Finally found some time to go through all the individual fly data. All groups lost some flies that had to many or too long flight pauses, strong preferences already before training or some other screw-up during the experiment that I either failed to notice or only showed themselves in this quality control phase. And it was the red group, where I had thought everything just worked so fine, that lost most flies. More than a third of all the flies I had tested in this group failed the very basic quality control criteria – double the number of any other group. This is very annoying as it means there now are a lot more flies for me to test. Nothing to do about that, than to carry on. Here is the result after the massacre, where red lost 10 flies, green lost 5 and purple lost 2:

But even with so many flies removed, the red group still has the flies with the strongest optomotor response:

Now two weeks of data collection, but so far no time for quality control. This means a bunch of flies will likely drop out after I have been able to check for the quality of the reocrdings. So far, one group (red) seems to learn really well, while the other two are too early to tell.

Raw data avilable on the publication server.

My previous JoyStick and T-Maze experiments revealed problems concerning our control groups. While control flies that were fed with ATR supplemented food avoided the light nicely, we also observed avoidance in our negative control who did not receive any ATR. In our experiments we use the CsChrimson channel, which should need ATR to be functional.
My hypothesis for why also our negative control now shows avoidance was, that some mutation in the gene encoding for the CsChrimson channel might have affected its sensitivity and now there was at least some residual activation, even without ATR.

As an attempt to identify the underlying problem I crossed new control flies, this time using NorpA;20xUAS-Chrimson flies from our stock. If there had been a mutation in the flies I used earlier the new flies should not show avoidance.

As of now, the results are hard to interpret. There seems to be also avoidance with the new flies, however this might also be due to low sample size. If increasing the sample size will only show more robust avoidance, we’ll have to think about other causes for the problem…

Update 17.09.25:

The flies used in the previous experiments unfortunately turned out to be not blind. I conducted a new set of experiments, this time with blind flies, and I also changed the light used to red light, at a peak intensity of 500 Lux.
With this setup the results look more like what we would expect from our negative control. Of course the sample size is too low for me to really be able to tell whether the negative control will not show a preference.

Update: 07/10/25: The results below were obtained from a new set of experiments. I reared new flies (this time the blind flies actually should be blind) and crossed them to Gr28bd-Gal4; TrpA1-Gal4 flies from our stock. If the problem was indeed the driver line, this negative control should not show avoidance. Its a bit early to say but for now it seems as I might be on the right track.

In this project, one of the two experimental groups inhibits the ellipsoid body ring neurons using TNT-E. The second uses RNAi to knock down the DopR2 receptor in these neurons and the third is the driver control. As I am blind to the groups and it’s only been one week, the data are not terribly revealing, yet.

The 1st cross produced eggs, but they did not hatch or become pupae. In all of the 10 vials, eggs were observed, but no pupae. On the other hand, the 2nd cross did not produce any eggs. (The first trial was kept up for 10 days. After 10 days, a new trial began from the same stock. ) Control and the other two crosses produced eggs and hatched new flies.

The same crosses were repeated to ensure the lethality. In the second trial, ‘T4/T5xWTB’ cross produced eggs and pupae. But there is still the possibility that the T4/T5xWTB is also a lethal crossing. ‘T4/T5xTNTE’ cross again produced only eggs but no pupae. The other two crosses produced pupae again and became dark, but haven’t hatched yet. The second attempt is 10 days old.