Name: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila (Video) | JoVE Journal
Uploaded: 2014-02-13T13:45:00.000+00:00
Duration: 6 min 18 s
Description: Learn a high-throughput method to measure flight performance in Drosophila using a flight cylinder and digital imaging. This technique enables rapid screening of genetic mutations and conditions affecting flight ability.

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This work was supported by National Institutes of Health grants F32 NS078958 (DTB) and R01 AG033620 (BG).

1. Assemble Flight Tester
<ol>
	<li>Secure flight cylinder to Ring Stand 1 using chain clamps. (Leave approximately 3 cm underneath the cylinder for weigh dish.) 
	(Note: The flight cylinder we use is 90 cm long with a diameter of 13.5 cm.)</li>
	<li>Insert weigh dish with a thin layer of mineral oil underneath the flight cylinder.</li>
	<li>Secure funnel to Ring Stand 2 using a ring clamp and claw clamp. Adjust the height of the funnel so that the bottom of the funnel is flush with the top of the flight cylinder. (Note: the tip diameter of the funnel must be less than the outer diameter of vials placed into the drop tubes so that the vials will not fall through.)</li>
	<li>Insert drop tube into the top of the funnel and secure using a claw clamp. 
	(Note: We use a drop tube that is 25 cm long. Dropping fly-containing vials from this height allows consistent ejection of all flies with uniform force. The inner diameter of the drop tube should be slightly larger than the outer diameter of the vial to allow the vial to drop freely.)</li>
	<li>Cut polyacrylamide sheet(s) to the proper size. (Note: To aid in inserting and removing the sheet, the width should be slightly smaller than the inner circumference of the flight cylinder).</li>
	<li>Apply a thin layer of Tangle-Trap to the sheet. Allow to sit for 1 hr before use. (Note: Leave enough room at the top and bottom of the sheet (approximately&#160;3 cm) uncoated to grasp the sheet for insertion/removal.)</li>
	<li>Insert the polyacrylamide sheet in the flight cylinder.</li>
	<li>Assemble the camera track using pine support brackets. (Note: ensure that the bottom of the track can support the camera without blocking the lens. Refer to Figure 1B.)</li>
	<li>Add stoppers and screw into place. (Note: place the stoppers in locations that will allow the camera to view the entire plastic sheet in panoramic mode.)</li>
</ol>
2. Run Experiment
<ol>
	<li>Collect vials of flies to be tested. For best results, use no more than 20 flies/vial.</li>
	<li>Gently tap flies to bottom of vial, unplug, then insert into drop tube and release vial. 
	(Note: The vial falls down the drop tube until it hits the narrow funnel opening. When the vial hits the funnel, the flies are ejected into the flight cylinder.)</li>
	<li>Lift the drop tube to remove the empty vial. 
	(Note: Multiple vials of flies of the same test group can be assayed on a single polyacrylamide sheet. We find that up to 200 flies (10 vials of 20 flies each) can be tested and imaged readily on a single sheet.</li>
	<li>Remove the plastic sheet and place it on a flat white surface. 
	(Note: white poster board may be used if bench tops are dark colored.)</li>
	<li>Assemble the camera track over the plastic sheet. The camera should be sufficiently high above the sheet to have both the top and bottom of the sheet in the field of view.</li>
	<li>Slide the camera along the track while holding the &#34;capture&#34; button to acquire a panoramic image.</li>
	<li>The number of flies landing in the oil may be counted manually for each trial.</li>
	<li>Repeat steps 2.2-2.7 for all conditions in a given experiment. The flies can be removed from the sheet between each trial. Alternatively, several sheets may be used, with a new sheet for each trial.</li>
</ol>
3. Data Collection
<ol>
	<li>Open image files using ImageJ software.</li>
	<li>Crop images if necessary to include only the landing surface area. (This is the area coated in Tangle-Trap.)</li>
	<li>Convert images to 8-bit grayscale.</li>
	<li>Create a &#34;Threshold&#34; to filter out the white background. 
	(Image &#8594; Adjust &#8594; Threshold).</li>
	<li>Set the parameters to identify each fly using the &#34;analyze particles&#34; menu. 
	(Analyze &#8594; Analyze Particles) Define the parameters used to identify a particle. With our set up, we find that using an area of 5-90 pixels2 and a circularity of 0.4-1.0 will accurately identify all samples.</li>
	<li>Measure the location of each fly using the generated list of coordinates for each particle. The x-coordinate in pixels can be converted to centimeters to calculate the landing height.</li>
	<li>Import table into a spreadsheet (such as Microsoft Excel).</li>
</ol>

<ol><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+dissection+of+behavior%5BTitle%5D)+AND+%22Sci.+Am%22%5BJournal%5D)">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li>
<li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assaying+locomotor%2C+learning%2C+and+memory+deficits+in+Drosophila+models+of+neurodegeneration%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504(2011).</li>
<li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192(2013).</li>
<li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+aggression+in+Drosophila+%28fruit+flies%29%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155(2007).</li>
<li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+to+assay+Drosophila+behavior%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795(2012).</li>
<li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proboscis+extension+response+%28PER%29+assay+in+Drosophila%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193(2007).</li>
<li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flightless+flies%3A+Drosophila+models+of+neuromuscular+disease%5BTitle%5D)+AND+%22Ann.+N.Y.+Acad.+Sci%22%5BJournal%5D)">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li>
<li>Atkinson, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li>
<li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li>
<li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li>
<li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+iterative+negative+geotaxis+%28RING%29%3A+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila%5BTitle%5D)+AND+%22Exp.+Gerontol%22%5BJournal%5D)">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li>
<li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visual+control+of+flight+speed+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li>
<li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li>
</ol>

The authors have no conflicts of interest to disclose.

