Classical Techniques in an Unconventional System: Investigation of the Insect Immune System and Gut Microbiome

Abstract

The role of the gut microbiome in development and function of the host immune system has long been an area of interest, investigated across taxa for decades. As mammalian systems can sometimes provide a prohibitive challenge in conducting germ-free studies in many laboratories, it has also been of increasing interest to generate germ-free insects to investigate interactions between the gut microbiota and host immune system. Insects such as Drosophila, a system in which methods have been described to generate axenic animals, have a relatively simple gut microbiome, consisting of only a few species. In contrast, the American cockroach (Periplaneta americana) has a gut complexity approaching that observed in mammalian systems, poising this organism as a promising venture for axenic studies. However, the current field of insect immunology lacks standardized methods of hemocyte naming conventions and a full understanding of immunology in hemimetabolous insects. Therefore, the purpose of this dissertation was to determine how we might employ modern immunological techniques to develop an understanding of conventional Periplaneta americana immune composition and function. As an additional project, I also progressed a working protocol for the generation of axenic cockroaches, as well as hemolymph collection of these insects, in our lab. Using flow cytometry, I characterized hemocytes based on cellular morphology (size and complexity), lectin binding capabilities, and production of reactive oxygen species. LEA lectin binding provided a consistent method of identifying and sorting three distinct hemocyte populations, and ROS assays revealed two populations of hemocytes producing ROS to different degrees. Gene expression is a readily accessible metric characterizing immunological function. Therefore, I also aimed to determine the transcriptional profile of both hemocyte and fat body tissues in response to various bacterial stimuli. I found that hemocytes did not have a robust immune response to heat killed S. aureus or E. coli; however, both hemocyte and fat body tissues exhibited an immune response to heat killed and live S. marcescens. My evidence of differential gene expression in these tissues marks the first transcriptional evidence of key components in the IMD pathway in P. americana. Collectively, my dissertation provides fundamental insight into the American cockroach immune system at a cellular and transcriptomic level, providing both novel findings and the necessary baseline for future axenic studies.