I tested more wtb, this time with the switch mode protocol. Here are the results:

With the modified laser settings I was able to test 3 more wtb. Here are the results:

–> yaw torque

Before collecting the actual data I make sure that the flight simulator in the basement does its thing ;) Here are the test runs with wtb flies for different protocols:

—>> yaw torque

–>> switch mode

–>> habit formation

Below you can find the data I collected from the offspring flies:

-> offspring from trained parents

-> offspring from untrained parents

(I left out data from flies that showed negative preference during two training periods in a row)

Since the results I got after training the parental flies looked a bit odd on first sight I decided to take a closer view…

First I excluded some weird animals that either showed a larger preference for one side than avoidance or showed no avoidance two training periods in a row:

Next I compared the behavior of flies that showed avoidance but no learning with the behavior of the remaining flies:

-> Avoidance is almost the same but note the first test period!

Lastly I split the data according to male and female flies. Here is what I got:

-> Looks a bit like there is negative learning in the male flies however I don´t have enough data to be sure…

As an overview here are all the flies (except for the excluded ones) together again:

Below you find the data from my experimental rounds A and B:

-> learning scores of the parental flies from experimental round A and B

-> learning scores of the trained parent´s offspring only from experimental round A

-> learning scores of the untrained parent´s offspring only from experimental round A

The results confuse me a lot and I am happy to discuss reasons :) However the offspring of the round-B will be ready for testing by the end of this week so there is still some data to collect…

DNA Assembly in a 1:2 ratio of vector to insert with gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using QuickCIP). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was wrongly used.

DNA Assembly in a 1:2 ratio of vector to insert with the gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using FastAP and 2 extraction steps). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was used in reaction.

The offspring of my first experimental fly cohort finally hatched! Below you find a few first pre-tests I ran last week :)

First, here are the results of a quick test to see if the offspring shows a preference for the parentally trained side after the first training period:

After that I played around with the laser a little bit to find the learning threshhold. I set the laser on 2,6V but the results I got look a bit weird:

-> untrained wtb flies

-> offspring of trained wtb flies

I´m optimistic however there is still a lot of work to do…

Via gDNA analysis and PCR was the specific area of the rsh gene extracted and amplified where the nucleotide substitution: C to T (Folkers et al., 2006) should be for the rsh¹ mutation. The amplicon was Sanger sequenced which proved the nucleotide substitution.