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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Feeding behavior of flies carrying a beetle (e.g. firefly) luciferase gene can be conveniently monitored in real-time by measuring bioluminescence in 96-well microplates. Beetle luciferases catalyze a bioluminescent reaction by oxidizing D-luciferin with molecular oxygen. This also requires ATP as second substrate to first convert D-luciferin into the activated lucyferyl-adenylate form. Luciferases are widely used as reporter genes, most commonly via lytic assays of cell cultures and tissues. D-luciferin is relatively stable is solution for many 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 by 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). </div><div class = "text-block"><span style = "font-style:italic;">Drosophila</span><span> 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 1 hour half-life and essentially returns to the baseline after 4 hours. Naturally, flies tend to 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.</span></div></div>

[STEPS]
?. [Placing flies into 96-well plates]
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)
Flies were anesthetized by cooling on ice.Note: an actual temperature on ice cooled flat plastic surfaces is a few degrees above 0°C. Flies kept at a room temperature (20-25°C) are rendered immobilized on such surfaces. However, it should be mentioned that flies kept at 5-16°C for at least a few hours undergo some cold adaptation and usually have an ability to slowly walk on such surfaces.
?. [Placing flies into 96-well plates]
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.
Videos of flies moving in microplate wells.1) single flies in wells2) 3 flies in some of the wells:
?. [Placing flies into 96-well plates]
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.
?. [Preparation of a 96-well plate with a luciferin-containing food]
Prepare a hot fly food mix and keep it unsolidified by placing a tube with food into a 48-50°C water bath.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 (Tataroglu & Emery, 2014). 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.
50 °C
?. [Preparation of a 96-well plate with a luciferin-containing food]
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. 3 mM luciferin is a commonly used concentration providing good signal levels. 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-anion, these metal ions may affect the food taste to some extent at high luciferin concentrations if used in a simple food like 5% sucrose.)
50 °C
[D-luciferin]
[D-luciferin]
Luciferin analogs such as Akalumine-HCl (cas # 1176235-08-7) or CycLuc1 (cas # 1247879-16-8) can also be used if needed. They are less soluble and much more expensive, so it is convenient to prepare food with a 0.1 mM concentration. UAS-luc flies (producing luc(+) P. pyralis luciferase) show about 10-fold lower signals with them compared to 0.1 mM D-luciferin. These substrates could be useful due to their red-shifted light emission (e.g. for dual-color measurements or for a better tissue penetration), particularly if coupled to mutant luciferases designed to have high activity with them (Iwano et al, 2018)
?. [Preparation of a 96-well plate with a luciferin-containing food]
Dispense 100-300 μl food into the wells of a 96-well plate.Wait 20-30 min for food to solidify. 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.
0 Room temperature
96-well plate filled with about 300 μl food per well: either a regular fly food (yellow-brown) or 5% sucrose / 1% agar (translucent).
100 μl food provide the highest amount of space for a fly to move inside the well and will increase the signal change when flies move between the food surface and the film cover.Higher volumes of food can be beneficial during multi-day recordings since they will dry out slower. Higher food volumes are also used in circadian protocols to minimize fly movements as much as possible (Stanewsky, 2006).
In some of the older papers there is a suggestion to first place 200-250 μl of luciferin-free food and then, after it solidifies, top it up with 50-100 μl of luciferin-containing food "to save expensive substrate" (Stanewsky, 2006). However, the luciferin from the top layer will get equilibrated by diffusion throughout the whole volume after several hours, so this additional step is unnecessary and will result in lower luciferin concentrations than intended.
?. [Examples of bioluminescence patterns due to feeding]
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 methoddifferent feeding behaviors depending on a food type, experimental conditions and fly differences
Bioluminescence returns close to the baseline after 4 hours if luciferin-fed flies are placed on luciferin-free food or simply refrain from eating.
Bioluminescence decays with about 1 hour half-life at various luciferin concentrations when flies refrain from eating.
Feeding behavior is very similar on regular (complex) fly food and on the simple 5% sucrose, 1% agar mix.
Interpretation of bioluminescence curves in terms of fly feeding and movement.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 (~ 1 hour half-time) and an increase if new food is ingestedAn 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.
The method nicely shows when a fly starts eating. When placed in wells (5% sucrose, 1% agar), flies can start eating right away or can voluntarily refrain from eating for >12 hours.
Compared to 5% w/v sucrose, flies do not eat plain agar food and prefer not to eat low sucrose (0.5% w/v) food until really hungry (>12 hours).Experiments with plain agar food show that bioluminescence monitoring can also be used to determine when flies become incapacitated or dead.Low sugar food (0.5% sucrose) forces flies to eat very frequently. This often leads to flat luminescence curves but the extent of the effect depends on fly genotypes: in many cases flies will still show intervals of non-feeding.
Individual flies can have different feeding patterns. Some individuals may show a very nice circadian feeding behavior with large fasting intervals.
