In: Experiments with Drosophila for Biology Courses. 2021. (eds: S.C. Lakhotia & H.A. Ranganath). 493
Indian Academy of Sciences, Bengaluru, India
Measuring aggressive behavior in Drosophila
R. Sai Prathap Yadav (firstname.lastname@example.org), Shreeraksha Upadhyaya
(email@example.com), Pranava Swaroopa (firstname.lastname@example.org.
edu), Pavan Agrawal* (email@example.com)
Centre for Molecular Neurosciences, Kasturba Medical College, Manipal, Manipal Academy
of Higher Education, Udupi-576104, Karnataka, India.
* For correspondence
Recommended Level: UG/PG/Research
Sub-discipline: Neurobiology/Behavioral Biology
Aggression is conserved across the animal kingdom and is crucial to ensure survival and
reproduction of animals. Due to its powerful genetic toolkit, Drosophila has emerged
as an attractive model in the last two decades to dissect neurons and genes involved in
aggressive behaviors (Kravitz & Fernandez, 2015). Environmental stressors such as social
isolation inuence animal behavior. For example, social isolation increases aggression
(Agrawal et al., 2020; Kim et al., 2018; Kravitz & Fernandez, 2015) in Drosophila,
whereas, social enrichment suppresses such behaviors. Aggression is a complex behavior
and its analysis requires use of sophisticated computational tools to carry out the assay in
a high throughput manner. Here we present a step by step protocol to measure aggressive
behaviors in Drosophila. The exercise described here is modied from a previously
published assay (Dankert et al., 2009; Dierick, 2007), and instead of using automated
machine vision setup, it utilizes smartphones for recording, and freely available software
for data analysis. This can be easily extended to study the eects of factors like dietary
changes, starvation, access to mates etc. on complex decision making in Drosophila.
Identication of various aggressive behaviors in Drosophila melanogaster and
quantication of male aggression under dierent social environments.
1. A 12 well aggression arena (16 mm diameter and 10 mm height for each well) with
Yadav, Upadhyaya, Swaroopa and Agrawal
lid, food tray and screws (Fig. 1A - H). The arena including top plates are fabricated
as described (Dierick, 2007). As an alternative, commercially available transparent
petri dishes (25 mm diameter) can be used by making a loading hole on each lid and
covering it with transparent tape (Fig. 2A).
2. Stereo-binocular microscope.
3. CO2 pad for anaesthetizing and sorting ies. Alternatively, ies maybe anaesthetized
by keeping on a cold plate (see Chapter 1 for methods of anaesthetizing ies by CO2
or cold plate).
4. Weighing balance.
5. Microwave/heater to prepare apple juice agar.
6. Autoclave for y food preparation and decontamination.
7. Timer or stopwatch.
8. Recording room with uniform light, controlled temperature (25oC) and humidity
9. Camera: Smartphone camera with stand, capable of recording at 30 frames per sec
10. Computer with at least 4 GB RAM.
11. White paper to serve as background for recording.
Note: after loading the test ies, the top lid is pushed gently to one side so that the loading
holes (circles and arrows, Fig. 1F, H) are no longer open.
Fig. 1. The 12 well aggression arena used for assaying y aggression. (A) Top view of the
components of 12 well aggression arena, (B) screws with studs, (C) Top plate with y loading
holes, (D) bottom food plate and, (E) food holding tray, (F) Top view of the 12 well arena
containing apple juice agar food (well diameter- 16 mm diameter and height- 10 mm) with
arrows showing loading holes (circles) on top of each individual well, (G) Side view of the
aggression arena. (H) Top view of aggression arena before recording.
Fig. 2. Experimental setup for rearing and handling flies for aggression assay. (A) A
transparent Petri dish (25 mm diameter) with apple juice agar and yeast paste at the
bottom, and a loading hole (arrow) drilled in its cap (several such Petri dishes can serve as
alternative to the aggression arena described in Fig. 1), (B) Fly aspirator used for transferring
flies into the arena: the rubber tube of the aspirator is covered at both ends using cheese cloth
to prevent flies from getting sucked inside the tube and restraining them in the initial part
of the tube, (C) Vials containing group-housed males (20 males/vial) with standard fly food,
(D) 1.5 mL micro-centrifuge tubes with fine holes (to facilitate air-exchange) on top and
food at the bottom are used to individually house male flies.
B. Other Materials & Reagents:
1. Sigmacote (Sigma: SL2-25 mL) to coat the lid of the 12 well arena to prevent the ies
from walking on the arena ceiling.
2. Fluon/PTFE (Insect-a-slip; Bioquip or equivalent) to coat the walls of the 12 well
aggression arena. Fluon makes the walls slippery and prevents ies from climbing.