Using the methods described here, we have been able to rapidly assess flight performance of a large number of Drosophila mutants, providing greater efficiency than previously possible. For our experiments, we routinely separate males and females and raise them at low density (less than 20 flies/vial) to limit aggression that could damage wings. Another important consideration is to control properly for differences in flight performance owing to differences in genetic background. We also find it helps to allow fl...

Drosophila has long been used to study the genetic basis of behavior1, and researchers have devised a number of ways to analyze various types of behavior2-6. Flies have been particularly useful in providing useful models of neuromuscular disorders7. A common assay used to study locomotor behavior is flight performance. The original flight tester is useful for identifying flight defective mutants and for quantitative assessment of flight ability1, but it has several shortcomings that limit its application for high throughput screens: the use of oil-coated cylinders is messy and cumbersome, certain features such as the length of the cylinder and introduction of flies into the tube with variable force reduce quantitative accuracy; and it is difficult to recover live flies from the tester. To overcome these limitations, we have modified the flight tester to include a number of improvements. We added a &#34;drop tube&#34; to introduce flies to eliminate variability between experiments and users. We use removable acrylic sheets coated with an adhesive that allows for easier cleanup and recovery of individual flies. We have increased the length of the flight tube to improve quantitative accuracy and reliability. Finally, we use a digital camera and imaging software to calculate the landing heights of flies. We believe these improvements will be useful to any laboratory interested in conducting large-scale genetic screens for defects in flight performance.

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an improved method for accurate and rapid measurement of flight performance in drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

Figure 1A shows a schematic of the updated flight tester assembly. Figure 1B illustrates the track design allow the camera to take a panoramic image without blocking the field of view. Representative results are shown in Figure 2, where the flight performance of slowpoke mutant flies, which have a known flight defect8-10, are compared to wild-type Canton-S flies. Control flies consistently land near the top of the cylinder, with very little s...

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

The overall goal of this procedure is to rapidly and accurately measure flight performance in Drosophila. This is accomplished by first dropping flies into a flight cylinder. The second step of the procedure is to image the landing surface with a digital camera.The final step is to analyze the data using imaging software by measuring the landing height of each fly. Ultimately, this procedure helps identify mutations and conditions such as aging or neurodegeneration, which impair flight ability and helps to quantify flight performance. The main advantage of this technique over existing methods, such as the original flight tester, is that it's more appropriate for high throughput screens for mutants and other conditions that impair flight.We first had the idea for this method when we realized how much time was involved with testing a large number of genotypes using the original flight tester. Secure the flight cylinder to a ring stand using chain clamps. Leave approximately three centimeters underneath the cylinder for the way dish.Load the way dish with a thin layer of mineral oil and slide it under the flight cylinder. Secure the funnel to a second ring stand. Adjust the height of the funnel so that the bottom of the funnel is flush with the top of the flight cylinder.The narrowest part of the funnel must be narrower than a fly vial. The diameter of the drop tube should be slightly wider than a fly vial, so the vial is able to drop freely. Insert the drop tube into the top of the funnel and secure it using a claw clamp.Next from a poly acrylamide sheet, cut a rectangular piece the length of the flight cylinder with a width slightly smaller than the inner circumference of the flight cylinder. Apply a thin layer of tangle trap adhesive to the sheet, leaving enough room at the top and bottom uncoated to hold the sheet. After the adhesive on the sheet has dried for an hour, slide the sheet into the flight cylinder with the adhesive inward.Next, assemble the camera track using pine support brackets. Ensure that the bottom of the track can support the camera without blocking the lens. Then screw the stoppers into place where they do not interrupt the camera's view of the plastic sheet.Collect vials of flies to be tested with no more than 20 flies per vial. Gently tap the flies to the vial bottom. Unplug the vial and insert it into drop tube.Release the vial. When the vial hits the walls of the narrowing funnel, the flies that are ejected into the flight cylinder. Lift the drop tube to remove the empty vial and load more flies if desired.Up to 200 flies loaded from 10 vials can be tested and imaged readily on a single poly acrylamide sheet. Now, remove the plastic sheet and place it on A flat white surface white poster board may be used if benchtops are dark colored. Assemble the camera track over the plastic sheet.The camera should be sufficiently high above the sheet to have both the top and bottom of the sheet in the field of view. Slide the camera along the track while holding the capture button to acquire a panoramic image. The number of flies landing in the oil in the dish below the flight chamber can be counted manually.For each trial between trials, flies can be removed from the sheet and the sheet can be reused. Open the image files in image J software crop the images if necessary to include only the landing surface area. The area coated entangle trap.Convert the images to eight bit gray scale and create a threshold. To filter out the white background. Set the parameters to identify each fly.Using the analyze particles menu, define the parameters. Used to identify a particle using the demonstration apparatus. An area of five to 90 pixels squared and a circularity of 0.4 to 1.0 will accurately identify all samples.Now, measure the location of each fly using the generated list of coordinates for each particle. The X coordinate in pixels can be converted to centimeters. To calculate the landing height, import the table into a spreadsheet and proceed with analysis.The flight performance of slow poke mutant flies, which have a known flight defect, was compared to wild type canton s flies. All the flies were three days old, raised at room temperature control flies consistently landed near the top of the cylinder. The spread of the landing was much greater for slow poke mutants.The average landing height for cantons at 73 centimeters was significantly greater than that of slow poke mutants at 44 centimeters. Following this procedure, additional methods such as histology can be used to answer additional questions regarding the structural integrity of flight muscles, motor neurons, or neuromuscular junctions. After watching this video, you should have a good understanding of how you can rapidly assess flight performance in oph.

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Learn a high-throughput method to measure flight performance in Drosophila using a flight cylinder and digital imaging. This technique enables rapid screening of genetic mutations and conditions affecting flight ability.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

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