The authors acknowledge continued funding the Natural Sciences and Engineering Council of Canada (NSERC) to MBS.

1. Prepare Grape Plates and Holding Bottles for Collection of Larvae
<ol>
	<li>To make holding bottles, cut holes in one side of 6 oz fly culture bottles, large enough to fit a fly vial plug for air supply (Fig. 1D).</li>
	<li>To make grape plates, prepare 250 ml&#160;of grape juice medium (1.8% agar, 45% grape juice, 2.5% acetic acid, 2.5% ethanol) by boiling the agar, grape juice and most of the water, cool down to 70 &#186;C (stir while cooling), then add acetic acid and ethanol and bring up to volume with water.</li>
	<li>Use a syringe (without needle) to fill 35&#160;x 10 mm Petri dish lids until the surface of the grape juice medium makes a dome above the lip of the lid (Fig. 1A).</li>
	<li>Store grape plates in a humid and airtight container at 4 &#186;C until use (up to a maximum of 2 weeks).</li>
</ol>
2. Preparation of Food Plates
<ol>
	<li>Prepare standard fly food recipe. 
	Note: To test rearing food quality effects on path-length we used low nutrient fly food: 10% sucrose, 1.3% agar, 0.8% potassium sodium tartrate, 0.1% monopotassium phosphate, 0.05% sodium chloride, 0.05% calcium chloride, 0.05% magnesium chloride, 0.05% ferric sulfate, 5% yeast and 0.5% propionic acid in water; and high nutrient fly food: 1.5% sucrose, 1.4% agar, 3% glucose, 1.5% cornmeal, 1% wheat germ, 1% soy flour, 3% molasses, 3.5% yeast, 0.5% propionic acid, 0.2% Tegosept and 1% ethanol in water. For both foods autoclave the solution and cool to 50 oC before adding the propionic acid, Tegosept and ethanol.</li>
	<li>Pour 40 ml of fly food into 100 x 15mm Petri dishes.</li>
	<li>Store food plates in a humid and airtight container at 4 &#186;C until use (up to a maximum of 2 weeks).</li>
</ol>
3. Prepare Test Plates
<ol>
	<li>Prepare 58 x 25 cm, 6 mm thick black Plexiglas plates with 10, 1 mm deep circular wells, 10 cm in diameter and arranged in a 2 x 5 design (Fig. 1C) to test larvae on. 
	Note: Up to 30 larvae can be tested at a time if 3 or more of the above Plexiglas plates are available. Alternatively, larvae can be tested in Petri dishes, 10 cm in diameter. This option is much more laborious and allows for lower numbers of larvae to be tested.</li>
	<li>Obtain a black 39 x 8 cm, 6 mm thick Plexiglas spreader for spreading the yeast paste on the Plexiglas test plates.</li>
	<li>Prepare 100 x 15 mm Petri dish lids for recording larval path-lengths by separating lids from bottoms in advance.</li>
</ol>
4. Setting up Parental Populations
<ol>
	<li>Keep rearing conditions constant throughout the entire assay (e.g., 25 &#186;C, 60% humidity and a 12:12 light:dark cycle on standard fly food).</li>
	<li>Raise parental population in same rearing conditions.</li>
	<li>Collect 100 - 150 females and 50 - 75 males and age them for 5 &#177; 1 days for optimal fecundity.</li>
	<li>Allow females and males to mate overnight in a 6 oz fly culture bottle with standard fly food.</li>
	<li>Transfer flies into a holding bottle and secure a grape plate (a small amount of fresh yeast paste on the grape plate will promote egg laying; Fig. 1B) over the top with masking tape (Fig. 1E).</li>
	<li>Leave bottles standing upside down (Fig. 1F), with the grape plate at the bottom to allow adults to lay eggs on the grape plate.</li>
	<li>Discard the first grape plate after 24 hr&#160;and place a new grape plate in the holding bottle. This will be the grape plate from which larvae are collected the next day. 
	Note: Grape plates will have to be cleared of larvae 22 hr&#160;after being set up, set up plates so that they can be cleared early next day (e.g., set up plate at 12 pm and clear at 10 am the next day). It is advisable to discard the first grape plate to avoid any eggs that females might have been withholding and that might be advanced in development.</li>
