819 The Amino Acids Used in Reproduction by Butterflies: A Comparative Study of Dietary Sources Using Compound-Specific Stable Isotope Analysis Diane M. O’Brien 1, * Carol L. Boggs 2 Marilyn L. Fogel 3 1 Institute of Arctic Biology, P.O. Box 757000, University of Alaska, Fairbanks, Alaska 99775-7000; 2 Department of Biological Sciences, Stanford University, Stanford, California 94305; 3 Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Road NW, Washington, DC 20015 Accepted 12/20/2004; Electronically Published 5/27/2005 ABSTRACT It is a nutritional challenge for nectar-feeding insects to meet the amino acid requirements of oviposition. Here we investigate whether egg amino acids derive from larval diet or are syn- thesized from nectar sugar in four species of butterfly: Colias eurytheme, Speyeria mormonia, Euphydryas chalcedona, and Heliconius charitonia. These species exhibit a range of life his- tory and differ in degree of shared phylogeny. We use 13 C dif- ferences among plants to identify dietary sources of amino acid carbon, and we measure amino acid 13 C using compound- specific stable isotope analysis. Egg essential amino acids de- rived solely from the larval diet, with no evidence for metabolic carbon remodeling. Carbon in nonessential amino acids from eggs derived primarily from nectar sugars, with consistent var- iation in amino acid turnover. There was no relationship be- tween the nonessential amino acids of eggs and host plants, demonstrating extensive metabolic remodeling. Differences be- tween species in carbon turnover were reflected at the molecular level, particularly by glutamate and aspartate. Essential amino acid 13 C varied in a highly consistent pattern among larval host plants, reflecting a common isotopic “fingerprint” associated with plant biosynthesis. These data demonstrate conservative patterns of amino acid metabolism among Lepidoptera and the power of molecular stable isotope analyses for evaluating nu- trient metabolism in situ. * Corresponding author; e-mail: ffdo@uaf.edu. Physiological and Biochemical Zoology 78(5):819–827. 2005.  2005 by The University of Chicago. All rights reserved. 1522-2152/2005/7805-4134$15.00 Introduction A striking aspect of animal diversity is dietary: all animals are heterotrophic and consume diets as diverse and specialized as animals are themselves. Differences between the elemental com- position of animals and their diets have provided a powerful framework for understanding aspects of animal ecology and life history (Elser et al. 2000; Sterner and Elser 2002). In par- ticular, there has been growing appreciation for the importance of specific elements (primarily N and P) in constraining growth (Elser et al. 1996). Requirements for growth and reproduction include an additional level of complexity, in that many of the pathways required to synthesize essential compounds have been lost in animals. Thus, an understanding of nutritional bio- chemistry may help refine diet-based ecological predictions even further. For example, the capability for ascorbic acid syn- thesis varies among bird and mammal species (Birney et al. 1980; Martinez del Rio 1997), and requirements for specific sterols as hormone precursors vary among insects (Nation 2002). A more fundamental example is the inability of animals to synthesize many of the amino acids required in proteins. Protein requirements are particularly complex because, of the 20 amino acids used to make proteins, nine cannot be synthesized by animals: methionine, tryptophan, threonine, va- line, leucine, isoleucine, lysine, phenylalanine, and histidine (Reeds 2000). A tenth, arginine, is essential in many (e.g., fish: Akiyama et al. 1997; insects: Dadd 1973; Nation 2002) but not all (e.g., humans: Laidlaw and Kopple 1987) lineages. Two more amino acids require essential amino acids as precursors: cys- teine and tyrosine are synthesized from methionine and phe- nylalanine, respectively (Berg et al. 2002). Thus, most animals are dependent on dietary sources for the majority of the amino acids found in their proteins (12 out of 20). The dietary re- quirement for these amino acids constrains the types of diets animals can rely on for growth and reproduction. Many insects feed on plant phloem or nectars for part or all of their life. These diets are rich in sugars but generally poor or unbalanced in amino acids (Baker and Baker 1973, 1986; Baker 1977; Douglas 1993). These insects exhibit diverse and sometimes bizarre strategies for meeting their protein budgets, from supplementing diets with pollen, carrion, or dung (Gilbert