This work was supported by the National Science Foundation under grant #IOS-1025627 and by the National Institutes of Health under grant #1R21NS078582-01 to J.A.W.

This protocol uses two setups (Figure 1) to acquire four different readouts collected from flies subjected to a bacterial infection. These outputs include 1) sleep/wake behavior; 2) survival outcome; 3) bacterial load in the fly; and 4) real-time measurement of NF&kappa;B reporter activity in vivo. In combination with the genetic tools that are available in Drosophila, these measurements provide mechanistic insight into the molecular link between immune function and behavior.
1. Measure Locomotor Activity and Sleep in Flies
<ol>
	<li>The setup used to measure locomotor activity and sleep in flies, which includes the Drosophila activity monitoring system (DAM2, Trikinetics), incubators, dark room, and the preparation of experimental animals and activity tubes, has been described previously6. The same approach is used here, with some minor modifications.
	<ol class="lalpha">
		<li>The lighting of the incubator is controlled by the incubator itself. Therefore, it is important to synchronize the time setting among computer and incubators.</li>
		<li>Activity tubes are cleaned for re-use by boiling on a hot plate in de-ionized water. A small amount of detergent is added for the first boil, tubes are well-rinsed and boiled twice more. If yarn is used to plug the tube, used activity tubes (containing food, wax, fly and yarn) can be cleaned without prior removal of the yarn.</li>
	</ol>
	</li>
	<li>Prior to initiating an experiment, place cultures containing late pupal staged flies in incubators for three to four days to adapt to experimental lighting and other environmental conditions. Light:dark or constant conditions have been commonly used to measure sleep behavior. In this example, flies are acclimated to constant light to eliminate the influence of the circadian clock on the immune response and behavior2,7,8 . As described in Figure 1A, load 1-4 day old flies into the Trikinetics DAM2 activity monitors as described6, and record for a minimum of three days prior to the infection.</li>
</ol>
2. Infect Flies with a Pathogenic Strain of Bacteria
<ol>
	<li>The protocol described here is specific for S. marcescens, which are easy to grow and maintain. Protocols for long-term storage and culture conditions for other bacterial species will vary. S. marcescens are stored in 15-50 % glycerol at -80 &deg;C. To prepare for long-term storage, mix 2 volumes of overnight bacterial culture (O.D. 600 = 0.5 - 1.0) to 1 volume of autoclaved 50% glycerol in 1.5 ml microcentrifuge tubes. This will result in a final glycerol concentration of 17%. Store tubes at -80 &deg;C. To prepare a short-term in-use source of bacteria, scrape the frozen stock with a sterile 200 &mu;l pipette tip and perform a three-way streak on an LB agar plate. Incubate the plate overnight at 37 &deg;C to get isolated colonies. Store the plate at 4 &deg;C. 
	One day before the scheduled infection, pick a single colony from the LB agar plate with a sterile 200 &mu;l pipette tip and submerge the tip into a culture tube containing 5 ml LB medium. Grow bacteria overnight, or up to 16 hr inside an incubator shaker at 37 &deg;C and 250 rpm until it reaches the exponential growth phase. Measure the concentration with a spectrophotometer at OD600. The concentration in this phase ranges from 0.5 to 1. If the concentration is too high, subculture the bacteria and grow for another several hours to get an optimal concentration. Perform the procedure near a flame source. Some bacterial strains are engineered for antibiotic resistance and are grown and selected for appropriate antibiotic added to the medium. S. marcescens (ATCC #8100) are not antibiotic resistant and are therefore grown in sterile medium without antibiotic. To verify sterile technique, do a mock culture without bacteria as a control. 
	The solution to infect flies contains bacteria (diluted to OD600=0.1) and 1% food coloring (Brilliant Blue FCF) in PBS. Prepare a solution for injection control by adding equivalent amount of LB medium used in infection solution and 1% blue food coloring in PBS. Store solutions on ice.</li>
	<li>Prepare glass needles for injecting the flies. Pull glass capillaries (o.d. = 1 mm, i.d. = 0.58 mm, WPI) to a fine tip using a micropipette puller (Narishige). Under a dissecting microscope, use fine forceps to break off the tip of the needle so that the opening is large enough to fill with injection fluid by applying suction with a syringe, but small enough to minimize damage to the fly during the injection process. After breaking the tip, the tip size should be around 40-50 &mu;m. The glass needle is attached to a 3 cc plastic syringe with a length of tubing. The flow of injection medium is controlled manually applying positive pressure or suction with the syringe. Avoid contaminating the rubber tubing with injection fluid, as the syringe apparatus is used for both infection and control injections.</li>
