TITLE: Monitoring fly feeding behavior and timing by beetle luciferase reporters
AUTHORS: Misha    Koksharov

[DESCRIPTION]
Monitoring Drosophila feeding behavior usually requires laborious procedures and custom-made equipment. This protocol describes how feeding behavior of individual flies carrying a beetle (e.g. firefly) luciferase gene can be conveniently monitored in real-time by measuring bioluminescence in 96-well microplates. 

Drosophila flies expressing a beetle luciferase gene produce bioluminescence if fed with a food containing luciferin. Bioluminescence in flies appears within a minute and peaks within 2-3 minutes after a food ingestion. When refraining from eating or placed on a luciferin-free food, fly bioluminescence decays with about 0.3-1 hour half-life and essentially returns to the baseline after 4 hours. Naturally, under non-stressed conditions, flies eat sporadically and often make intervals of many hours between eating. This makes beetle luciferase bioluminescence a very convenient system to monitor fly feeding timing and, to a considerable extent, food intake in real time for many days (see the Abstract Figure). In this protocol I describe one of the possible procedures to monitor fly feeding using commonly available plate readers, outline a historical background on recording bioluminescence in live flies to study circadian gene expression, illustrate several examples of feeding behaviors that can be analyzed by this procedure and discuss some potential applications.

Bioluminescence signal in live freely moving flies is usually overwhelmingly dominated by fly feeding and movement. This makes firefly luciferase reporters poorly suitable to report changes in gene expression or other intracellular parameters in live flies despite their historically popular application for this purpose. Some approaches to address this problem are also discussed.

[BEFORE_START]
Read the Guidelines and Materials sections.

[GUIDELINES]
INTRODUCTION

Beetle luciferases catalyze a bioluminescent reaction by oxidizing D-luciferin with molecular oxygen (Carrasco-Lopez et al, 2020). ATP is required as a second substrate to first convert D-luciferin into the activated luciferyl-adenylate form. Luciferases are widely used as reporter genes, most commonly via lytic assays of cell cultures and tissues. D-luciferin is relatively stable in solution for several days, so the beetle luciferase reaction can also be monitored in real time in live cell and tissue cultures for many days. Bioluminescence imaging is often used to track tumor development in live mice if cancer cells are labeled with a luciferase. When injected into mice, D-luciferin is relatively quickly eliminated from the bloodstream (~30 min half-life). 

  
Monitoring Drosophila feeding behavior usually requires laborious procedures and custom-made equipment (Deshpande et al, 2014; Ro et al, 2014). The described method (Fig. 1) allows to conveniently and precisely monitor the timing of food intake (as well as general movement of flies between the two horizontal surfaces within a well) for transgenic luciferase-containing flies as they freely move within wells of a single 96-well plate. While the provided well space is small, flies can still move around and behave relatively naturally (see the videos in Step 8). When the D-luciferin substrate reaches a beetle luciferase enzyme produced inside fly cells, bioluminescence is emitted which is detected by a luminescence platereader.

The method relies on the following aspects of Drosophila fly biology and behavior:
luciferin quickly (within 1-2 min) enters the fly circulatory system (and reaches a luciferase enzyme in cells where it is produced) after an ingestion of D-luciferin containing food (Itskov et al, 2014)
it is relatively quickly removed from the circulatory system unless replenished in flies and in mice
when flies do not ingest food or ingest food without luciferin, bioluminescence decays with about 0.3-1 hour half-life and essentially returns to the baseline after 4 hours
under normal conditions flies eat sporadically, often with >1 h intervals; food pass-through time can be as short as 15-20 min (Deshpande et al, 2014)
intervals between eating can be up to many hours and vary quite a lot between individual flies and between different genetic backgrounds (e.g. see FLIC measurements in Fig. 2.3 in the PhD Thesis of Qi Zhang (Zhang, 2016))
repetitive lateral plate movement which occurs cyclically many times during measurements in luminescence plate readers (to place wells being measured under one or more PMT detectors) doesn't seem to disturb flies much and affect their behavior

The bioluminescence intensity measured from a well with an individual fly varies essentially because of the two factors: 
Gradual signal decay due to luciferin excretion (0.3-1 hour half-time depending on a tissue) and an increase if new food is ingested
An abrupt 2-fold or higher change when a fly moves from the well bottom onto the film covering the well (since it is closer to the light detector and the ventral fly side generally absorbs less emitted light). 
As long as the sampling rate of the recording is at least 5-10 minutes, these events can be clearly and easily discriminated in the recorded bioluminescence curves (Fig. 1).

