Most animals on our planet are herbivores, using plants as their food. Plants have evolved a wide variety of toxic chemicals to protect themselves from being eaten. In return, many herbivores have evolved resistance to toxins, and some are even able to use plant toxins to protect themselves against their own enemies, including predators and parasites. One such herbivore is the monarch butterfly, whose caterpillars feed on milkweeds, which contain toxins called cardenolides. Previous work has shown that monarchs do not only use toxic milkweed as food, but also as medicine against a debilitating parasite. However, it remains unknown whether cardenolides directly kill parasites, or whether they change the monarch gut microbiome (all the micro-organisms that form the insect’s gut flora) so that specific bacteria or fungi kill the parasite. In this project, the researchers will carry out a series of experiments to find out how toxic milkweeds provide medicine to monarch butterflies. Do their toxins act like drugs? Do they make the gut flora inhospitable to parasites? Or do the milkweeds themselves provide probiotic bacteria to the monarch so they can withstand infection? As part of the research, the researchers will train a postdoc and undergraduate students, and work with the Fernbank Science Center in Atlanta to develop lesson plans for fifth grade public school students. Lesson plans will focus on the question how diet can change the gut flora of animals and thereby change susceptibility to disease. Experimentally validating the indirect link between diet quality, gut community composition, and infectious disease has proven challenging given the complex nature of many animal gut microbial communities and disease pathologies. In this project, the researchers will use monarch butterflies and their protozoan parasites as a focal host-parasite interaction to investigate the interplay between host plant chemicals and microbes, herbivore gut microbiomes and parasite resistance. This project provides an ideal opportunity to fully connect these interrelated pieces of host ecology to disease outcomes. In testing these predictions, the proposal will also expand our understanding of how medicinal metabolites interact with “probiotic” gut microbial communities to influence infection. The researchers will address the following specific aims: (1) Determine the role of phytochemistry and plant microbes in shaping the assembly of gut microbial communities towards an anti-parasitic state; (2) identify key traits of the microbial communities that are critical to gut microbial protection against parasitic infection; (3) determine the extent to which spatial and chemical attributes of gut microbial communities increase resistance to parasitism. Building upon preliminary results, the researchers predict that: (i) ingesting a high diversity of toxic secondary metabolites and plant microbes selects for a more diverse gut microbial community; and (ii) a more diverse microbial community provides hosts protection from infection through both physical and chemical interference of parasites. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.