Finn Phillips- Blog Post 3

      I am nearing the end of the insecticide resistance surveillance project that my partner Fernando and I have been working on. It has been really cool to see the process go full circle and to have moved through the entire process myself. I had six sites I collected from, where I was able to sort out a sufficient number of Culex Tarsalis to bioassay with sufficient replicate numbers. I caught the mosquitoes, sorted them, performed bottle assays, froze them, extracted their DNA, and then qPCR'd them. While I initially thought I might find heterozygote genotypes for a kdr mutation because a number of the mosquitoes were recovering after being knocked down, the qPCR results showed that 99% of the mosquitoes caught were homozygotes for the wild-type allele that is susceptible to the insecticide permethrin.

     I spoke to my Karla (my PI) about this and she said that the behavior of the Culex Tarsalis, and their ability to recover, but the lack of kdr mutations in their genotype makes her wonder if the diagnostic dose of insecticide is outdated, or if the mosquitoes have another mode of resistance such as a surplus of the enzyme cytochrome P450, which would allow the mosquitoes to metabolize the insecticide fast enough to survive and recover after exposure. I found this idea particularly interesting because I have been interested in the idea of a reduction in vector competence due to the abundance of these metabolic enzymes. Regardless, the results of this surveillance project showed a lack of kdr mutation, which is good for the public health of Fort Collins, but somewhat uninteresting as well.

     Most recently, I have been having to troubleshoot a problem with the DNA samples from a collection. One of my partners performed the DNA extraction for a collection I made, and when I attempted to PCR the DNA, it did not amplify. I nano-dropped the samples and found 260/230 ratios consistently between 0.1 and 0.2, which means the samples were heavily contaminated, and this is likely what has been preventing the amplification of the DNA. To troubleshoot this, I first tried using a filtration and concentration kit; however, the amount of DNA lost over the course of several filtrations using the kit resulted in too little DNA being left over afterwards. I then pivoted and made a solution of sodium acetate and used this alongside 100% ethanol to try to re-precipitate the DNA and go through the process of rinsing the DNA that would usually be performed in an extraction. I have been able to reproduce the pellets of DNA and am trying to rinse them, although they are very delicate because this is their second time going through this process, presumably. Hopefully, I will be able to produce DNA samples that have sufficient DNA concentrations and a 260/230 ratio of < 1; otherwise, we will likely have to redo this collection from the start of the process. I will say, however, that I have really enjoyed the troubleshooting part of this problem. It has been cool to not have a scripted protocol to follow, and to be able to apply my knowledge of molecular genetics and chemistry to try and recover these samples. 

     Once I have either cleaned up these samples or collected new ones, I will be done with the hands-on part of the project, and I will have all the data processed. The only thing left to do after this would be to write about it! I feel like I really learned a lot in this project, especially when it comes to species identification. I went from struggling to have any idea what I was looking at to creating a simple guide that can help RaHP vec students in the future with getting started with ID. I am excited to be finishing up this project and potentially be moving on to the next soon.