click above or below the reading interval option to select different time frames in which data will be stored. For example, select reading interval times from 1 sec to 1 hr.</li>
	<li>To select varying parameter for recording data, choose preferences and then select the corresponding boxes under output data type (counts, moves, positions and dwell, see Table 1). Each parameter provides a unique analysis of the larval activity within the tube.</li>
	<li>To begin recording, connect the monitor to the Power Supply Interface Unit (PSIU) using CAB6 telephone cable. Connect the PSIU to a power outlet. A green light will indicate appropriate connection.</li>
	<li>Connect the PSIU through a Universal Serial Bus (USB) cable to a Macintosh or Windows PC for data recording. Open the DAM SystemMB1v6x program on the computer to automatically start data recording.</li>
	<li>After the desired collection time is complete, select Quit, and then Quit now on the DAM activity screen; data will then be automatically saved under DAM System Data. Take this data file (e.g., monitor 1) and drag into a separate folder. This will store the raw data from the DAM software so that they can later be processed.</li>
</ol>
4. Preparing Data for Processing (DAM FileScan)
<ol>
	<li>To process the raw data into an understandable format open the DAM FileScan application and select Select Input Data Folder. Then choose the data folder and the desired file, and select the scan option. Lastly select the bin length to the desired reading period. This will organize the raw data into parameters set by the operator and the program will report the data collected in that particular range.</li>
	<li>Select the output data type to analyze the desired motion setting (monitor counts, moves, dwell). Under the extra readings menu, select sum into bin (this is if the bin length is greater than the selected system reading interval).</li>
	<li>Name the file appropriately and save. The file will be stored in the same location as the folder from step 3.9.</li>
</ol>
5. Accessing Data for Analysis
<ol>
	<li>In order to collect average moves, process the raw data generated from step 4. To generate a table in a spreadsheet program, open the file that was previously saved (step 4.3), and when the text import wizard window appears, select finish to open the data files in a spreadsheet format. 
	Note: Depending on the data type analyzed, the spreadsheet will be read differently. Typically to measure monitor moves or counts, the time period of the study will be recorded numerically, while each individual reading tube will receive a designated letter. When viewing the data in the spreadsheet, columns K-Z represent the slots for assay tubes 1-16 respectively. The rows represent the data points collected.</li>
	<li>For example, if moves were collected for 20 min in 1 min intervals, average the 20 rows to calculate an average number of moves/minute and other measurements.</li>
</ol>

