266V Poster - 02. Immunity and the microbiome
Wednesday April 06, 4:00 PM - 7:00 PM

Authors:
Paige E Bonnette ¹; Gerald B Call ²

Affiliations:
1) Department of Biomedical Sciences, College of Graduate Studies, Midwestern University, Glendale, AZ; 2) Department of Pharmacology, College of Graduate Studies, Midwestern University, Glendale, AZ

Keywords:
m. microbiome; q. other (microbiome)

Parkinson’s disease (PD) is a neurodegenerative disease commonly associated with motor symptoms. Additionally, PD patients often suffer from non-motor symptoms, including gastrointestinal issues such as constipation and gut dysbiosis. These gut manifestations in a neurodegenerative disease support the hypothesis of a gut-brain-axis, a bi-directional communication pathway between the central nervous system and the gut. To investigate this relationship, our lab has been performing bacterial mono-associations in a PD model (park²⁵) Drosophila melanogaster. The park²⁵ flies are an excellent PD model as they possess many similar phenotypes to PD, such as dopaminergic neuron loss and impaired motor function. To perform a mono-association experiment, the flies must first be made germ-free, or axenic. The most frequently used method of rearing axenic Drosophila is by embryo dechorionation. This process generally consists of rinsing embryos with a 0.6% bleach solution followed by sterile water rinses and placement onto a sterile diet. Following this, our approach to collect homozygous park²⁵ axenic flies involved transferring sterile, homozygous pupae (identified by the absence of the Tubby phenotype) to new sterile diet tubes. To create gnotobiotic adult flies, we performed two bacterial inoculations: the first with the embryos and the second onto the sterile diet that the pupae were transferred on to. While this method did produce successfully mono-associated park²⁵ flies, the adult survival rate was low. Therefore, we wanted to see if there was a more efficient and effective method to perform mono-association experiments. We began by maintaining axenic fly stocks of both our control (w¹¹¹⁸) and park²⁵ flies. Axenic status of the stocks was checked periodically throughout the experiments. To perform a mono-association from the axenic stocks, adult axenic flies were allowed to deposit embryos for three days on new, sterile food. When the adults were removed, the embryos and larvae present on the food were inoculated with Lactiplantibacillus plantarum or maintained axenic. Pupae were transferred from these tubes and placed into new sterile diet tubes without a second inoculation. Approximately 5-6 days post-eclosion, flies were homogenized, and bacterial colonies were cultured and counted to determine the average CFUs/fly. Despite there being one less inoculation event compared to the standard protocol, this method showed that the flies colonized at a similar level to previous experiments in our lab. Compared to our initial protocol, the axenic stock method with the single inoculation event colonized equally in w¹¹¹⁸ flies (96.4%), or even better in park²⁵ flies (266%). Preliminary results suggest that this new method is similar to the traditional dechorionation method with regard to eclosion, adult survival, and motor function (climbing ability), which will be reported at the meeting.