title: Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.
abstract: 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. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. 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). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. 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.
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66046, USA.
authors.forename: Desiree
authors.initials: D
authors.lastname: Harpel
authors.affiliation: Zoological Institute, KU Leuven, Naamsestraat 59, Box 2465, BE-3000 Leuven, Belgium.
authors.forename: Darron A
authors.initials: DA
authors.lastname: Cullen
authors.affiliation: Department of Biology, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH, UK.
authors.forename: Swidbert R
authors.initials: SR
authors.lastname: Ott
authors.affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.
authors.forename: Chris D
authors.initials: CD
authors.lastname: Jiggins
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66046, USA. Electronic address: jrwalters@ku.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Walters
mesh_terms: D000818:Animals; D002080:Butterflies / Q000737:chemistry*; D005247:Feeding Behavior; D006868:Hydrolysis; D019476:Insect Proteins / Q000032:analysis*; D011058:Pollen; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000032:analysis*
chemical_list: D019476:Insect Proteins; D020543:Proteome; D012471:Salivary Proteins and Peptides
keywords: Heliconius; Lepidoptera; Pollen feeding; Proteomics; Saliva