Quantitative Measurement of the Immune Response and Sleep in Drosophila

Summary

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.

Abstract

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 system^3,4. 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 NFκB, 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 NFκB 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.

Protocol

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 NFκB 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

1. The setup used to measure locomotor activity and sleep in flies, which ....

Results

1. Infection promotes sleep. In this example, Canton-S (CS) wild type flies and mutant flies lacking an NFκB gene, Relish (Rel^E20) 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 infection^2,7,8. The Rel^E20 mutants were isogenized to CS as described previously¹¹. Both sets of flies were infected w.......

Discussion

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 NFκB 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. Rel^E20 muta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Incubators	Percival Scientific, Inc.	I30BLLC8 I36VLC8	Any incubator capable of running programmed light/temperature schedules is appropriate.
Drosophila Activitiy Monitors	Trikinetics Inc., Waltham, MA	DAM2	As described elsewhere6, this system requires a computer interface, software, and other accessories.
Pyrex Glass Tubes	Trikinetics Inc., Waltham, MA	PGT-5x65
Microplate scintillation and luminescence counter	Perkin Elmer	TopCount NXT 12 detector	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.
FluorChem 8900	Alpha Innotech		Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient.
Micropipette Puller	Tritech Research, Inc.	Narishige PC-10
Supplies
Borosilicate Glass Capillaries	World Precision Instrument Inc.	1B100F-4
3 ml Syringe	Fisher Scientific	BD 305482
Syringe Needles	Fisher Scientific	BD 305196	18 G - cut off the tip of the needle to prevent damage to the tubing.
Silicone Tubing, i.d. (0.030") o.d. (0.065") Wall Thickness (0.018")	VWR	60985-706	Used for attaching glass capillary needles to a syringe
3 Way Stopcock	American Pharmaseal Company	K75
Kontes Pellet Pestle Cordless Motor	Fisher Scientific	K749540-0000
Kontes Pellet Pestle	Fisher Scientific	K749521-1590
Glass balls 3mm	VWR	26396-630
Microplate Microlite 1+	Thermo Scientific	7571	Select 96-well plates that are appropriate for luminescence - they must be opaque.
TopSeal-A:96-well Microplates	PerkinElmer	6005185	Microplate Press-On Adhesive Sealing Film
D-Luciferin, Potassium Salt	Gold BioTechnology, Inc.	LUCNA
Software
Insomniac2	Available upon request to the authors	custom; written by Lesley Ashmore, Ph.D. (Westminster College)	Matlab based software that has been used routinely for analysis of sleep2,6,11
Drosonex	Available upon request to the authors	custom; written by Thomas Coradetti (Sidewalk Software)	A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system
Photoshop CS3	Adobe		Useful for obtaining numbers of cfu/plate from digital images (optional)