<ol>
	<li>Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=painless,+a+Drosophila.gene+essential+for+nociception.">painless, a Drosophila.gene essential for nociception.</a> Cell. 113, 261-273 (2003).</li><li>Ainsley, J. A., Kim, M. J., Wegman, L. J., Pettus, J. M., Johnson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+mechanisms+controlling+the+timing+of+larval+developmental+and+behavioral+transitions+require+the+Drosophila.DEG/ENaC+subunit.">Sensory mechanisms controlling the timing of larval developmental and behavioral transitions require the Drosophila.DEG/ENaC subunit.</a> Pickpocket1. Dev. Biol. 322 (1), 46-55 (2008).</li><li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila.+melanogaster.and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila. melanogaster.and the role of the fly in therapeutic drug discovery.</a> Pharmacol. Rev. 63 (2), 411-436 (2011).</li><li>Cattaert, D., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+central+generator+of+locomotor+rhythm+by+noncompetitive+NMDA+receptor+antagonists+in+Drosophila.larvae.">Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila.larvae.</a> J. Neurobiol. 48 (1), 58-73 (2001).</li><li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+larval+locomotion+in+Drosophila.+hordotonal+organ+mutants.">Dynamic analysis of larval locomotion in Drosophila. hordotonal organ mutants.</a> PNAS. 100 (26), 16053-16058 (2003).</li><li>Nichols, C. D., Bechnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. (61), (2012).</li><li>Godoy-Herrera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+genetics+of+digging+behavior+in+Drosophila.+arvae.">The development and genetics of digging behavior in Drosophila. arvae.</a> Heredity. 56, 33-41 (1986).</li><li>Sinadinos, C., Cowan, C. M., Wyttenbach, A., Mudher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+throughput+assays+of+locomotor+dysfunction+in+Drosophila.+larvae.">Increased throughput assays of locomotor dysfunction in Drosophila. larvae.</a> Journal of Neuroscience Methods. 203 (2), 325-334 (2012).</li><li>Homyk, T., Sheppard, D. 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Science Education Database Available from: <a target="_blank" href="https://www-jove-com.remotexs.ntu.edu.sg/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans">https://www-jove-com.remotexs.ntu.edu.sg/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans</a> (2015)</li><li>Woods, J. K., Kowalski, S., Rogina, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+spontaneous+locomotor+activity+in+Drosophila+melanogaster.">Determination of the spontaneous locomotor activity in Drosophila melanogaster.</a> J. Vis. Exp. (86), e51449 (2014).</li><li>Troncoso, B., Godoy-Herrera, R., Waldo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+larval+movement+patterns+in+Drosophila).">The development of larval movement patterns in Drosophila).</a> Heredity. 58 (1), 321-329 (1987).</li><li>Helfrich, C., Engelmann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+the+locomotor+activity+in+Drosophila.+melanogaster.and+its+mutants+&#8216;sine+oculis&#8217;+and+&#8216;small+optic+lobes.">Circadian rhythm of the locomotor activity in Drosophila. melanogaster.and its mutants &#8216;sine oculis&#8217; and &#8216;small optic lobes.</a> Physiological Entomology. 8 (3), 257-272 (1983).</li><li>Johnson, A. W., Carder, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+ociceptors+mediate+larval+aversion+to+dry+surface+environments+utilizing+both+the+painless+TRP+channel+and+the+DEG/ENaC+subunit,+PPK1.">Drosophila, ociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1.</a> PLoS ONE. 7 (3), (2012).</li></ol>

The authors declare that they have no competing financial interests.