	<li>Anesthetize flies by putting them on a CO2 pad. CO2 is passed through a sealed container of water to humidify the pad and to reduce static electricity, which can complicate manipulation of flies on the pad. Inject flies by poking the glass needle into the region above the scutellum of the dorsal thorax. The passing of injection medium into the fly is verified by the food coloring - flies turn blue as the food coloring spreads through the system. The dose of bacteria that flies receive can be quantified as described in Section 3, below. Some experimental designs include a control group that receives aseptic injury by injecting flies with PBS and food coloring but without bacteria.</li>
</ol>
3. Determine the Bacterial Load
One approach to evaluating the immune response against bacterial infection is to determine the bacterial load post infection. D. melanogaster is a great model to determine this parameter because the whole fly can be homogenized to estimate the total bacterial numbers within an individual. The rationale behind this protocol is that when grown on a solid medium such as Luria broth (LB) agar on a Petri dish (LB plate), a single bacterium forms a visible distinguishable colony. Therefore, by homogenizing infected flies in LB liquid medium, generating serial dilutions of the homogenate, and spreading the diluted homogenate onto LB plates, the number of bacterial cells infecting a fly can be determined. A control group of flies injected with PBS and food coloring but without bacteria should be used to verify that the infection was not contaminated with other bacterial species. There should be no colonies on the LB agar plate in this condition.
<ol>
	<li>Autoclave all materials that will be used for this procedure before performing the actual homogenization step. This includes 200 &mu;l pipette tips cut with scissors, LB media, and pestles used for the homogenization. Prepare plates by pouring autoclaved LB/agar medium into 10 cm sterile Petri dishes at least 1 day before homogenizing flies.</li>
	<li>Anesthetize and collect flies in 1.5 ml microcentrifuge tubes and store tubes on ice.</li>
	<li>Perform the homogenization near a flame to prevent contamination. A control homogenization containing flies without infection is recommended especially when using a bacterial strain such as S. marcescens, which are not antibiotic resistant. Homogenize a minimum of 2 groups of 10 flies each per experimental condition. The number of flies used per homogenate is determined by the experimenter. Some groups have used one fly per homogenate, which is useful for evaluating variation in bacterial load between individual flies8, while others have used from 3-10 flies per homogenate to compare across genotype or experimental condition9-12.
	<ol class="lalpha">
		<li>Add 400 &mu;l LB medium to each microcentrifuge tube containing flies and homogenize using a small motor and pestle (Kontes).</li>
		<li>Using sterilized cut pipette tips, serial dilute the homogenate 1:10 by adding 20 &mu;l homogenized LB/bacterial medium to 1.5 &mu;l microcentrifuge tube containing 180 &mu;l LB medium. Cutting pipette tips prevents blockage from fly debris and ensures that an appropriate volume is being transferred.</li>
		<li>Dilution factor is determined empirically and depends on the genotype of the fly, the bacterial strain used, and experimental condition. For wild type flies infected with S. marcescens, use dilutions of 1:102 or 1:103 for flies homogenized immediately post-infection, and dilutions of 1:104 or 1:105 for flies homogenized 24 hr post-infection.</li>
		<li>Add 100 &mu;l LB/bacterial medium from the microcentrifuge tubes containing the final dilution and spread the medium on an LB plate using glass balls (VWR) to ensure an even distribution.</li>
		<li>Discard the glass balls into 100% EtOH solution. Place the LB plates in a 37 &deg;C incubator overnight.</li>
		<li>As an optional step, use an imaging system with visual light to obtain an image of the LB plates (FluorChem 8900; Alpha Innotech). Count the numbers of colonies either on the plate itself or from the image of the plate using the counting tool on Photoshop software (Adobe Photoshop CS3).</li>
		<li>Calculate the number of colony-forming units per fly using the formula: [n /(N *D *v)]*V; where n= number of colonies grown on the LB plate; N=the number of the flies pooled in one microcentrifuge tube; D= dilution factor; and v = volume of solution spread onto each plate; V = initial volume of the homogenate.</li>
	</ol>
	</li>
</ol>
4. Evaluate Sleep and Survival Duration After Infection
<ol>
	<li>For flies that are not harvested for measuring bacterial load, continue monitoring behavior for another 7-10 days. Survival duration is influenced by genotype of the fly, environmental conditions, and bacterial species used for infection.</li>