This insert illustrates and discusses typical results when recordings are performed according to the standard circadian reporter protocol. In this case multiple plates are measured by an automatic loading and transfer between two vertical plate stacker columns with each plate periodically being loaded into the platereader detection chamber.In this circadian reporter protocol, several features induce flies to eat more often which is a premise for using this method to record circadian gene expression reporters in live flies. Three main factors seem to induce flies to eat frequently:1) Periodical loading the plate into the vertical stacker outside the instrument seems to disturb flies and stimulate them to eat more often. 2) In some protocol implementations flies are placed under small plastic domes inside wells (Stanewsky, 2006; Yu & Hardin, 2007) which induces flies to eat more frequently (likely, again by disturbing them). 3) Likely, the formation of a water condensate on the film (if occurs) also can disturb flies and stimulate them to eat more often.The efficiency of all these factors can vary depending on genotypes and not all fly individuals may be affected to the same extent.More often than not the feeding rhythms still seem to persist through this procedure. Quite commonly, under these conditions typical separate feeding events in individual flies merge into a continuous bioluminescence curve which has two 12h peaks and was reported in early papers on the method (see Fig. 6A in Plautz et al (1997) and Fig. 2A in Brandes et al (1996)).Such 12-hour patterns are often observed for Gal4xUAS-luc flies when recorded using the stacker method. See the Figure below.When switching from a continuous recording to the stacker method, the bioluminescence profile (for constant luciferase levels in Gal4 lines) tend to switch from separate bioluminescence peaks into a continuous curve - from non-oscillating (an ideal result) to having high amplitude 12 hour rhythms (a commonly observed artifact).   It seems that some fly genotypes are more prone to maintain constant frequent feeding than others under these conditions. For example, the circadian BG-luc reporter flies indeed tend to display intrinsic circadian expression rhythms when switched to a stacker-based recording protocol.   Overall, very rigorous controls and analyzing individual flies are required to discriminate feeding and gene expression when using the original method or interpreting results obtained by it.
?. [Placing flies into 96-well plates]
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.
Ventilation holes in adhesive film above wells:
?. [Placing flies into 96-well plates]
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.
Flies are placed into a 96-well plate (evry other well) and covered with an adhesive film.
Filling and sealing the plate lane by lane:
When using adhesive films covered with an unprotected adhesive, care should be taken that flies don't stick to it with their wings during placement. This can happen due to an attraction by a static charge or if the free volume above food is very small. Flies trapped this way are often not able to release themselves. Otherwise, such films are perfectly compatible with flies living in wells for 1-2 weeks. The commonly used sealing film of this type is TopSeal-A Plus (Perkin Elmer, USA) (Stanewsky, 2006; Yu & Hardin, 2007)
?. [Record the plate luminescence in the platereader]
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.
Details of suitable software settings for TopCount instruments (Perkin Elmer, USA) were described in methods papers on using bioluminescent circadian reporters in live flies (Stanewsky, 2006; Yu & Hardin, 2007).
Flies inside the plate reader are in a constant darkness.If light/dark cycles are needed, most readers can be programmed to remove the plate from the reading chamber for a specified time period before the next measurement. Then for the time outside the platereader the flies will be subject to the outside light/dark conditions.
Flies generally seem to quickly accustom to regular horizontal plate movements which occur during measurements or during loading/unloading plate into the reader. They do not seem to change their position inside wells or get agitated in response to these movements.in an ideal technical set-up the plate should be static while the detectors would move but this is not available in typical plate readersit is crucial to use a single plate and not to use vertical stacker columns to load/unload multiple or even a single plate. As shown in examples, the use of a stacker disturbs flies and causes them to eat more frequently (likely, by abrupt shakes and taps during loading a plate into a stacker). This effect should be avoided unless desired for a specific purpose.
The TopCount NXT instrument (Perkin Elmer, USA) equiped with 12 PMT units permits very fast rates of continuous plate measurements, for example:all 96 wells: measurement every 3 min (15 sec integration time per well)every other well (48 wells): measurement every 2.5 min (15 sec integration time per well)12 wells only: measurement every 18 sec (7 sec integration time per well)
Some plate readers may have features useful for certain kind of experiments. For example, programmed plate shaking, delivery of excitation or transmission light to wells, selection of excitation and emission wavelengths, etc (e.g. Biotek plate readers).
Water condensation on the inside of the film sometimes becomes a problem, especially if the plate is removed frequently from the chamber. This happens when the film is consistently colder than the plate.This can be addressed by several approaches:use an anti-condensation feature if the plate reader has it (it keeps the temperature above the plate slightly higher)poke more holes (e.g. 4 instead of 2)try to tweak the cooling or heating systems of your platereadertweak the temperature and humidity outside the platereaderuse food with a higher osmolarity (e.g. 30% sucrose, 15% glucose, 13% xylitol, addition of NaCl, combinations thereof, etc) which reduces the vapor pressure inside wells
?. [Process, plot and/or analyze the data]
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.
A bioluminescence curve from an individual fly reflecting its feeding activity.