This method can be used to:
monitor feeding behavior per se under different conditions (e.g. how it depends on food types or genotypes; to look at circadian or other temporal questions)
to simply detect when flies have ingested food for the first time or have refrained from eating after that (e.g. in experiments involving fly fasting or when flies need to consume various drugs and effectors in food which can be followed by subsequent experimental steps such as looking how the behavior or other parameters have changed).

HISTORICAL BACKGROUND and WARNINGS

The described protocol is a based on a technique developed by groups investigating circadian gene expression (Stanewsky, 2006; Yu & Hardin, 2007) to monitor circadian luciferase transcriptional reporters in live flies. Many useful experimental details on fly and plate preparation are described in these two referenced papers. This protocol can serve as an additional visual guide to prepare flies for this kind of experiments as well but, as can be seen from examples, extreme care should be taken not to interpret various feeding artifacts as intrinsic activity of these luciferase reporters.

In the current described method flies are recorded in a single plate at high temporal resolution to obtain detailed bioluminescence traces from individual flies (1-5 min resolution). In the referenced technique multiple plates are recorded in succession (by cyclical loading from a vertical stacker) with a low temporal resolution (30-60 min between measurements) and data from many flies (e.g. n=20-100) is averaged out without analyzing individual flies.

 


Measurements of bioluminescent transcriptional reporters in live flies are orthogonal to monitoring feeding as it assumes and requires flies to eat continuously and uniformly, so that there is no change in luciferase signal due to feeding patterns which is otherwise dramatic (up to 10-fold and more) under more natural conditions as illustrated in this method (Fig. 1). 

To some extent, the use of plate stackers and additional plastic domes inside wells indeed forces flies to eat more frequently but even then they usually still show feeding patterns: either more continuous regions with discrete gaps or, quite commonly, two continuous peaks during the 24 h with maximums around the middle of day and night and a more than 10-fold decrease of activity between these peaks (see Fig. 12, 13 and the Note 4 in the Steps section). This "two 12 hour peaks" pattern was reported for individual flies in early papers on this circadian technique where bioluminescence amplitude changes 10-fold or more in a circadian manner (see Fig. 6A in Plautz et al (1997), Fig. 2A in Brandes et al (1996)). This 12-hour pattern generally persists into many averaged out resultant curves and due to averaging the amplitude decreases to about 2-fold thus resembling typical circadian reporters (e.g. see Fig. 7 in (McDonald et al, 2001) and less pronounced in Fig. 2 in (Johnstone et al, 2022)). These 12 h rhythms are commonly interpreted as a gene expression in many publications using this technique. However, Ralf Stanewsky correctly ascribed them to a some kind of behavioral artifact that still depends on circadian rhythms (Stanewsky et al, 1997) but it was not clear at that time that this is a feeding activity. 

Overall, it seems that for most fly genotypes the feeding rhythms in bioluminescence cannot be really and reliably suppressed, so any luciferase reporters in live flies require very rigorous controls to show that any observed bioluminescence rhythms are not due to feeding or movement behavior. 

This need for the rigorous verification by independent methods generally makes the use of luciferase transcriptional reporters in live flies unreliable as an independent method on its own (e.g. to screen for gene expression phenotypes). Some fly genotypes, however, may be more susceptible to showing continuous feeding under such conditions and, if properly controlled, can be used to monitor gene expression reporters. For example, XLG-luc flies seem to produce similar circadian bioluminescence rhythms both in separated fly organs in a liquid culture (where feeding timing is not an issue) and in live moving flies (Glaser & Stanewsky, 2005).

 


 


 



[STEPS]
SECTION: Preparation of a 96-well plate with a luciferin-containing food
1. Prepare a hot fly food mix and keep it unsolidified by placing a tube with food into a 48-50°C water bath.50 °C 

a) It can be a regular complex cooked fly food (such as BDSC Cornmeal Food).
b) It can be various synthetic and semi-defined formulations with 1% agar.

5% sucrose, 1% agar mix is commonly used when studying fly circadian behavior and sleep (Versteven et al 2020). Healthy flies live on it and retain normal activity often for more than 2 weeks. (To some extent this seems to be dependent on microbes which colonize the food, live off sucrose and trace elements in this food then supplying some nutrition to flies.)

