In order to get the most accurate behavior tracking, we create environments that are very artificial to flies. We also need to get very good light/dark contrast to easily visualize the bodies and extremities of the flies to reduce tracking errors. One of the ways to do this is to use intense lighting conditions. Does this create a stressful environment for flies?
Of course light is important for maintaining circadian rhythms and it’s important to maintain L/D cycles. I write more about that in “How does the Light:Dark cycle affect fly behavior”. Here I’m addressing the lighting conditions in the behavior arena.
Sun is a stressor in nature, but it’s difficult to know if that’s because of the light or the intense heat. Larvae prefer areas that are dark and avoid areas that are light (Imambocus et al, 2022), although they can be trained to prefer light (Gerber et al, 2004). Drosophila typically pupate in the dark as well (Manning & Markow, 1981). Larvae reared in intense light conditions develop more slowly and can be more small (we learned this by trial and error when we switched incubators).
Adult flies are prone to desiccation and in orchards they prefer shady spots as opposed to full-sun areas (Silva-Lopez et al, 2023). When given a choice between bright light and dim light in a small arena, Drosophila prefer to spend most of their time in the dimly lit area, even if the food is in the brightly lit area (Rieger et al, 2007). Constant intense light in a behavior arena can result in observing behaviors you wouldn’t otherwise see such as male-male courtship in wild-type flies (Ueda et al, 2023). Pulsed white light alters walking behavior in w1118 flies (Qiu & Xiao, 2018). Flashing red light alters activity in a number of different wild-type flies (Scaplen et al, 2019).
What does this mean for running behavior assays?
When tracking flies, I recommend using an infrared (IR) camera with an IR backlight (more on that on “How do I build a simple tracking system for fly behavior?”) instead of a regular camera and brightly lit background.
Perform your experiments in a dimly lit environment chamber (more on that on “How do I build an environment chamber?”) using lights that don’t increase temperature (like LEDs).
Use an arena-specific surround to block any shadows, or passing objects and to unify the light dispersion in the arena (usually a white sheet of mat fabric or plastic should do it).
Do you have other methods you use to ensure continual low lighting? I’d love to hear them!