</ol>
5. Collect L1 (First Instar) Larvae from Grape Plates
<ol>
	<li>Remove grape plates from bottles 22 hr after they were set up and place in a 60 x 15 mm Petri dish.</li>
	<li>Place a new grape plate with fresh yeast paste on the holding bottle.</li>
	<li>Clear and discard all larvae from the seeded grape plate using a dissecting probe.</li>
	<li>Incubate cleared grape plates for 4 hr&#160;in standard conditions.</li>
	<li>After 4 hr, pick 100 L1 larvae per strain from the grape plates and place on food plates.
	<ol>
		<li>Place larvae on the food gently (without perforating the food and burying the larvae) and avoid crowding by distributing the larvae across the food plate.</li>
	</ol>
	</li>
	<li>Incubate food plates until test animals are approximately 10 hr&#160;before the start of wandering. 
	Note: At 25 &#186;C, 10 hr&#160;before wandering generally corresponds to 72 - 96 hr&#160;after hatching. The exact amount of incubation time should be determined before starting the experiment by collecting an initial set of larvae for each strain and recording the number of hours until the start of wandering (wandering larvae will move out of the food onto the sides and lid of the food plates). 
	Note: If needed, the same parents can be used to collect larvae for 3 - 4 days of testing by repeating steps 4.7 to 5.5. Change the order in which strains are set up and tested across days to avoid a test order effect on path-length. Test the same &#34;control&#34; strain/strains each day of testing to control for day effects.</li>
	<li>If many strains are tested, stagger the set up and collection times of the larvae to allow for the testing of each strain at the appropriate time point later on.</li>
</ol>
6. Larval Path-length Test
<ol>
	<li>Dissolve dry-active baker&#39;s yeast in water at a 1:2 ratio (weight to volume). The finished yeast paste should be thinner than pancake batter, in a way that when lifting a spoon out of the paste, it drips and leaves behind ribbons on the surface of the paste that quickly melt away. 
	Note: The consistency of the yeast paste is important and should be constant throughout the experiment. The exact yeast:water ratio might have to be adjusted depending on the brand and batch of yeast.</li>
	<li>Gently pick larvae of the first strain to be tested from the food plate using a paint brush.</li>
	<li>Collect larvae in a Petri dish and gently wash with water to remove all food.
	<ol>
		<li>Quickly wash larvae with 1 - 2 ml of water and gently dab larvae dry with a laboratory wipe to avoid transferring any water when placing the larvae on the test plate. To avoid stress, keep larvae humid but not submersed in water.</li>
	</ol>
	</li>
	<li>Arrange three test plates side by side and set a 5 min timer for each test plate.</li>
	<li>Apply yeast paste to the top of the test plates and using the spreader, spread a thin layer of yeast paste on each test plate so that all the wells have an even layer of yeast.</li>
	<li>Gently place one larva in the center of each well, starting the 5 min timer after placing the first larvae on each plate. Note: Three plates can be done at a time, yielding a sample size of 30/strain/test day, up to a total of 500 larvae.</li>
	<li>Place a Petri dish lid on top of each well.</li>
	<li>When the timer runs out, without removing the larvae from the plate, start tracing the path-lengths left behind in the yeast using a permanent marker. Trace path-lengths in the same order as larvae were placed on the test plates.
	<ol>
		<li>Perform tracing at the same speed as larvae were placed on the plates. For consistency in later data analysis, use the same marker for all path-lengths. Permanent marker is recommended.</li>