In case of synthetic and semi-defined formulations with 1% agar, dissolve the components in water, then add 1% agar and prepare a hot liquid mix by heating in a microwave. Heat sensitive components can be added when cooled down to 50-60°C.
SECTION: Preparation of a 96-well plate with a luciferin-containing food
2. While the liquid mix is kept in a 50°C water bath, add luciferin from a 100 mM stock to have a desired concentration (0.1 mM - 3 mM, or higher) and mix thoroughly. 50 °C 

 3 millimolar (mM) 
3 mM luciferin is a commonly used concentration providing good signal levels. 

0.1 millimolar (mM) 
0.1 mM luciferin leads to a 2-3-fold lower signal compared to 3 mM concentration which is not an issue in most cases. This very low concentration could be used as likely non-interfering with fly behaviors and to conserve the luciferin substrate. (Since luciferin has sodium or potassium as a counter-ion, these metal ions may affect the food taste to some extent at high luciferin concentrations if used in a simple food like 5% sucrose.)

 

SECTION: Preparation of a 96-well plate with a luciferin-containing food
3. Dispense 100-300 μl food into the wells of a 96-well plate.

Wait 20-30 min for food to solidify. Room temperature 

In experiments where a minimal signal cross-talk between wells is important, the luminescence carry-over between neighboring wells can be minimized by filling and using every other well for the fly recordings.
 

 

 

SECTION: Placing flies into 96-well plates
4. 
Anesthetize flies by cooling them on ice or by using a CO2 pad. The CO2 pads are the most common method used in fly labs while cooling is the simplest. "In addition, it [cooling] is the only method which will not affect fly neurology,  therefore behavior studies may begin after the flies have warmed up sufficiently." (See the manual from the Berg Lab)

 

SECTION: Placing flies into 96-well plates
5. As anesthetized flies are laying on the cooled box (plate) or on a CO2 pad, take a chosen fly by the wing with tweezers (or take it by a small bush) and put it into a plate well.

The transfer of all flies should be done quickly so that flies won't resume moving and escape from uncovered plate wells.
SECTION: Placing flies into 96-well plates
6. Load flies into wells of a 96-well plate and seal the plate with an adhesive plate sealing film. Several approaches can be used:

a) After quickly loading the whole plate, carefully cover the plate with a press-on transparent adhesive film. 

Care should be taken to avoid static electricity when detaching the film from its backing liner and handling. Otherwise, flies can get pulled out from wells by a static charge.

Firmly attach the adhesive film by applying pressure across the whole surface either by fingers or by a plastic sealing pad. The use of a sealing pad is more important in case of films that have an encapsulated adhesive which is released only under pressure.
 


b) Alternatively, the flies can be loaded lane by lane gradually sealing each lane after flies are loaded. In this case, there is less risk than flies will start moving and leave the wells. Also, the problem of static electricity is less likely to appear.
 


c) The whole plate can be cooled and kept on ice to keep flies anesthetized.

 

SECTION: Placing flies into 96-well plates
7. Poke 2-4 small holes in a film above each well for ventilation (for example, by a tip of a syringe needle).
This is necessary to provide a fly with air. Otherwise, a fly will run out of oxygen in a couple of hours and die.
 

SECTION: Placing flies into 96-well plates
8. Flies move in the wells within the free space between the food layer and the transparent film cover. While the provided well space is small, it is sufficient to move around and flies behave relatively naturally. In terms of spacing, 3-4 flies can easily be kept in a single well if desired (but this would rarely make experimental sense in feeding experiments). Overall, this is likely not dramatically different from common crowded conditions often occurring in fly breeding tubes.
 



SECTION: Record the plate luminescence in the platereader
9. Program the luminescence plate reader for continuous measurements with at least one measurement per fly every 5-10 min. Typically, a signal is integrated during 15-30 sec for each well (fly); this can be adjusted depending on a signal strength.
Load the plate into the luminescence plate reader and start measurements. The experiment can be conducted for many days.
 

 

 


 

 

 


SECTION: Process, plot and/or analyze the data
10. The obtained readings data can be further processed to obtain "luminescence - time" curves and analyzed by different approaches depending on the purpose of experiments.

Flies are being recorded in real time, so in relevant experiments flies that have eaten or refrained from eating for a defined time can be identified and collected for further processing.
 

SECTION: Examples of bioluminescence patterns due to feeding
11. This section lists examples of representative bioluminescence patterns reflecting different feeding behaviors of individual flies at various experimental conditions. 

They serve to illustrate:
the principles and assumptions of the method
different feeding behaviors depending on a food type, experimental conditions and fly differences