	<li>After ~10 days, terminate the experiment, download and process behavioral data as previously described6. Insomniac2 custom software, which is based in Matlab, is used to analyze sleep parameters. It is important to eliminate dead flies from the behavioral analysis. Normally, this restricts analyses to within the first day after infection, depending on the type of infection and fly genotype. Death in the assay is indicated by the time all activity counts have reached zero. To facilitate analysis of survival, Drosonex, custom software written in Microsoft Visual C++ 6.0, is used to process raw data files from the Trikinetics DAM system. The software reports as Excel files, survival duration of each fly (in hours), compiles activity data from all monitors into a single spreadsheet, and reports the percent flies surviving over time as set by the user. The Excel files are designed to integrate into other statistical software packages for further analysis.</li>
</ol>
5. Measure NF&kappa;B Activity During Infection Using a Luciferase Reporter Assay
Transgenic &kappa;B-luc flies used in this assay were generated previously as described in Kuo et al., 20102. Briefly, the &kappa;B-luc reporter contains 8 repeats of an NF&kappa;B binding sequence that were inserted into a promoter upstream of a luciferase open reading frame.
<ol>
	<li>Adjust flies to experimental lighting conditions as described in section 1.2. In this example, 1-4 day old &kappa;B-luc flies are housed in vials containing a 5% sucrose, 2% agar food medium and placed in constant light (LL) for 2 days to reach the same age as other flies during infection.</li>
	<li>Prepare a 96-well microplate for flies. Each well contains 2 layers of food medium (Figure 2); the top layer contains luciferin, the substrate of luciferase. Add 300 &mu;l of a 5% sucrose, 2% agar solution to each well and allow to solidify. Next, add a 50 &mu;l top layer to each well containing 5% sucrose, 1% agar, and 2 mM luciferin (Gold BioTechnology, Inc.). Luciferin concentrations used for measuring reporter activity in Drosophila have varied in the literature, and have been as low as 100 &mu;M13. The concentration required can be determined empirically. Between dispensing the food layers, cover the plate with a fine mesh cloth to facilitate drying while maintaining sterility. The plate should be allowed to dry thoroughly, up to one hour, in order to prevent flies from getting stuck in condensation droplets. Because luciferin is light-sensitive, avoid exposing plates to light more than necessary.</li>
	<li>Apply a clear adhesive film (Top-Seal-A; Perkin Elmer) to a 96-well microwell plate and perforate using a fine needle at a quantity of 2 holes per well. These holes will not only allow air exchange to each well, but will also provide a way to anesthetize flies on an individual basis. Using a sharp blade and straight edge, introduce a cut between each column. This will provide an easy way to load/unload flies in groups of 8 at a time. 
	To load flies into the microwell plate, remove the vials from the incubator and anesthetize flies on a CO2 pad. Load flies one-by-one to each well column-by-column. Should a fly inadvertently get stuck to the adhesive seal, leave the fly alone as they are often able to free themselves without intervention. Return the microwell plate to the incubator in LL for 8-24 hr to adjust to the new environment and to consume the luciferin substrate.</li>
	<li>Culture bacteria, S. marcescens, and prepare a solution for infection as described above.</li>
	<li>Anaesthetize the flies by using a micropipette tip attached to a low-pressure CO2 line. Place the micropipette tip directly above the ventilation holes made for each well. Take caution to ensure that the CO2 pressure is high enough to anaesthetize the fly, but low enough to avoid injury to the fly. In groups of eight, individually transfer each fly to a CO2 pad, and infect as described above. After infection, return each fly to its original well and reseal the microplate.</li>
	<li>Measure luminescence (TopCount-NXT Luminescence and Scintillation Counter; Perkin-Elmer). The TopCount luminescence counter contains a stacking cassette for plates, which allows for programmed and automated readings continuously at desired increments for any length of time (usually up to five days). This feature is not available on all instruments, and data collection for experiments performed with other instruments may therefore be less frequent. The luminescence counter is housed in a room with a controlled temperature and lighting schedule. When loading plates into the stacking cassette, stack the plates containing the flies between clear blank plates to ensure that flies receive light. Program the luminometer according to manufacturer's specifications to collect readings every hour for a minimum of 24 hr. In this example, the detectors are programmed to read each well for 10 sec and to express the result as counts (arbitrary units) per second. This value is averaged across 3 readings per well. Export the data files to a spreadsheet and perform a standard analysis, graphing the results of luminescence.</li>
</ol>