3. Apple juice (without added sugars and preservatives).
4. Sugar (organic sulphurless sugar or equivalent).
5. Agar Powder, Bacteriological grade.
6. Micro-centrifuge tubes- 2 mL.
7. Micro-centrifuge tube rack.
8. 5 mL syringe needle (Dispovan or equivalent).
9. Autoclave and microwave safe gloves.
Yadav, Upadhyaya, Swaroopa and Agrawal
1. Newly eclosed wild type Drosophila melanogaster males or mutants, as required.
2. Fly vials with cotton plugs (25 mm diameter X 90 mm height).
1. VLC (Windows/Mac/Linux compatible) for video le tagging to keep a record of
2. Microsoft oce excel/GraphPad Prism or equivalent for plotting and statistical analysis.
3. HitFilm Express (or equivalent software)- this is a suggested free video editing software
to segment videos from 12 well aggression arena into single chamber videos.
A. Fly stock rearing and handling:
Maintain Drosophila melanogaster (Canton-S) or wild caught strains under standard
conditions, i.e., ~25oC and ~60% relative humidity with a 12-Hr light/dark cycle on standard
y food. In the absence of a B.O.D. incubator with light cycle control, light/dark cycle can be
achieved by keeping vials in a naturally lit environment and air conditioning can be used to
provide constant 25oC environment. The room temperature must not rise beyond 30oC as this
can reduce baseline aggression.
B. Manipulation of social experience:
i. Create holes in the 1.5 mL microcentrifuge tubes with a syringe needle to allow air
exchange to house single-housed flies.
ii. Anesthetize 24-48 Hr old flies on fly pad with CO2 or on a metal pad cooled with ice and
iii. Place individual male in 1.5 mL microcentrifuge tube containing 0.5 mL of standard
food for a period of 4-5 days to generate single housed males. Group-housed flies are
generated by keeping 20 males in standard sized fly vials with standard fly food for a
period of 4-5 days (Fig. 2, C,D).
C. Aggression arena preparation:
i. The aggression arena to record aggressive behaviors contains a desired resource, i.e. sugar
and nutrient rich food which motivates flies to fight and defend their territory.
ii. Prepare apple juice agar (2.5% w/v sucrose and 2.24% w/v agar in apple juice) as
1. Apple juice 300 mL
2. Agar 9 gm
3. Apple juice 100 mL
4. Sugar 10 gm
Each part is separately heated until boiling in a microwave or on a heater to fully dissolve
the contents, following which they are mixed together. Recipe makes 400 mL of food.
Unused food is covered in aluminium foil and placed in refrigerator which can be reheated
for reuse within the next 2-3 days.
Note: It takes longer to dissolve agar in microwave, Therefore, mix occasionally with a
steel spatula and monitor continuously to prevent any spillage. Heat Part B for ~2 min.
Allow it to cool down a bit and mix with Part A. Microwave the mixture again for 1 min
and allow to cool down a bit before pouring into petri-dishes or the bottom food tray of the
arena. Ensure that there are no air bubbles; if any are trapped, remove them with the help
of a steel spatula.
iii. The bottom food tray of arena is lled with approximately 40 mL apple juice agar,
covered with cheese cloth and allowed to dry at room temperature for a day. Alternatively,
add approximately 4-5 mL of apple juice agar to 25 mm diameter Petri dishes.
iv. Remove the bottom plate from the food tray next day with the help of a spatula and clean
any extra apple juice agar from the side using a blade. Put the 12 well arena with screwed
lid on top of the food plate (Fig. 1 B-D).
D. Aggression Assay:
i. Place the fully assembled 12 well aggression arena (Fig. 1D) on a white sheet of paper to
allow simultaneous recording from multiple arenas from their top view. Write identity of
housing condition on paper to clearly distinguish between group- and single-housed ies in
the arena. Tighten screws so that the top lid stays rmly in place.
ii. If the alternative small Petri-dishes (25 mm diameter) with loading hole on top plate
covered with transparent tape are used (Fig. 2, A), they may also be labelled and arranged
serially on a white paper sheet. Increased diameter of the Petri-dish will lead ies to move
away from each other, which can decrease the frequency of aggressive encounters. Adding
yeast paste in the center of arena on top of apple juice agar (Fig. 2, A) mitigates this eect
to some extent.
iii. Gently introduce a pair of male ies (either from single- or group-housed ies) in each
well of the aggression arena/Petri dish with the mouth aspirator (Fig. 2B).
iv. After introducing the pair of single- or group-housed ies into the aggression arena,
close the loading holes by covering with pipette tips. In case of Petri dishes, quickly cover
the loading holes with a transparent plastic sheet to prevent ies from escaping.
v. Flies are allowed to acclimatize to the arena for 5 min following which the video recording
is started and continued for 20 min. Any smartphone or a digital camera xed on a stand, and
capable of 30 fps recording can be used. Camera view should cover entire arena from the top.
vi. Various aggressive behaviors are annotated and analysed manually by playing back the
Yadav, Upadhyaya, Swaroopa and Agrawal
videos on a computer. Follow the steps listed below to annotate the dierent aggressive
behaviors using the VLC software (Windows/Mac/Linux compatible).