	</ol>
	</li>
	<li>Repeat steps 6.2 to 6.8.1 for each strain to be tested. Pick each strain from the food immediately before testing to avoid starvation effects on path-length.</li>
</ol>
7. Food Deprivation
<ol>
	<li>If larvae are to be food deprived prior to testing, collect larvae as described in steps 6.2 to 6.3, but the desired amount of food deprivation time earlier (e.g., if larvae are to be food deprived for 4 hr, collect larvae 4 hr&#160;before test time).</li>
	<li>Divide washed larvae into two groups and place the food deprived group in a 60 x 15 mm Petri dish with 5 ml 1% agar and the control group in a Petri dish with 5 ml standard food.</li>
	<li>Place a weight on top of the inverted lid of the Petri dishes (otherwise larvae are able to crawl out when searching for food).</li>
	<li>Incubate for the previously established food deprivation period and proceed with testing as described in steps 6.2 to 6.9.</li>
	<li>If many strains are to be tested, stagger the food deprivation start times to allow for testing of each strain at the appropriate time point.</li>
</ol>
8. Data Analysis
Note: This section describes a method for data analysis using ImageJ7 or Fiji8 for processing of path-lengths, although other software can be used.
<ol>
	<li>Use a light table to transfer all traced path-lengths from the Petri dish lids to white paper (lids can be washed with ethanol and reused for further path-length recording).</li>
	<li>Trace a 50 mm straight reference line on one of the sheets of paper.</li>
	<li>Scan the path-lengths at a resolution of 300 dpi and save as bitmap or tiff image files.</li>
	<li>Open the image file, select the &#34;Process&#34; tab &#62; &#34;Binary&#34; &#62; &#34;Make binary&#34;. Then under the &#34;Process&#34; tab, select &#34;Binary&#34; &#62; &#34;Skeletonize&#34;. This produces a one pixel width line, regardless of thickness of trace.</li>
	<li>Make sure each path-length appears as a continuous single line with no crossovers.</li>
	<li>Separate any intersections using the &#34;Straight line tool&#34; and coloring the line white (the continuity of the path can be checked with the &#34;Wand&#34; (tracing) tool, the entire path should be highlighted yellow (Fig. 2).</li>
	<li>Select the &#34;Analyse&#34; tab &#62; Set measurements and select &#34;Perimeter&#34; and &#34;Display label&#34;.</li>
	<li>Select the &#34;Analyse&#34; tab &#62; &#34;Set scale&#34; and select &#34;Remove scale&#34;.</li>
	<li>On the image, select the reference line only, select the &#34;Analyse&#34; tab &#62; &#34;Analyse particles&#34; and select &#34;Show: Outlines&#34;, &#34;Display results&#34; and &#34;Clear results table&#34;. Note: The output will show the number of pixels corresponding to the 50 mm scale line.</li>
	<li>Select &#34;Analyse&#34; tab &#62; &#34;Set scale&#34; and fill in the pixel number in &#34;Distance in pixels&#34; and the corresponding distance (50 mm) in &#34;Known distance&#34;.</li>
	<li>Finally, select all the path-length drawings of one genotype at a time, select the &#34;Analyse&#34; tab &#62; &#34;Analyse particles&#34; and select &#34;Show: outlines&#34;, &#34;Display results&#34; and &#34;Clear results table&#34;.
	<ol>
		<li>Remove any written labels or obvious extraneous markings (they will be recognized as path-lengths) within the selected area by selecting the area and filling it white.</li>
	</ol>
	</li>
	<li>Visually inspect the &#34;Outlines&#34; output file to ensure the perimeters were correctly ascertained. The output will show a path-length in mm for each path-length drawing in the selection. This can easily be exported to a spreadsheet and analyzed with any statistical software.</li>
</ol>