<ol>
	<li>Majde, J. A., Krueger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+the+innate+immune+system+and+sleep.">Links between the innate immune system and sleep.</a> J. Allergy Clin. Immunol. 116, 1188-1198 (2005).</li><li>Kuo, T. H., Pike, D. H., Beizaeipour, Z., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+triggered+by+an+immune+response+in+Drosophila+is+regulated+by+the+circadian+clock+and+requires+the+NFkappaB+Relish.">Sleep triggered by an immune response in Drosophila is regulated by the circadian clock and requires the NFkappaB Relish.</a> BMC Neurosci. 11, 1471-2202 (2010).</li><li>Preston, B. T., Capellini, I., McNamara, P., Barton, R. A., Nunn, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+resistance+and+the+adaptive+significance+of+sleep.">Parasite resistance and the adaptive significance of sleep.</a> BMC Evol Biol. 9, 7 (2009).</li><li>Imeri, L., Opp, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+(and+why)+the+immune+system+makes+us+sleep.">How (and why) the immune system makes us sleep.</a> Nat. Rev. Neurosci. 10, 199-210 (2009).</li><li>Ayres, J. S., Schneider, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Signaling+Protease+Required+for+Melanization+in+Drosophila+Affects+Resistance+and+Tolerance+of+Infections.">A Signaling Protease Required for Melanization in Drosophila Affects Resistance and Tolerance of Infections.</a> PLoS Biol. 6, e305 (2008).</li><li>Chiu, J. C., Low, K. H., Pike, D. H., Yildirim, E., Edery, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Locomotor+Activity+to+Study+Circadian+Rhythms+and+Sleep+Parameters+in+Drosophila.">Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila.</a> J. Vis. Exp. (43), e2157 (2010).</li><li>Lee, J. E., Edery, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+Regulation+in+the+Ability+of+Drosophila+to+Combat+Pathogenic+Infections.">Circadian Regulation in the Ability of Drosophila to Combat Pathogenic Infections.</a> Curr. Biol. 18, 195-199 (2008).</li><li>Stone, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock+protein+timeless+regulates+phagocytosis+of+bacteria+in+Drosophila.">The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila.</a> PLoS Pathog. 8, e1002445 (2012).</li><li>Hill-Burns, E. M., Clark, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-linked+variation+in+immune+response+in+Drosophila+melanogaster.">X-linked variation in immune response in Drosophila melanogaster.</a> Genetics. 183, 1477-1491 (2009).</li><li>Short, S. M., Lazzaro, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+and+male+genetic+contributions+to+post-mating+immune+defence+in+female+Drosophila+melanogaster.">Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster.</a> Proc. Biol. Sci. 277, 3649-3657 (2010).</li><li>Williams, J. A., Sathyanarayanan, S., Hendricks, J. C., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+sleep+and+the+immune+response+in+Drosophila:+a+role+for+the+NFkappaB+relish.">Interaction between sleep and the immune response in Drosophila: a role for the NFkappaB relish.</a> Sleep. 30, 389-400 (2007).</li><li>Ramsden, S., Cheung, Y. Y., Seroude, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+Drosophila+immune+response+during+aging.">Functional analysis of the Drosophila immune response during aging.</a> Aging Cell. 7, 225-236 (2008).</li><li>Williams, J. A., Su, H. S., Bernards, A., Field, J., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circadian+output+in+Drosophila+mediated+by+neurofibromatosis-1+and+Ras/MAPK.">A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK.</a> Science. 293, 2251-2256 (2001).</li><li>Hedengren, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relish,+a+central+factor+in+the+control+of+humoral+but+not+cellular+immunity+in+Drosophila.">Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.</a> Mol. Cell. 4, 827-837 (1999).</li><li>Leulier, F., Rodriguez, A., Khush, R. S., Abrams, J. M., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+caspase+Dredd+is+required+to+resist+gram-negative+bacterial+infection.">The Drosophila caspase Dredd is required to resist gram-negative bacterial infection.