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Studies of animal movement and migration over large geospatial scales have long relied on natural continental-scale hydrogen isotope (δ 2 H) gradients in precipitation, yet the physiological processes that govern incorporation of δ 2 H from precipitation into plant and then herbivore tissues remain poorly understood, especially at the molecular level. Establishing a biochemical framework for the propagation of δ 2 H through food webs would enable us to resolve more complicated regional-scale animal movements and potentially unlock new applications for δ 2 H data in animal ecology and eco-physiology. Amino acid δ 2 H analysis offers a promising new avenue by which to establish this framework. We report bulk tissue δ 2 H, δ 13 C, and δ 15 N data as well as amino acid δ 2 H and δ 13 C data from three Pipevine swallowtail ( Battus philenor ) tissues—caterpillars, butterfly bodies, and wings—as well as their obligate plant source: pipevine leaves ( Aristolochia macrophylla ). Insects are often dominant herbivores in terrestrial food webs and a major food source for many higher-level consumers, so it is particularly important to understand the mechanisms that influence insect tissue δ 2 H values. Our data reveal extensive δ 2 H variation within and among individuals of a relatively simple plant-herbivore system that cannot be explained by temporal or geospatial gradients of precipitation δ 2 H or dietary differences. Variations in essential amino acid δ 2 H and δ 13 C indicate that  B. philenor  acquire these compounds from an additional source that is isotopically distinct from pipevine leaves, potentially gut microbes. We also found multiple isotopic carryover effects associated with metamorphosis. This study emphasizes the strong influence of physiology on consumer-diet δ 2 H discrimination in a local population of pipevines and swallowtails and provides a template that can be broadly applied to Lepidoptera—the second most diverse insect order—and other holometabolous insects. Understanding these physiological mechanisms is critical to interpreting the large degree of δ 2 H variation in consumer tissues often observed at a single collection site, which has implications for using δ 2 H isoscapes to study animal movement. Further investigation into amino acid δ 2 H holds promise to elucidate how subsets of amino acids may be best utilized to address specific ecological and physiological questions for which bulk tissue δ 2 H is insufficient.

Physiology Drives Reworking of Amino Acid δ2H and δ13C in Butterfly Tissues