Drosophila melanogaster has been a versatile tool for thousands of researchers for a long-long time. Yet, its star shone so brightly in genetics and molecular biology while many other application fields have been overseen and perhaps underrated. Nowadays, fruit flies slowly make their way in the research of oxidative stress and the step-by-step building of the bridge between the nutritiology and the in vivo bioactivity research of the potential drugs and food&feed additives. As it happens with everything new, the need for the methods’ optimization and standardization arises. While I was browsing through the multiple studies involving D. melanogaster as a model for oxidative stress research, I found a vast variety of stressors and a big gap in detailed methodology. This short behind-the-paper story recaps my nearly year-long journey of stressing the fruit fly in a reproducible way and the troubleshooting that accompanied me all the way.

Trouble 1. Eat or die? Easy! I guess, I will die now

Administering the stressor to a fruit fly should not be rocket science. Just mix it in the food and you are good to go. Or so the theory was… There are multiple chemical stressors used to induce the detectable increase of the reactive oxygen species (ROS) in the fruit fly: hydrogen peroxide, paraquat, iron sulfate, copper sulfate, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). And there are multiple ways to administer food to the fly: solid agar-based nutrition, natural fruit mesh, liquid solution in a glass capillary, and liquid solution on a paper filter – all with their advantages and disadvantages. First, I started the screening of these stressors except paraquat in the form of liquid saccharose solution on a paper filter. This method worked flawlessly when dextran sodium sulfate (DSS) was administered to the fruit flies to simulate the leaky gut syndrome and required sensibly less material, time, and energy resources than the preparation of solid food. Yet, the first results left me puzzled. The more aggressive stressor I used and the higher concentration I applied, the lower the ROS level of the fruit flies was. I repeated the experiment over and over in various configurations just to see the seemingly antioxidant effect of the published oxidants. And suddenly, it occurred to me that fasting is one great way to reduce oxidative markers in your body. I dyed the fly food just to confirm what I had already suspected. My test subjects would rather starve to death than eat a sugary beverage with a fine note of hydrogen peroxide. Following that bitter realization, I gave up the liquid stressor administration and moved on to the solid agar-based medium, which included enough nutrients to cover up the bad stressor flavor. Due to the addition of the stressor to the still liquid and quite hot agar solution, I had also given up on AAPH and H₂O₂, due to their questionable stability in such conditions. For the first time, I have succeeded – the ROS signal went up.

Trouble 2. Don’t you just get stressed?

Once I found a way to stress my lab gourmets, I moved on to the optimization of the fine details, such as “how much?” and “how long?”. By the time I have selected my champions, copper and iron sulfates, and have been up- and down-scaling their concentrations to see the visible biochemical impact while still having zero mortality during the treatment time. It seemed that the perfect configuration was within my grasp, but once the experiments were repeated, the outcome was completely different and the stress seemed much weaker at that. Were flies not eating again? No, they had quite a healthy appetite. After tracing my steps back, I have realized that the stressors tend to lose their effect within 1-2 weeks of the food storage at refrigerated conditions. Whether the stressor interacts with a food matrix or chemically transforms to a less aggressive form remained unclear. But I could clearly see that freshly prepared food with stressors induced higher ROS response, so the next troubleshooting solution was to cook&stress as fresh as possible.

Trouble 3. Pattern or random numbers?

Pass the troubles one and two I have formed a fixed schedule (almost like in the Cure song): Monday – I am transferring flies, Wednesday sorting to the groups, Thursday – cook the food for them, and Friday – let them stress. 30 mM iron sulfate seemed to do the job. Fruit flies preferred iron-enriched food over copper-food, and 30 mM lay well above physiological concentrations of the harmless iron. Yet, I was not completely satisfied with the experimental setting. Though I could consistently score significant ROS increase in the stressed flies over the control group, the absolute values were different all the time, and this feeling of quantitative randomness was leaving a bitter aftertaste. What could I do differently? What could I do better? How can I make my results more consistent and reproducible? The answer turned out to be simple yet massively overseen by all the topic-relevant methods I have read so far. It was the time. And to be precise, time of the day. Whether I pushed the flies to the food at 9:30 or 11:30 played a big role when it came to the bold numbers. Even though each time I stressed them the same number of hours and each time they were indeed stressed, I paid for neglecting the importance of the circadian rhythm in the physiology of the model organism with big error bars.

Troublefree conclusion

Once I had finalized the method and confirmed the reproducibility of the stressor effect, I moved to the actual experiments with the biotransformed flavanones and was rewarded beyond expectations. From experiment to experiment, I have seen the stable and reproducible results. The lessons I have learned:

1. When testing an orally administered compound (which is new in your lab routine) in the model organism, confirm the consumption with a detectable method. Nobody likes bitter food.
2. Confirm the stability of your compound in the food matrix over the time of the experiment and aim to cut down the storage time of experimental food if the interaction of compound, time, temperature, and humidity is unknown.
3. Respect the circadian rhythm of living organisms to optimize reproducibility, and do not neglect to include it in your method description.