</a> EMBO Rep. 1, 353-358 (2000).</li><li>Wu, L. P., Choe, K. -. M., Lu, Y., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Immunity:+Genes+on+the+Third+Chromosome+Required+for+the+Response+to+Bacterial+Infection.">Drosophila Immunity: Genes on the Third Chromosome Required for the Response to Bacterial Infection.</a> Genetics. 159, 189-199 (2001).</li><li>Dionne, M. S., Ghori, N., Schneider, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+is+a+genetically+tractable+model+host+for+Mycobacterium+marinum.">Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum.</a> Infect. Immun. 71, 3540-3550 (2003).</li><li>Romeo, Y., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+immunity:+methods+for+monitoring+the+activity+of+Toll+and+Imd+signaling+pathways.">Drosophila immunity: methods for monitoring the activity of Toll and Imd signaling pathways.</a> Methods Mol. Biol. 415, 379-394 (2008).</li><li>Lu, Y., Wu, L. P., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+arm+of+the+Drosophila+innate+immune+response+requires+an+I{kappa}B+kinase.">The antibacterial arm of the Drosophila innate immune response requires an I{kappa}B kinase.</a> Genes Dev. 15, 104-110 (2001).</li><li>Koh, K., Evans, J. M., Hendricks, J. C., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+age-associated+changes+in+sleep:wake+cycles.">A Drosophila model for age-associated changes in sleep:wake cycles.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 13843-13847 (2006).</li><li>Bushey, D., Hughes, K. A., Tononi, G., Cirelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep,+aging,+and+lifespan+in+Drosophila.">Sleep, aging, and lifespan in Drosophila.</a> BMC Neurosci. 11, 1471-2202 (2010).</li><li>Allee, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The social life of animals. , (1938).</li><li>Stanewsky, R., Jamison, C. F., Plautz, J. D., Kay, S. A., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+circadian-regulated+elements+contribute+to+cycling+period+gene+expression+in+Drosophila.">Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila.</a> Embo. J. 16, 5006-5018 (1997).</li><li>Levine, J. D., Funes, P., Dowse, H. B., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+analysis+of+behavioral+and+molecular+cycles.">Signal analysis of behavioral and molecular cycles.</a> BMC Neurosci. 3, 1 (2002).</li><li>Ayres, J. S., Schneider, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anorexia+in+resistance+and+tolerance+to+infections+in+Drosophila.">The role of anorexia in resistance and tolerance to infections in Drosophila.</a> PLoS Biol. 7, e1000150 (2009).</li><li>Plautz, J. D., Kaneko, M., Hall, J. C., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+photoreceptive+circadian+clocks+throughout+Drosophila.">Independent photoreceptive circadian clocks throughout Drosophila.</a> Science. 278, 1632-1635 (1997).</li><li>Huber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+homeostasis+in+Drosophila+melanogaster.">Sleep homeostasis in Drosophila melanogaster.</a> Sleep. 27, 628-639 (2004).</li><li>Zimmerman, J. E., Raizen, D. M., Maycock, M. H., Maislin, G., Pack, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+video+method+to+study+Drosophila+sleep.">A video method to study Drosophila sleep.</a> Sleep. 31, 1587-1598 (2008).</li><li>Toth, L. A., Rehg, J. E., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+in+sleep+and+other+pathophysiological+sequelae+of+influenza+virus+infection+in+naive+and+immunized+mice.">Strain differences in sleep and other pathophysiological sequelae of influenza virus infection in naive and immunized mice.</a> J. Neuroimmunol. 58, 89-99 (1995).</li><li>Hendricks, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rest+in+Drosophila+is+a+sleep-like+state.">Rest in Drosophila is a sleep-like state.</a> Neuron. 25, 129-138 (2000).</li><li>Wu, M. N., Koh, K., Yue, Z., Joiner, W. J., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+for+sleep+and+circadian+mutants+reveals+mechanisms+underlying+regulation+of+sleep+in+Drosophila.">A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila.</a> Sleep. 31, 465-472 (2008).</li></ol>

