The Source of Firefly Chemical Defenses: Endogenous Biosynthesis of Lucibufagins from Cholesterol

Fireflies, also known as lightning bugs, are beetles in the family Lampyridae. Several species of North American fireflies have been investigated to determine their toxic components. The primary molecules that act as chemical defenses are steroids in the bufadienolide class, called lucibufagins (LBGs). However, whether fireflies sequester LBGs from a food source or biosynthesize them de novo has remained unclear. Here, we utilize a laboratory culture of a North American firefly, <i>Pyractomena borealis</i>, to determine whether LBGs are synthesized from cholesterol. We used mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) combined with a paired feeding assay to detect the incorporation of doubly ¹³C-labeled cholesterol in two LBGs produced by <i>P. borealis</i> larvae, providing direct evidence to show the production of LBGs from dietary cholesterol.