E. Analysis of aggressive behaviors:
The analysis of behavioral data is divided into three parts (i) video segmentation, (ii) manual
annotation, and (iii) plotting and statistical analysis. These are described below:
(i) Video segmentation:
We recommend using Hit Film Express, a free video editing software, for segmenting
videos into individual arena les, Follow these steps:
a. Import the video into the software and use the ‘Composite Shot’ feature to resize.
Position the video such that only one arena is visible in the frame.
b. Apply ‘Black and White’ eect. This makes the video annotation part easy by allowing
easier dierentiation between the ies and the background.
c. While exporting the video, ensure that the output videos retain the same frame rate as
the original recording (30 fps) and the bit rate is sucient i.e. not much lower than the
input bit rate for the selected video dimensions. Lower bit rate will cause deterioration
in the video quality.
(ii) Manual annotation:
Aggressive behaviors like lunge and chase should be precisely identied. Lunge
is dened as a y raising its forelegs and snapping downward and forward on
its opponent (an example of a lunge in slow motion, can be seen at https://www.
5326238e8509/mmc2.avi;Hoyer et al., 2008). Chase is dened as one y running after
the other. During manual annotation segmented videos are played back on computer
and annotated by the experimenter either manually or with help of a video tagging
software. It is recommended that each annotated video is cross-checked by a second
experimenter to minimize individual bias. Steps for using software to annotate video
les are described below:
Video tagging using VLC
Download VLC extension “Time”(v3.2 as tested) from https://addons.videolan.
a. Install the extension to VLC and restart the application.
b. Open the segmented video in VLC media player and open the extension menu from
the “view” tab (Fig. 3 A,B). This may be congured to t the conditions.
c. Use an excel spreadsheet to simultaneously record your observations.
d. The spreadsheet should list: time stamp of the start of an observation, behavior type
(Lunge or Chase) and duration of the behavior in milliseconds.
e. Use keypress ‘E’ to go ahead frame by frame.
Note: VLC does not support going back to the previous frame but manually moving the
slider can achieve this.
(iii) Statistical analysis:
Data generated from aggressive encounters are usually non-normally distributed. Therefore,
use non-parametric tests for statistical analysis with appropriate post-hoc correction e.g.,
Mann-Whitney U test. Statistical analysis of behavioral data can be performed using Prism
8 (GraphPad Software) or Microsoft excel. See (Agrawal et al., 2020) for examples.
Fig. 3. VLC interface showing steps for video tagging to aid in manual annotation:
(A) Conguration for selection of time extension, (B) VLC Interface showing suggested
QUESTIONS FOR SELF-STUDY AND INTERPRETATION:
1. Would aggressive behavior be dierent between male and female Drosophila?
2. Single housing (Fig. 2 D) for 4-6 days is known to increase aggression in males compared to
Yadav, Upadhyaya, Swaroopa and Agrawal
group housing (10-20 males/vial, Fig. 2C, also see reference (Agrawal et al., 2020)). What
will be the effect of varying the duration of social isolation vs. social enrichment on
aggression? Will longer duration of social isolation increase aggressive behaviors?
3. What will be the eects of varying diet components, e.g., starving ies for a few Hr to a day
by providing only agar lacking any sugar or nutrients before conducting aggression assay?
4. Would starvation increase aggression by increasing ‘motivation’ to ght for the coveted
food resource? If yes, what duration of starvation will be sucient to motivate the ies e.g., 6
Hr, 12 Hr, 24 Hr etc.?
REFERENCES AND FURTHER READING:
Agrawal, P., Kao, D., Chung, P., and Looger, L. L. (2020) The neuropeptide Drosulfakinin
regulates social isolation-induced aggression in Drosophila. Journal Experimental Biology.
Dankert, H., Wang, L., Hoopfer, E. D., Anderson, D. J., & Perona, P. (2009) Automated
monitoring and analysis of social behavior in Drosophila. Nature Methods. 6, 297–303. https://
Dierick, H. A. (2007) A method for quantifying aggression in male Drosophila melanogaster.
Nature Protocols. 2: 2712–2718. https://doi.org/10.1038/nprot.2007.404
Kim, Y. K., Saver, M., Simon, J., et al., (2018) Repetitive aggressive encounters generate a
long-lasting internal state in Drosophila melanogaster males. Proceedings National Academy
Sciences USA.115: 297-303. https://doi.org/10.1073/pnas.1716612115
Kravitz, E. A. and Fernandez, M. de la P. (2015) Aggression in Drosophila. Behavioral
Neuroscience. 5: 549–563. https://doi.org/10.1037/bne0000089
INDIAN ACADEMY OF SCIENCES, Bengaluru
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