No conflicts of interest declared.

This protocol outlines an approach to investigate how behavior, particularly sleep, is linked to immune response parameters. These parameters include bacterial load, survival outcome, and NF&kappa;B activity as measured by a luciferase reporter in vivo. Together these parameters provide information about how well a fly can fight an infection. Bacterial load and survival outcome are immune response parameters that involve a straightforward measurement in Drosophila. RelE20 muta...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </td></tr><tr><td>Incubators</td><td>Percival Scientific, Inc.</td><td>I30BLLC8&lt;br/&gt; I36VLC8</td><td>Any incubator capable of running programmed light/temperature schedules is appropriate.</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Activitiy Monitors</td><td>Trikinetics Inc., Waltham, MA</td><td>DAM2</td><td>As described elsewhere&lt;sup&gt;6&lt;/sup&gt;, this system requires a computer interface, software, and other accessories.</td></tr><tr><td>Pyrex Glass Tubes</td><td>Trikinetics Inc., Waltham, MA</td><td>PGT-5x65</td><td></td></tr><tr><td>Microplate scintillation and luminescence counter</td><td>Perkin Elmer</td><td>TopCount NXT&lt;br/&gt; 12 detector</td><td>Any microplate reader capable of detecting luminescence can be used for this type of reporter assay. TopCount contains multiple detectors and an automated stacker; it is capable of being programmed to read continuously from multiple plates. </td></tr><tr><td>FluorChem 8900</td><td>Alpha Innotech</td><td></td><td>Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient.</td></tr><tr><td>Micropipette Puller</td><td>Tritech Research, Inc.</td><td>Narishige PC-10</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Supplies&lt;/strong&gt; </td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>3 ml Syringe</td><td>Fisher Scientific</td><td>BD 305482</td><td></td></tr><tr><td>Syringe Needles</td><td>Fisher Scientific</td><td>BD 305196</td><td>18 G - cut off the tip of the needle to prevent damage to the tubing. </td></tr><tr><td>Silicone Tubing, i.d. (0.030&quot;) o.d. (0.065&quot;) Wall Thickness (0.018&quot;)</td><td>VWR</td><td>60985-706</td><td>Used for attaching glass capillary needles to a syringe</td></tr><tr><td>3 Way Stopcock</td><td>American Pharmaseal Company</td><td>K75</td><td></td></tr><tr><td>Kontes Pellet Pestle Cordless Motor</td><td>Fisher Scientific</td><td>K749540-0000</td><td></td></tr><tr><td>Kontes Pellet Pestle </td><td>Fisher Scientific</td><td>K749521-1590</td><td></td></tr><tr><td>Glass balls 3mm</td><td>VWR</td><td>26396-630</td><td></td></tr><tr><td>Microplate Microlite 1+</td><td>Thermo Scientific</td><td>7571</td><td>Select 96-well plates that are appropriate for luminescence - they must be opaque.</td></tr><tr><td>TopSeal-A:96-well Microplates</td><td>PerkinElmer</td><td>6005185</td><td>Microplate Press-On Adhesive Sealing Film</td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Gold BioTechnology, Inc.</td><td>LUCNA</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt; </td></tr><tr><td>Insomniac2</td><td>Available upon request to the authors</td><td>custom; written by Lesley Ashmore, Ph.D. (Westminster College)</td><td>Matlab based software that has been used routinely for analysis of sleep&lt;sup&gt;2,6,11&lt;/sup&gt; </td></tr><tr><td>Drosonex</td><td>Available upon request to the authors</td><td>custom; written by Thomas Coradetti (Sidewalk Software)</td><td>A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system</td></tr><tr><td>Photoshop CS3</td><td>Adobe</td><td></td><td>Useful for obtaining numbers of cfu/plate from digital images (optional)</td></tr></tbody></table>

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.

<ol>
	<li>Infection promotes sleep. In this example, Canton-S (CS) wild type flies and mutant flies lacking an NF&kappa;B gene, Relish (RelE20) 14, were loaded into two DAM2 activity monitors (n=32 for each genotype) and infected as described above. Flies were maintained in constant light to eliminate the influence of the circadian clock on behavior and infection2,7,8. The RelE20 mutants were isogenized to CS as described previously11. Both sets of flies were infe...

Watch this Scientific Journal Video about Quantitative Measurement of the Immune Response and Sleep in Drosophila at JoVE.com

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.

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 ...

quantitative-measurement-immune-response-sleep

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.3K Views.  University of Pennsylvania Perelman School of Medicine. 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, NFκB.

Video: Quantitative Measurement of the Immune Response and Sleep in Drosophila

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

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

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 ...

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Behavior

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

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.

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

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 ...

Biology

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents &#62;95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation.

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Measurement of Larval Activity in the Drosophila Activity Monitor

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

Measurement of Larval Activity in the Drosophila Activity Monitor

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

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Summary

Explore More Videos

Chapters in this video

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

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

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

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Larval Activity in the Drosophila Activity Monitor

Oral Bacterial Infection and Shedding in Drosophila melanogaster