Activity of Drosophila larvae is influenced by a variety of factors including genotype8, age13 and ambient temperature2. Although powerful videographic methods capable of highly detailed analysis have been developed by those who study locomotion5, this level of detail may be superfluous for those who wish to determine basic parameters of activity. The method described here employs a device that is available in many laboratories, is easy to operate, generates highly reproducible results, and is manageable even for those whose primary research focus is not locomotion. The example results demonstrate that this assay can be used to detect significant changes in motility in larvae subjected to different temperatures (Figure 1) and of different genotypes (Figure 2).
When larvae were measured at temperatures ranging from 5 &#176;C &#8211; 35 &#176;C, their activity increased with temperature, except for a break in the trend between 20 &#176;C and 25 &#176;C (Figure 1). It has been shown by Ainsley and coworkers that foraging early third instar larvae prefer temperatures within +/- 2 &#176;C of the typical 25 &#176;C culture temperature. However, when larvae enter wandering mid-third instar phase they prefer somewhat cooler temperatures2. That finding is consistent with the observation that locomotor activity for third-instar larvae is greater at 20 &#176;C than 25 &#176;C, suggesting that some portion of animals assayed were in the wandering stage and more active at the cooler temperatures, and less so at the normal culture temperature of 25 &#176;C.
This method offers simplicity, objectivity and robust throughput, but there are limitations. The applications described above represent assays occurring over a relatively short time frame, in part because the current set-up does not provide larvae with a food source. Ensuring adequate nutrition would be necessary to study changes in activity over extended periods of time or to measure circadian rhythm in the longer term. The 4% agar plugs may restrict gas exchange between the chamber and the external environment, which could result in the larvae experiencing hypoxic conditions. However this does not appear to affect activity within a 20&#160;min assay period, because when average moves per minute of larvae during each minute of the period were analyzed, larvae did not appear to show any change in activity over the recording period (Figure 4).
Because the device records position continuously it represents an improvement in capturing more motion compared to the non-automated methods cited, however some motion does escape detection. Very small movements of animals might not trigger a response from this device, and larvae can move in a circumferential fashion within one infrared beam without breaking neighboring beams, resulting in inaccurately low readings. However, since this type of error would be expected to occur in all treatment groups, it is unlikely to cause misleading results. Although third instar larvae are the primary focus of this analysis, the device is capable of measuring the motion of the much smaller first and second instar larvae as well (Figure 3). As expected, the number of moves recorded per minute in the younger animals is lower than that of the larger third instar animals.
Although the full range of uses for this device has yet to be demonstrated, there are a variety of other adaptations that could diversify the uses of this apparatus for studies involving larvae. For instance, the device allows a &#8216;dwell&#8217; measurement, which represents time spent in a determined region of the tube. This may provide valuable information when employed in various Drosophila larval taxis assays. By placing the apparatus on its side so that the tubes are oriented vertically instead of horizontally, one could measure larval geotaxis. To measure phototaxis, a light gradient could be established in the tubes, testing whether larvae have a preference for light or its absence. To study chemotaxis, a test chemical could be placed on one of the agar plugs and the position of the larvae might then reveal determine their preference for or avoidance of the chemical.
The monitor system allows the analysis of various motion parameters, summarized in Table 1. By selecting all parameters during the pre-assay setup (see step 3.6), the experimenter can choose which parameter to analyze after the assay. However, if any setting is not selected, that data will not be available for post hoc analysis. It should be noted that after each selected period of time, data are frozen at current counts and saved to the host computer. Data collection then resets to zero after this period and begins again, providing a series of time interval data points. One must manually quit to end data recording.
Future studies involving this method will focus on the use of the dwell parameter and its various applications. Also it may be possible to develop a protocol that would allow for studies to occur over a longer period of time, such as circadian studies, by providing food and exchanging agar plugs for a more gas-permeable material14. Moisture levels would need to be controlled as well, as dry conditions inhibit locomotion15. Currently, this protocol provides an accurate, simple, cost-effective method to evaluate basic parameters of larval activity under a variety of experimental conditions.

The use of Drosophila as a genetic tool has transformed scientific knowledge of biological systems. Drosophila larvae have been used in a variety of studies including nociception1, development2 and as a model for the study of human disease genes3. Drosophila activity encompasses a range of behaviors that vary under different conditions including temperatures2, exposure to drugs4 and amongst different genotypes. Yet, despite the significant use of the larva as a model organism, a simple, standardized method to analyze larval activity has not been available. Presently, many larval locomotion studies employ sophisticated video analysis software5. While powerful, the complexity of such automated tools may discourage labs that are not already equipped to study locomotion from including analysis of informative activity parameters in their studies. In other current non-automated methods, such as the grid crawling analysis, motion is scored by a human observer, which introduces the possibility of subjectivity and limits throughput to one animal at a time6-7. A similar study used a 5-lane crawling assay, which measured the time it took larvae to travel a certain linear distance8. In such non-automated assays, displacement is measured but this does not account for non-linear travel between the start and end points. As discussed below, the method described here accounts for more of the actual larval movement, is objective, easy to operate, and offers robust throughput.
To easily study larval activity behavior without the compromise of accuracy, efficiency, or cost, this method employs the TriKinetics Drosophila Activity Monitor (DAM), a device often used to study adult activity. Using one device to study both adult and larval activity is cost-effective, and allows direct comparison of motion by animals at these two life stages. The system, featuring the manufacturer&#8217;s highest level of resolution, makes use of 17 infrared detection beams per assay tube, which record larval activity when sensor beams are broken within the 16 individual tubes. The system then automatically saves recorded information to a computer, making it available for manipulation with basic graphing software. The data obtained represents the beams that were broken by individual larvae (which can be converted into a rate), movement when a larva stays within a detector beam and the position of the animals within the assay chambers during a recording period (allowing one to calculate position preference). The system is efficient and relatively simple to operate, and brings highly reproducible basic activity analysis within the reach of any laboratory studying Drosophila larvae.
To demonstrate the power of this assay, data are presented that show its use to verify differences in activity resulting from varying ambient temperatures, as well as through the comparison of a mutant previously described as hypoactive (iav1)9 with a widely analyzed control (w1118).

