Isolating a new scaleless strain will be the focus of the next chapter of my project. In theory, three approaches can be used to isolate new mutations, I can choose to 1) collect E. kuehniella from the field and screen them for natural occurring mutations, 2) induce random mutations in the genome and hope for the best or, 3) target specific genes which I know (or suspect) to be involved in the development of wing scales. The first two options will include screening of lots of adults and hoping that I can find some individuals without wing scales. If I am lucky enough, I can find one or several adults that have scaleless wings and use those to start rearing a new strain. The next step would be to find out which gene has been mutated and is responsible for the development of scaleless wings which again will cost quite some time. Because luck and time aren’t always on my side (let’s be honest, they generally aren’t), I decided to go with the third option. By inducing gene-specific mutations I can easily predict where the mutation will occur and I don’t have to worry much about additional mutations occurring that I don’t want. The only problem is finding the right gene(s) to target and selecting the best technique to induce the mutations.
Whether the use of a technique will be successful or not is mostly dependent on extensive preparations which is what I am working on right now. Due to the high specificity of the method I finally chose the CRISPR/CAS9 method. Determining the ideal target regions, developmental stage and injection concentrations from literature was the first step. The next step will be to optimize the method for my species of interest. E. kuehniella have a relatively long development time (about 2 months), which makes testing the method using a gene that causes scalelessness (that can only be seen in adults) a very long and inefficient process. Therefore, I also selected a gene that, once knocked-out, should change the appearance of larvae which will be used as a “proof of principle” of the method in E. kuehniella. I will optimize the method for E. kuehniella during my secondment in the lab of Prof. Dr. Leo Beukeboom in Groningen in October this year and with some luck maybe even isolate a new scaleless strain.