<ol><li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Behavioral+Genetics+of+the+Fly+%28Drosophila+melanogaster%29%22">Behavioral Genetics of the Fly (Drosophila melanogaster)</a>. Dubnau, J. , Cambridge University Press. New York. 173(2014).</li>
<li>Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foraging+strategies+of+Drosophila+melanogaster%3A+a+chromosomal+analysis%5BTitle%5D)+AND+%22Behav+Genet%22%5BJournal%5D)">Foraging strategies of Drosophila melanogaster: a chromosomal analysis.</a> Behav Genet. 10, 291-302 (1980).</li>
<li>de Belle, J. S., Hilliker, A. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+localization+of+foraging+%28for%29%3A+A+major+gene+for+larval+behavior+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genetic localization of foraging (for): A major gene for larval behavior in Drosophila melanogaster.</a> Genetics. 123, 157-164 (1989).</li>
<li>Osborne, K. A., Robichon, A., Burgess, E., Butland, S., Shaw, R. A., Coulthard, A., Pereira, H. S., Greenspan, R. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+behavior+polymorphism+due+to+a+cGMP-dependent+protein+kinase+of+Drosophila%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila.</a> Science. 277, 834-836 (1997).</li>
<li>Kalderon, D., Rubin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cGMP-dependent+protein+kinase+genes+in+Drosophila%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">cGMP-dependent protein kinase genes in Drosophila.</a> J Biol Chem. 264 (18), 10739-10748 (1989).</li>
<li>Reaume, C. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cGMP-dependent+protein+kinase+as+a+modifier+of+behavior%5BTitle%5D)+AND+%22Handb+Exp+Pharmacol%22%5BJournal%5D)">cGMP-dependent protein kinase as a modifier of behavior.</a> Handb Exp Pharmacol. 191, 423-443 (2009).</li>
<li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
<li>Pereira, H. S., MacDonald, D. E., Hilliker, A. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chaser+%28Csr%29%2C+a+new+gene+affecting+larval+foraging+behavior+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Chaser (Csr), a new gene affecting larval foraging behavior in Drosophila melanogaster.</a> Genetics. 140, 263-270 (1995).</li>
<li>Shaver, S. A., Riedl, C. A. L., Parkes, T. L., Sokolowski, M. B., Hilliker, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+larval+behavioral+mutants+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">Isolation of larval behavioral mutants in Drosophila melanogaster.</a> J Neurogenet. 14, 193-205 (2000).</li>
<li>Graf, S. A., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rover%2Fsitter+Drosophila+foraging+polymorphism+as+a+function+of+larval+development%2C+food+patch+quality+and+starvation%5BTitle%5D)+AND+%22J+Insect+Behav%22%5BJournal%5D)">The rover/sitter Drosophila foraging polymorphism as a function of larval development, food patch quality and starvation.</a> J Insect Behav. 2, 301-313 (1989).</li>
<li>Gonzalez-Candelas, F., Mensua, J. L., Moya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Larval+competition+in+Drosophila+melanogaster%3A+effects+on+development+time%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Larval competition in Drosophila melanogaster: effects on development time.</a> Genetics. 82, 33-44 (1990).</li>
<li>Durisko, Z., Kemp, R., Mubasher, R., Dukas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+social+behavior+in+fruit+fly+larvae%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Dynamics of social behavior in fruit fly larvae.</a> PLoS One. 9 (4), e95495(2014).</li>
<li>Sawin, E. P., Harris, L. R., Campos, A. R., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensorimotor+transformation+from+light+reception+to+phototactic+behavior+in+Drosophila+larvae+%28Diptera%3A+Drosophilidae%29%5BTitle%5D)+AND+%22J+Insect+Behav%22%5BJournal%5D)">Sensorimotor transformation from light reception to phototactic behavior in Drosophila larvae (Diptera: Drosophilidae).</a> J Insect Behav. 7, 553-567 (1994).</li>
<li>de Belle, J. S., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heredity+of+rover%2Fsitter%3A+alternative+foraging+strategies+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Heredity%22%5BJournal%5D)">Heredity of rover/sitter: alternative foraging strategies of Drosophila melanogaster.</a> Heredity. 59, 73-83 (1987).</li>
<li>de Belle, J. S., Sokolowski, M. B., Hilliker, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+the+foraging+microregion+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genome%22%5BJournal%5D)">Genetic analysis of the foraging microregion of Drosophila melanogaster.</a> Genome. 36, 94-101 (1993).</li>
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</ol>

