An investigation of novel traits in the order Lepidoptera through the use of proteomic methods

Abstract

This thesis investigates two biological novelties found in Lepidoptera through the use of shotgun proteomics. The first chapter delves into an adaption of pollen feeding found in Heliconius butterflies. We used shotgun proteomics to discover the saliva proteome, and furthermore, discover protein candidates that could be used in digestion and degradation of pollen. The second chapter explores the phenomenon of sperm heteromorphism. Although sperm heteromorphism occurs sporadically throughout many species, this heteromorphism is uniquely found in all Lepidoptera besides the most basal species. Using shotgun proteomics, we id and functionally annotated the sperm proteome for D. plexippus, the monarch butterfly. Both of these projects furthered research about novel Lepidopteran traits through the use of a molecular approach. The first thesis chapter reports an initial shotgun proteomic analysis of saliva from Heliconius melpomene. While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Using liquid-chromatography tandem mass-spectrometry, we confidently identified 31 salivary proteins. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and “housekeeping”(4). These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty. The second chapter explores the phenomenon of sperm heteromorphism in D. plexippus. Lepidoptera have sperm heteromorphy, in which males produce both a fertilizing (eupyrene) and non-fertilizing sperm (apyrene). Apyrene sperm lacks DNA and a nucleus and is produced in higher quantities than eupyrene sperm. The recent availability of several completely sequenced Lepidopteran genomes presents a novel opportunity to apply proteomic methods to characterize the protein content of these two heteromorphic sperm morphs. Here I report an analysis of the sperm proteome from Danaus plexippus, the monarch butterfly. I have analyzed a “commixed” sample of combined apyrene and eupyrene sperm as well as isolated fractions each sperm type. Notably, this study is the first to use shotgun proteomics to independently characterize the protein content of purified apyrene and eupyrene sperm. Comparison of the commixed sample to comparable proteomes of commixed Manduca sexta (Hornworm moth) sperm and monomorphic Drosophila melanogaster sperm revealed few functional differences but high levels of gene turnover. Functional annotations were assessed through the use of Blast2GO software. Next, we delved into the differences in proteins found between the apyrene and eupyrene sperm types, and found few differences functionally and molecularly between the two proteomes. These results reveal a first pass analysis of the sperm proteome found in D. plexippus, and offer insights into the next steps to be taken to further our knowledge on the phenomenon of sperm heteromorphism.