Microbiome analysis of monarch butterflies reveals effects of development and diet.

Diet profoundly influences the composition of an animal's microbiome, especially in holometabolous insects, offering a valuable model to explore the impact of diet on gut microbiome dynamics throughout metamorphosis. Here, we use monarch butterflies (Danaus plexippus), specialist herbivores that feed as larvae on many species of chemically well-defined milkweed plants (Asclepias sp.), to investigate the impacts of development and diet on the composition of the gut microbial community. While a few microbial taxa are conserved across life stages of monarchs, the microbiome appears to be highly dynamic throughout the life cycle. Microbial diversity gradually diminishes throughout the larval instars, ultimately reaching its lowest point during the pupal stage and then recovering again in the adult stage. The microbial composition then undergoes a substantial shift upon the transition from pupa to adult, with female adults having significantly different microbial communities than the eggs that they lay, indicating limited evidence for vertical transmission of gut microbiota. While diet did not significantly impact overall microbial composition, our results suggest that fourth instar larvae exhibit higher microbial diversity when consuming milkweed with high concentrations of toxic cardenolide phytochemicals. This study underscores how diet and developmental stage collectively shape the monarch's gut microbiota.