The CRC for Plant Biosecurity has funded us to explore how fruit flies respond to environmental stresses delivered at lethal doses. The grant is managed by Wei Xu (Murdoch Uni) via a grant conceived and written by Alexie. It’s only been less than a month but Kay, being ever efficient, has already started spearheading pilot experiments using heat and cold stresses.
As part of the Science Exchange of our funding body (Plant Biosecurity CRC), we met in Adelaide and discussed her findings. As expected there were a good number of technical problems so we went into the classic troubleshooting mode…
Our first issue was that the larvae were ‘disappearing’ during the bioassay! Kay would inoculate the diet with say 10 larvae but only a handful would remain after a few days. Our previous experience with moths is that cannibalism can occur when larvae are stressed or starving but no one that we know of has observed (or tested) this in fruit flies. Alternatively, maybe they went into hiding into our diet-soaked sponge? Or they somehow they evolved teleportation skills? Anyway, we decided to solve it by placing individual larvae into PCR tubes (actually a plate).
Our next issue was the details of our cold treatment. Our initial attempts were to use melting ice water (i.e. about 0’C) but the issue we had was that it would take more than 10 days of cold stress before we saw any effect. Since the larvae can survive for many dies like that, Alexie thought that we’re not really looking at the effects of cold stress but the effects of starvation. Enzymes and metabolism are both slowing down meaning that while not as much energy is used, the insects are less likely to be able to eat and digest their food properly. So if we wanted to look at the which genes are switched on and off after the stress was removed then our data would be swamped by starvation-response genes! After considerable debate on its value on post-harvest entomology we decided to use significantly lower temperatures such as -70’C using melted salt-water. The stress would only last for a couple minutes meaning that we could identify not only stress early response genes but potentially also recovery ones.
Then had a brief discussion about the value of controlling the temperature in our cold and heat treatments. For heat we went for the first solution a molecular biologist would think of (a PCR machine, which we first have to find without inconveniencing everyone else since we have move it to our quarantine facility). For cold it was harder. Thanks to Google we verified that salt water can indeed be used to control the temperature. Also since our cold treatment is now very short (instead of 10 days) we don’t really need to control it as long as we can measure it and block the bioassays appropriately.
We left for dinner quite excited and agreed we will have lots of data by December and start crunching numbers 🙂