Abstract:
Drosophila melanogaster is an efficient genetic model to study immunity, and by understanding the fly’s innate immunity we can draw similarities and figure out the homologies in humans, which will help us understand our own innate immune system. There are candidate genes with unknown functions suspected to be involved with D. melanogaster’s innate immune responses based on previously conducted microarray studies. RNA interference (RNAi) was utilized to knockdown the candidate genes’ expression and test for the effects of their silencing on the flies’ innate immunity. For this, the survival is assayed after different microbial infections and bacterial proliferation assays are performed. The expression levels of antimicrobial peptides (AMPs) are determined by real-time polymerase chain reaction (RT-PCR). The results allow us to determine whether a candidate gene is a component the Toll or the IMD pathway or if it acts independently of these pathways. Thirty-one different genes were selected based on previous microarray studies and were screened by conducting survival assays using Gram-positive bacteria, Gram-negative bacteria and fungi as the immune challenges. Based on the survival analysis results, 14 genes were selected and subjected to bacterial proliferation assays to study the bacterial clearance. Interestingly enough, the Gram-negative bacteria did not affect the survival rate nor the bacterial clearance rate of the candidate genes. For the Gram-positive bacteria, however, some genes were shown to overlap in the positive selection for the survival assay and bacterial clearance. Statistical analysis method proved that there is a significant correlation between survival rate and CFU count using. Although the RT-PCR runs on control flies were successful, the runs for testing the candidate genes were not completely finalized due to the malfunctioning of the RT-PCR machine. Regardless, the samples were saved so they can be tested in the future.