<table><tbody><tr><td>Drosophila Activity Monitor, Multibeam, 16 tubes, including wires</td><td>TriKinetics Inc.&amp;nbsp;</td><td>MB5</td><td></td></tr><tr><td>Power Supply Interface for Activity Monitor&amp;nbsp;</td><td>TriKinetics Inc.&amp;nbsp;</td><td>PSIU24</td><td></td></tr><tr><td>Glass 80 x 5 mm tubes for Activity Monitor (100)</td><td>TriKinetics Inc.&amp;nbsp;</td><td>PGT 5x80</td><td></td></tr><tr><td>DAMsystemMB1v6x Data Acquisitions Software for Macintonsh OSX (Intel)</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>DAMFileScan 108x software for Macintosh</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>USB software (PSIUdrivers.zip).</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>DAMSystem Notes 308</td><td>&lt;a href=&quot;http://www.trikinetics.com/&quot;&gt;www.trikinetics.com&lt;/a&gt;</td><td></td><td>free download</td></tr><tr><td>Zeiss Stemi 2000C- Stereo Microscope</td><td>Spectra Services</td><td>SP-STEMI2000C-BS</td><td></td></tr><tr><td>Carbon Dioxide</td><td>Maine Oxy</td><td></td><td>anaesthesia</td></tr><tr><td>Fly Pad</td><td>Genesee</td><td>59-114</td><td>surface for sorting anaesthetized flies</td></tr><tr><td>Small paint brush&amp;nbsp;</td><td>Winsor &amp;amp; Newton</td><td>#2 ROUND</td><td>or similar, used for sorting anaesthetized flies</td></tr><tr><td>Silk Screen Printing Mesh (160)</td><td>msj-gallery.com</td><td>SM160W63-3YD</td><td>pore sized used in this protocol was ~ 0.1 mm</td></tr><tr><td>Tegosept</td><td>Genesee</td><td>20-258</td><td>preservative</td></tr><tr><td>Ethanol (190proof)</td><td>Pharmco</td><td>111000190</td><td>used to dissolve Tegosept</td></tr><tr><td>6 oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td></td></tr><tr><td>&quot;Flugs&quot; for Plastic Fly bottles</td><td>Genesee</td><td>49-100</td><td></td></tr><tr><td>Drosophila Vials, Wide (PS)</td><td>Genesee</td><td>32-117</td><td></td></tr><tr><td>Flugs for wide plastic vials</td><td>Genesee</td><td>49-101</td><td></td></tr><tr><td>Yellow Degerminated Corn Meal</td><td>Gold Medal</td><td></td><td></td></tr><tr><td>Drosophila agar</td><td>LabScientific</td><td>FLY 8020</td><td></td></tr><tr><td>Baker&#039;s Yeast - Red Star</td><td>King Arthur Flour</td><td>1270</td><td></td></tr><tr><td>Granulated Sugar - Extra Fine</td><td>Domino</td><td></td><td></td></tr></tbody></table>

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.