The authors have nothing to disclose.

The path-length assay outlined here offers a robust and simple measure of foraging behavior of Drosophila larvae. The protocol follows the general methodology described in Sokolowski2, but has since been improved in regards to efficiency and experimental controls. To the best of our knowledge this method is the only available method for measuring larval path-length. The original version of the path-length protocol2, 3, 15, 16 tested larvae on Petri dishes with a thin layer of yeast paste ap...

Since the discovery of the white gene in Thomas Hunt Morgan&#39;s laboratory in 1910, the fruit fly, Drosophila melanogaster (D. melanogaster), has been used as a model for the study of the molecular and physiological underpinnings of various biological processes. The popularity of D. melanogaster largely stems from the considerable quantity and variety of genetic tools. Drosophila&#39;s small size, relative ease of handling and short generation time render it an ideal model for genetics studies. Equally important is Drosophila&#39;s capacity to demonstrate many of the phenotypes expressed by more complex organisms including mammals. This includes complex phenotypes such as behavior that stand at the interface between the organism and its environment. As such, behavioral studies on the fruit fly have contributed substantially to our understanding of how genes and the environment mediate behavior1.
One of the first studies of D. melanogaster larval behavior investigated individual differences in larval foraging strategies by measuring the path-lengths of larvae2 while feeding. Path-length was defined as the total distance travelled by a single larva on yeast, within a five minute period. Both laboratory strains and flies from a natural population in Toronto varied in their foraging behaviors and there was a genetic component to individual differences in path-length. Two larval foraging morphs were described from the quantitative path-length distributions and they were called rover and sitter. Rovers exhibit longer path-lengths while traversing a larger area while on a food substrate than sitters. Using this path-length assay, de Belle et al.3 mapped the foraging (for) gene underlying these individual behavioral differences to a discrete location on chromosome-2 (24A3-24C5). The D. melanogaster&#160;for gene was later cloned4 and revealed to be a cGMP dependent protein kinase5, a modulator of physiology and behavior in Drosophila and other organisms6.
Here we outline the current protocol for the larval path-length assay originally developed in Sokolowski2. Although some aspects of the assay have changed over the years, the concept behind the design has not. We also provide data to illustrate the assay&#39;s potential to assess genetic and environmental contributions to individual differences in the foraging behavior of Drosophila larvae. The larval path-length assay is simple, efficient, and yet robust. A single person can test up to 500 larvae with ease in four hours and results can be obtained with a high level of reproducibility. Originally developed to localize for, it can be used in genetic screens, quantitative trait locus mapping, and in studies of gene-by-environment (GxE) interactions. Moreover, its simplicity and reproducibility make it a great resource for undergraduate teaching.

<table><tbody><tr><td>6 oz&amp;nbsp;fly culture bottles&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>AS355&amp;nbsp;</td><td></td></tr><tr><td>Fly vial plugs</td><td>Droso-Plugs</td><td>59-201</td><td></td></tr><tr><td>35 x 10 mm Petri dishes&amp;nbsp;</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>100 x 15 mm Petri dishes&amp;nbsp;</td><td>Fisher</td><td>875712</td><td></td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-168&amp;nbsp;</td><td></td></tr><tr><td>Dissecting probes</td><td>Almedic</td><td>2325-58-5300&amp;nbsp;</td><td></td></tr><tr><td>Yeast</td><td>Lab Scientific</td><td>FLY-8040-20F</td><td></td></tr></tbody></table>

foraging path-length protocol for drosophila melanogaster larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Differences in path-length of the rover and sitter for strains and the effect of food deprivation on path-length are illustrated in Fig. 3. Data collected over three consecutive days of testing showed a significant strain effect (F(1,421)&#160;= 351.89, p &#60; 2.20 x 10-16; Fig. 3A), with rovers traveling farther than sitters. In addition to the strain effect, there was also a significant food treatment effect (F(1, 42...

Watch this Scientific Journal Video about Foraging Path-length Protocol for Drosophila melanogaster Larvae at JoVE.com

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

foraging-path-length-protocol-for-drosophila-melanogaster-larvae

Research

JoVE Journal

Neuroscience

9.3K Views.  University of Toronto. We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Video: Foraging Path-length Protocol for Drosophila melanogaster Larvae

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Lifespan Protocol for Drosophila melanogaster: Generating Survival Curves to Identify Differences in Fly Longevity

Source: Bovier, T. F., et al. <a href="https://www.jove.com/video/59535/" target="_blank">Methods to Test Endocrine Disruption in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
Scientists use the Drosophila lifespan protocol to measure how different experimental conditions affect flies&#39; longevity and generate survival curves in the process to quantify the experimental outcomes. The sample protocol features the assay being applied to study the effects on longevity from exposure to varying concentrations of endocrine disruptor chemicals, or EDCs, such as 17-&#945;-ethinylestradiol (EE2).

Source: Bovier, T. F., et al. Methods to Test Endocrine Disruption in Drosophila melanogaster. J. Vis. Exp. (2019).
Scientists use the Drosophila lifespan ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www.jove.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

Samenvatting

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