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

Abstract

Studies of animal movement and migration over large geospatial scales have long relied on natural continental-scale hydrogen isotope (δ2H) gradients in precipitation, yet the physiological processes that govern incorporation of δ2H from precipitation into plant and then herbivore tissues remain poorly understood, especially at the molecular level. Establishing a biochemical framework for the propagation of δ2H through food webs would enable us to resolve more complicated regional-scale animal movements and potentially unlock new applications for δ2H data in animal ecology and eco-physiology. Amino acid δ2H analysis offers a promising new avenue by which to establish this framework. We report bulk tissue δ2H, δ13C, and δ15N data as well as amino acid δ2H and δ13C 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 δ2H values. Our data reveal extensive δ2H variation within and among individuals of a relatively simple plant-herbivore system that cannot be explained by temporal or geospatial gradients of precipitation δ2H or dietary differences. Variations in essential amino acid δ2H and δ13C 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 δ2H 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 δ2H variation in consumer tissues often observed at a single collection site, which has implications for using δ2H isoscapes to study animal movement. Further investigation into amino acid δ2H holds promise to elucidate how subsets of amino acids may be best utilized to address specific ecological and physiological questions for which bulk tissue δ2H is insufficient.

Central Metabolism and Growth Rate Impacts on Hydrogen and Carbon Isotope Fractionation During Amino Acid Synthesis in E. coli

“…However, this offset is larger in the batch cultures. The WT Ileu δ 2 H value is the most negative (∼-310‰) of all amino acids, which is consistent with what has been measured previously for δ 2 H values of Ileu (Fogel et al, 2016;Newsome et al, 2020;Morra et al, 2021). The -pgi and -zwf mutants have Ileu and Val δ 2 H values that are quite similar.…”

“…More recently, exploiting systematic differences in the carbon isotope composition of amino acids to tease apart the sources of amino acids in consumer tissues has been termed "isotope fingerprinting" (Larsen et al, 2009). Stable hydrogen isotope analyses of amino acids, on the other hand, have only been conducted recently and have yet to be fully exploited due to a lack of fundamental understanding of how these isotopes are distributed in amino acids (Fogel et al, 2016;Newsome et al, 2020;Morra et al, 2021). In a first paper examining the distribution of hydrogen isotopes in amino acids from biomass (Fogel et al, 2016), Escherichia coli was grown on either glucose or a complex protein source (tryptone) in batch culture.…”

A pilot study of stable isotope fractionation inBombyx morirearing

“…This complex life history can be analyzed through stable isotope technology. Stable isotope technology is widely used in feeding ecology to study the fractionation mechanisms of stable isotopes, in different organisms or within different tissues of the same organism, that can correlate with various ecological factors (Hood-Nowotny and Knols, 2007; Hyodo, 2015; Morra et al, 2021). The fractionation and enrichment characteristics of 13 C and 15 N can be used to determine an animal’s food source (Chávez-Solís et al, 2020), study its nutritional health (Burian et al, 2020; Ishikawa et al, 2020) and explore complex life history processes (Ishikawa et al, 2020).…”

Examination of amino acid hydrogen isotope measurements of scalp hair for region‐of‐origin studies

“…42,43 While δ 2 H analysis of individual amino acids may be a valuable approach for studying animal movement and diet, 44 few studies have used this approach to date. [45][46][47] Additionally, we examined if there were specific AA ESS and AA NESS that incorporated a greater proportion of hydrogen from drinking water than bulk hair tissue. Based on our understanding of amino acid synthesis and incorporation into keratin tissues, we predicted that latitudinal relationships among amino acid δ 2 H would persist due to regional drinking water isotope variation across the region of interest.…”

“…While δ 2 H analysis of individual amino acids may be a valuable approach for studying animal movement and diet, 44 few studies have used this approach to date 45–47 . Fogel et al used amino acid δ 2 H analysis to examine how hydrogen from growth media and environmental waters was incorporated into the bacteria Escherichia coli .…”