Project Felipe’s blog
By Lewis, Zach, Jai, Ben, Sena, Bridget & Luke
Our study was focused on researching how mutations in Drosophila Fruit Flies affect their resistance to various insecticides. Our first experiment involved exposing 3 types of Fruit Fly larvae to 3 different insecticides. Our control larvae were a wild type, completely natural, and others had been exposed to low and high levels of radiation. We exposed these larvae to Chlorantraniprole, Spinozad and Imidacloprid, 3 different insecticides. We then took short 10 seconds films of all the larvae at 15 minute increments, with the aim of observing differentiations in their movement as the insecticides began to affect them. Overnight the observational movement was analysed by a complex computational algorithm and converted into numerical data.
Insecticides are designed to specifically bond to the receptors of flies with the aim of incapacitating the target and not affecting other species that may come into contact with the insecticide. Receptors are protein molecules that receive chemical signals from areas such as the brain, interpreting the electrical impulses from the brain that tell a muscle to move. When an insecticide bonds to its appropriate receptor it corrupts and changes it, which leads to electrical impulses being processed incorrectly or not at all by the receptor. This leads to loss of muscle movement and control over muscles, inevitably leading to starvation and death.
Specific receptors can receive only specific messages, like a lock and key. When the insecticide bonds to the receptor, the receptor is corrupted. This is akin to changing a lock, meaning the key will no longer fit. The messages can no longer be received by the receptor.
Occasionally flies are born with inherent mutations that heighten their level of resistance to certain insecticides. A common mutation is that they possess a different kind of receptor, one that the insecticide is not designed to target. This allows it to continue sending electrical impulses from the brain to its muscles because it is unaffected by the insecticide. Radiation often develops these mutations in Drosophila flies, which allows us to perform experiments on them.
This can be a problem for farmers that use insecticides. They will work well initially, killing off large numbers of insects. However, the sole surviving insects that contain the immune mutation will begin to breed, unaffected by the insecticide. This leads to a new species that is entirely immune to the insecticide that was previously so effective.
Our results showed that in two of the three insecticides, EMS 1 (the irradiated species) showed higher resistance than the other species. The graphs show that by the last increment at 120 seconds, in two of the graphs the EMS irradiated species show the highest level of movements. An anomaly might be the increased movement that is shown in the first measured increment of 2 of the graphs, however this is a regular occurrence as pesticides often lead to an initial spike in movement which drops off quickly.
The next part of our experiment involved making use of GTACs Scanning Electron Microscope (SEM). This microscope functions by initialising a vacuum in the chamber, then firing electrons at its target. It then measures the rebound of its electrons, forming a crisp black and white picture of the objects hills and troughs. It allowed us to view our fly specimens with up to 5000 times magnification.
We scanned 3 different flies using this device. One was a natural wild species, one was an irradiated species and the last was an offspring of the two. We searched for phenotypic differences between the flies and to see if there were any visible mutations indicating the fact that the irradiated fly had mutated as a result of the radiation exposure.
The last part of our experiment involved investigating the mutations at a genetic level. The first part of our experiment involved isolating the DNA from the sample flies. This involved multiple steps and making use of various solutions designed to break down proteins, the lipid fats of the cell membranes and everything we didn’t need.
Once we isolated the DNA, the last part of the experiment involved attempting to find whether or not our fly species contained the mutations that are responsible for higher insecticide resistance. We inserted a primer into the DNA and then subjected it to various temperatures designed to bond the DNA with the primer. A primer is the complementary strand of DNA to the mutation that we are looking for. The temperatures that the DNA was subjected to were designed to first split the DNA strand, bond the DNA with the primer and then sew the DNA back together. If the mutation was present in the genetic material, then the primer (complementary strand) would bond to it. If it was not present then the primer would not bond to anything. We were looking for 2 mutations, one that should be present in the mutant fly and one that should be present in the wild fly.
After this process was complete we needed to see if the primer had bonded with the genetic material. To do this we subjected the DNA samples to electrophoresis. We dyed the DNA so that it would be visible in the jelly it was suspended in. We then ran a current through it. Hypothetically, if the primer had bonded with the DNA we should see a mark of DNA partway pulled through the jelly by the current, as the current was running through was positive and DNA is negatively charged. This is because the segment of DNA that bonded with the primer would be separate from the larger segments of DNA and would therefore be light and travel further down the jelly. Unfortunately, our experiment experienced an error and the primers did not bond with the DNA, this meant every sample showed the primer that had been pulled through the jelly. If this experiment was successful then we should have seen lines in the jelly indicating that the primer had bonded with the DNA and the mutation was present.
Project Felipe’ would like to thank to thank GTAC and the evolved staff for volunteering their time so that 10 Yea High school students and 11 work experience students could have an invaluable experience that afforded us an insight into future careers in sciences. Special thanks to Tony for volunteering his time in supervising us through the 5 days and organising the 5 day experience.