First of all, I gained experience in handling and rearing D. pseudoobscura and learned how to incubate bacteria, how to infect flies with bacteria, how to measure bacterial load, and how to quantify gene expression levels using RT-qPCR. I also determined which bacteria species and which concentration of bacteria are suitable to use for the following experiments. Then, to examine a relationship between the level of polyandry and female immune defence, I conducted survival assay using 10 gentypes of virgin females that genetically differ in the level of polyandry. I inoculated female flies with either Enterococcus faecalis (a Gram-positive bacteria) or Pseudomonas entomophila (a Gram-negative bacteria) and then checked the survival of female flies. Furthermore, to examine how mating affects female immune defence, I conducted survival assay, bacterial load assay and quantification of immune gene expression levels using virgin and one-mated females of 6 genotypes that genetically differ in the level of polyandry. I inoculated female flies with either E. faecalis or P. entomophila in the same manner as the above and checked their survival. For bacterial assay, I homogenized female flies in PBS at a certain time point after the inoculation and plated the homogenate on lysogeny broth plates to count colonies. For quantification of immune gene expression levels, I homogenized female flies in Trizol at a certain time point after the inoculation. After extracting RNA from all samples, I conducted reverse transcription and then performed qPCR for the all samples. I statistically analyzed the data obtained in this research project. Finally, I found that there is no correlation between the level of polyandry and female immune defence, which rejected my hypothesis that the level of polyandry positively correlates with the efficiency of female immune defence. Instead, I found that there is genetic variation in female immune defence and that effects of mating on female immune defence are different among the female genotypes. I have given poster presentations with these results at conferences for academics three times. I will give presentations on this research project whenever I have opportunities. I will also integrate the results into two original papers and publish them in peer-reviewed scientific journals.