Figure 1 shows the results from a temperature response study of control third instar larvae, w1118, using the monitoring device to detect differences in larval locomotion at seven different temperatures. Larvae were washed and placed into the DAM activity device as described above, and placed in an incubator set to the desired temperature. The apparatus was then allowed to acclimate to the environment for 5 min before recording began. Each larva was individually analyzed for locomotion over a 20&#160;min period and the average number of moves per minute was calculated for every animal and averaged for each set of 32 animals. Data were analyzed and graphed using a spreadsheet program. Larvae exhibited significantly increasing activity as temperature increased correspondingly from 5-35 &#176;C in 5-degree increments, except for a break in this trend at 20 &#176;C and 25 &#176;C.
To verify that differences could be detected between a control and a mutant previously described as hypoactive, inactive larvae (iav1) were tested. Data were analyzed as moves/minute for each of the 32 animals and an average was then calculated. As shown in Figure 2, the analysis indicates that inactive larvae were significantly less mobile than a control. While they are much smaller than third instar larvae, activity of first and second instar larvae was also measurable, as shown in Figure 3. Activity of third instar larvae in each minute of the 20&#160;min&#160;assay was shown to remain relatively consistent throughout the period (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/52684/52684fig1.jpg" /> 
Figure 1.&#160;w1118 larval locomotion was recorded at temperatures varying from 5 &#176;C &#8211; 35 &#176;C. Each column represents the average motion of 32 third instar animals with individual moves per minute averaged amongst the set. *All averages are significantly different from each other except 10 &#176;C and 15 &#176;C (p=0.116) (Different letters indicate a significant difference, Student t-test).
<img alt="Figure 2" src="/files/ftp_upload/52684/52684fig2.jpg" /> 
Figure 2.&#160;Third instar control larvae compared to inactive (iav1) larvae at 20 &#176;C. iav1 larvae exhibit significantly less mobility compared to control.
<img alt="Figure 3" src="/files/ftp_upload/52684/52684fig3.jpg" /> 
Figure 3. Measurement of activity of first, second and third instar larvae over a 20 min recording interval at 20 &#176;C. N=32 for each larval stage.
<img alt="Figure 4" src="/files/ftp_upload/52684/52684fig4.jpg" /> 
Figure 4.&#160;Histogram displaying average number of moves occurring in each minute of the 20 min recording interval. 32 third instar larvae were assayed at 20 &#176;C. The red line represents the number of moves per minute averaged for the entire 20 min&#160;interval.
<img alt="Figure 5" src="/files/ftp_upload/52684/52684fig5.jpg" /> 
Figure 5. Diagram illustrating the Drosophila Activity Monitor set-up and its connectivity to the PSIU interface unit and a desktop computer. The inside of the incubator is depicted, but during recording the incubator is to be shut.
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>Description</td>
			<td>Parameter</td>
		</tr>
		<tr>
			<td>Records data each time a larva crosses a beam, and additional counts are recorded if the larva moves while within a single beam.</td>
			<td>Counts</td>
		</tr>
		<tr>
			<td>Records data only when a fly repositions between separate beams, it does not record movement within a beam.</td>
			<td>Moves</td>
		</tr>
		<tr>
			<td>Records the animals position at each second over a recording interval. This data reveals position preference as a function of time spent at each sensor over the course of an assay.&#160;</td>
			<td>Dwell</td>
		</tr>
		<tr>
			<td>This setting analyzes the animal&#8217;s general position within the tube, indicating which sensor the animal is triggering during movement.&#160;</td>
			<td>Position&#160;</td>
		</tr>
		<tr>
			<td>This setting determines the frequency at which data collected during a set time frame is saved to the desktop computer.&#160;</td>
			<td>Recording Interval&#160;</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Description of measurement parameters (Counts, Moves, Dwell and Positions).
<img alt="Video 1" src="/files/ftp_upload/52684/52684vid1.jpg" /> Video 1.&#160;DAM system assay tube prior to insertion into operating device. Moisture is provided by condensation from breath. This level is sufficient to maintain larval locomotion throughout the study period without causing the animal to float or swim.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52684/McParlandlarvamovinginmoistassaytube.mov" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Measurement of Larval Activity in the Drosophila Activity Monitor at JoVE.com

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.

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 ...

measurement-of-larval-activity-in-the-drosophila-activity-monitor

Nanyang Technological University

Research

JoVE Journal

Biology

9.3K Views.  University of New England. 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.

Video: Measurement of Larval Activity in the Drosophila Activity Monitor

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Neuroscience

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Genetics

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Behavior

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Immunology and Infection

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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 ...

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Measurement of Larval Activity in the Drosophila Activity Monitor

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