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  • Writer's pictureKaun Lab

What controls should I use for behavior experiments?

Updated: 6 days ago

When I first started working on flies, I had no knowledge of genetics or insects. My limited undergraduate research experience included a handful of experiments on rats, helping the departmental veterinarian with daily cleaning and feeding needs (ie cleaning mostly rat and pigeon poo), and two years of work watching C. elegans move around in food and respond to taps. My undergraduate degree was in biopsychology and I barely remembered the central dogma (genes -> RNA -> proteins) from my biology classes. So, when I first started working on Drosophila behavior genetics experiments, I totally messed up the controls and had to redo all of my experiments. Although I totally recognize that sometimes you need to try things yourself and mess up in order to really learn something, I’m hoping in writing this down, I can save some of you some time. Below I outline some of the controls you should think about when designing and performing fly behavioral neurogenetics experiments.

Genetic controls: 

Mutations: Different genetic background have different behavioral responses. For example, wild-type Canton-S acts differently than wild-type Berlin on a number of ethanol experiments. For more info on this, check out ‘Which wild-type should I use’. This means when you test a mutant, you must compare it to the genetic background on which the mutant was made. For example, if you are using a MiMIC (Minos mediated integration cassette) you probably want to use y* w* for your genetic control because that was the line used for injections (Venken et al, 2011). There is a small chance that the y+ added to the transgene affects behavior, but you probably don’t need to test this unless you suspect it might (for example yellow affects courtship and mating success, Massey et al 2019).

Making sure you have the correct background for your mutant comparison is easiest if you are making your own mutants because you can pick whichever genetic background you want to use. If you can’t choose and the background the mutant was made on affects your behavior, or if you don’t know what background the mutant was made on, I recommend you backcross/outcross your line to your favorite genetic background. This means you take your mutant and cross it to your favorite background, then choose females of the F1 that have the mutation and cross these back to males of your favorite background (because you want recombination to occur and this doesn’t occur in males in Drosophila) and keep doing this for 5 generations or so, then homozygous your mutants on the new genetic background to maintain this line as a stock. Sometimes you need to outcross for more than 5 generations (for example when I studied foraging we did this for 9 generations), depending on how much of the new background you need to get your behavior of interest. All that being said, sometimes you get giant chunks of chromosomes recombining, and I’ve heard reports of people sequencing after outcrossing and not that much is replaced, so maybe recombination isn’t occurring the way we theorize it should.

GAL4/UAS transgenes: Typically you need to test your GAL4>UAS cross against both the UAS control and GAL4 control. This is because sometimes the insertional position of the UAS transgene or GAL4 transgene has effects on behavior (ie they could accidentally be making a mutant by being popped into a gene that we didn’t know affects our behavior of interest). This was common in the days of making transgenic flies by random insertion, and these days most of the transgenes are made in attp landing sites, so this is less of a problem. In any case, you don’t want to test the GAL4>UAS flies to the parental GAL4 and UAS lines because the GAL4>UAS line has one copy of the GAL4 and one copy of the UAS. So the best control is to make heterozygous controls to compare to your GAL4>UAS line. To do this you cross the UAS line to the genetic background of the GAL4 and the GAL4 line to the genetic background of the UAS. So you’ll have three lines to test: 1) GAL4>UAS, 2) GAL4>background(UAS), 3) UAS>background(GAL4). 

Often the backgrounds of the UAS and GAL4 won’t match. So, should you outcross everything to a common genetic background before doing your GAL4>UAS experiments? Not necessarily. We often procure the background for the UAS and the GAL4 and make the crosses with the correct backgrounds as described above. BUT, sometimes one of the heterozygote controls will be significantly different from the GAL4 control and look more like the GAL4>UAS experimental flies (or vice-versa). In cases like this, the best practice is to outcross both the UAS and GAL4 lines to a common genetic background and do the experiment again. OR find a different UAS or GAL4 you can use in backgrounds that aren’t so disparate in behavior. 

Empty-cassette transgenes: If you can get a so-called ‘empty cassette’ transgene, this is awesome because it means you may not need to run both the UAS and GAL4 heterozygote controls. By ‘empty cassette’ I mean a line that has an ‘empty’ transgene cassette (ie the same transgene minus the thing that affects gene expression) inserted in the same attp site as your UAS or Gal4. What you do is cross your UAS line to this empty GAL4 (or vice-versa crossing your GAL4 line to the empty UAS) and compare that directly to your UAS>GAL4. And voila, background and transgenes are all controlled. 

Environment controls for thermogenetic experiments: 

Temperature can affect behavior (for more info on this see ‘Does temperature affect Drosophila behavior’). If you are performing experiments with thermogenetic tools (like shibire-ts, TRPA1, Gal80-ts) you need to perform experiments with your experimental lines (ie GAL4>UAS) and heterozygote controls (ie emptyGAL4>UAS) at both the permissive and restrictive temperature. You need to do this to see if your temperature manipulation impacts behavior independent of neuronal/genetic manipulation.

Environment controls for optogenetic experiments: 

Light can affect behavior (for more info on this see ‘Does lighting affect behavior?’ and ‘How do I perform optogenetic experiments?’). If you are performing experiments with optogenetic tools (like CsChrimson, ChR2, GtACR) you need to perform experiments with your experimental lines (ie GAL4>UAS) and heterozygote controls (ie emptyGAL4>UAS) with the optogenetic light conditions. You need to do this to see if your light manipulation impacts behavior independent of neuronal/genetic manipulation. Alternatively, if you know that all-trans retinal (ATR), which you feed to flies to ensure activation of your light-gated ion channel, you can run just the experimental line GAL4>UAS with (experimental), and without (control) ATR and use the flashing light for all experiments.

Behavior controls: 

When you are designing your behavior experiments, you need to think carefully about how your mutation or transgenic manipulation can affect all aspects of the behavior. For example, if you are designing a sucrose memory experiment in which flies pair an odor with a sweet or bitter taste, you also need to test whether that mutant/manipulation affects the ability of the flies to smell and taste, since changing the ability to smell or taste will affect the ability to learn. You also need to make sure that when you food deprive or isolate your flies, all of your controls are food deprived / isolated in the same conditions for the same amount of time.

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