Aug 29, 2024
Behavior monitoring of D. melanogaster using ethoscopes
- Natalie Kaempf1,
- Jorge S. Valadas1,
- Antonio Ortega1,
- Sha Liu1,
- Patrik Verstreken1
- 1VIB-KU Leuven Center for Brain & Disease Research, 3000 Leuven, Belgium
Protocol Citation: Natalie Kaempf, Jorge S. Valadas, Antonio Ortega, Sha Liu, Patrik Verstreken 2024. Behavior monitoring of D. melanogaster using ethoscopes. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qyqjl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 104443
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000430
EMBO long-term postdoctoral fellowship
Grant ID: ALTF_299-2019
Research project, FWO Vlaanderen
Grant ID: G0A5219N
Research project, FWO Vlaanderen
Grant ID: G0B8119N
Methusalem project
Grant ID: METH/21/05 (3M210778)
Research project, KU Leuven Parkinson Fonds
Grant ID: EQZ-PARFON-O2010
Opening the Future grant, Leuvens Universiteitsfonds (LUF)
Grant ID: EQZ-OPTFUP-O2010
Research project, FWO Vlaanderen
Grant ID: G031324N
Abstract
This protocol describes the practical steps to perform behavioral monitoring using ethoscopes. The cleaning and preparation of glass tubes as well as loading the flies into ethoscopes is outlined.
Materials
glass tubes: Pyrex glass, PGT5x65, Trikinetics
wax: Surgipath Paraplast Plus, 39602004, Leica
Setup of ethoscopes
Setup of ethoscopes
please follow publications:
Geissmann, Quentin, Luis Garcia Rodriguez, Esteban J. Beckwith, Alice S. French, Arian R.
Jamasb, and Giorgio F. Gilestro. 2017. “Ethoscopes: An Open Platform for High-Throughput Ethomics.” PLOS Biology 15 (10): e2003026. https://doi.org/10.1371/journal.pbio.2003026.
Geissmann, Quentin, Luis Garcia Rodriguez, Esteban J. Beckwith, and Giorgio F. Gilestro. 2019. “Rethomics: An R Framework to Analyse High-Throughput Behavioural Data.” Edited by Jessica C. Flack. PLOS ONE 14 (1): e0209331. https://doi.org/10.1371/journal.pone.0209331.
Gilestro, Giorgio F. 2012. “Video Tracking and Analysis of Sleep in Drosophila Melanogaster.” Nature Protocols 7 (5): 995–1007. https://doi.org/10.1038/nprot.2012.041.
cleaning of glass tubes
cleaning of glass tubes
1. day: used glass tubes are cooked in soap-water mix in a big pot for multiple hours and let cool down overnight
2. day: take off the wax-food layer at the top, rinse tubes multiple times with water, wash tubes with 75% vinegar in demineralized water to remove scale from the glass tubes and incubate for 2-3h
wash again with demineralized water multiple times
3. day: wash tubes in 70% EtOH thoroughly, incubate them minimum 1h in EtOH and leave the tube to dry on tissue
group glass tubes in bundles of 10-20 tubes with rubber bands
filling glass tubes with fly food
filling glass tubes with fly food
warm up fly food (standard corn meal and molasses food) and pour it in weighing boats (around 1 cm high). Place multiple of the bundles of glass tubes immediatly in the fly food. Ensure that every tube is touching the ground and ensure that in every tube (also the one in the middle of the bundle) the fly food is rising up to about a 1 cm by turning them a bit or moving them slightly up and down.
let the tubes rest for at least 1h and cover them with tissues to avoid flies landing on/in tubes
when the fly food has cooled down and is solid, carefully remove the tube bundles by turning them slighly while pulling them from the fly food to avoid loosing the fly food from the tubes
remove the rubber bands and clean individual tubes thoroughly with tissues from the outside
seal the cleaned tubes with wax by dipping the end of the tube with fly food in melted wax 0.5 cm deep (not too deep, as it will interfere with the ethoscope arenas), let it solidify a couple of seconds by holding the tubes straight, which allows the wax to form an even drop at the end.
place the sealed fly food tubes in a humidified box and store it at 4 °C for not more than 2 weeks
loading flies
loading flies
collect and count 1-4 days old flies from the cross reared in a temperature and light-controlled incubator
at 25 °C and 12-hour light-dark cycle
prepare metadata file: specify the genotype and the location of the genotype (in which ethoscope number and which side of the arena) (arena should be labelled with the same number of the ethoscope to avoid confusion). If you use arena from Fig 3A Geissmann Q et al. (2017) Ethoscopes: An open platform for high-throughput ethomics. PLoS Biol 15(10): e2003026. https://doi.org/10.1371/journal.pbio.2003026
then the side with one dot is number 1-10, the side with 2 dots is 11-20.
Do not place the same genotype on both sides but distribute it over multiple ethoscopes, in case an ethoscope fails during the recording.
next day: load the flies according to the metadata table with a brush into the tubes (check that the tubes are well sealed and don't have condensation in which the flies can get stuck) and seal the open end with small cotton plugs.
push the tubes gently into the arena with the food facing to the outside of the arena.
put the arena in the corresponding ethoscope in incubators at 25 °C with 12:12 light-dark
conditions (the same ZT0 as the flies were reared in), double check that all dots on the arena are present and start the ethoscopes.
Ensure all ethoscopes are working and after a couple of mins are recognizing all the flies
after 6 days stop the recording and analyse the data
Protocol references
Geissmann, Quentin, Luis Garcia Rodriguez, Esteban J. Beckwith, Alice S. French, Arian R.
Jamasb, and Giorgio F. Gilestro. 2017. “Ethoscopes: An Open Platform for High-Throughput Ethomics.” PLOS Biology 15 (10): e2003026. https://doi.org/10.1371/journal.pbio.2003026.
Geissmann, Quentin, Luis Garcia Rodriguez, Esteban J. Beckwith, and Giorgio F. Gilestro. 2019. “Rethomics: An R Framework to Analyse High-Throughput Behavioural Data.” Edited by Jessica C. Flack. PLOS ONE 14 (1): e0209331. https://doi.org/10.1371/journal.pone.0209331.
Gilestro, Giorgio F. 2012. “Video Tracking and Analysis of Sleep in Drosophila Melanogaster.” Nature Protocols 7 (5): 995–1007. https://doi.org/10.1038/nprot.2012.041.