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UC Riverside UC Riverside Electronic Theses and Dissertations Title Characterization and Evolutionary Analyses of Silk Fibroins from Two Insect Orders: Embioptera and Lepidoptera Permalink https://escholarship.org/uc/item/0jg8m909 Author Collin, Matthew Aric Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Characterization and Evolutionary Analyses of Silk Fibroins From Two Insect Orders: Embioptera and Lepidoptera A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Genetics, Genomics and Bioinformatics by Matthew Aric Collin June 2010 Dissertation Committee: Dr. Cheryl Y. Hayashi, Chairperson Dr. David N. Reznick Dr. Julia N. Bailey-Serres Copyright by Matthew Aric Collin 2010 The Dissertation of Matthew Aric Collin is approved: _______________________________________________________________ _______________________________________________________________ _______________________________________________________________ Committee Chairperson University of California, Riverside Acknowledgements The text of this dissertation, in part or in full, is a reprint of the material as it appears in Insect Biochemistry and Molecular Biology, February 2009, and Biomacromolecules, August 2009. Co-author Edina Camama listed in the Biomacromolecules publication provided research assistance. Co-author Janice S. Edgerly listed in those publications provided embiopteran cultures and technical expertise. Co-author Cheryl Y. Hayashi listed in those publications directed and supervised the research that forms the basis for this dissertation. Co-authors Jessica E. Garb and Brook O. Swanson listed in those publications provided technical expertise. Justin Bastow, Daniel Gruner, John Mclaughlin, May Roberts, and Don Strong provided larval Hepialus californicus. I thank Edina Camama, Shou-Wei Ding, Samer Elkashef, Stuart Le, and Arthur Omura for laboratory assistance; Dan Borchardt, Richard Debus, and Bohdan Schatschneider for assistance with FTIR techniques; and Shun-ichi Sasanuma for the technical part of H. californicus EST sequencing. Nadia Ayoub, Dan Borchardt, Merri Lynn Casem, Richard Debus, Janice Edgerly, Jessica Garb, John Gatesy, Michael McGowen, Morris Maduro, Frantisek Sehnal, James Starrett, Brook Swanson, and anonymous reviewers contributed helpful comments. Research was supported by NSF awards DEB-0515868 to Cheryl Y. Hayashi, DEB-0515865 to Janice S. Edgerly, Czech grant ME32 to Frantisek Sehnal. H. californicus EST sequencing was supported by a grant from Japan’s Ministry of Agriculture, Forestry and Fisheries to KM. I thank UCR GGB and UCR for financial support through Teaching and Graduate Student Researcher Assistantships and a College Fellowship. Additional Graduate iv Student Researcher support was provided by NSF award DEB-0515868 to Cheryl Y. Hayashi. Chapter 1 reproduced with permission from Collin, M.A., Garb, J.E., Edgerly. J.S., Hayashi, C.Y. 2009. Characterization of silk spun by the embiopteran, Antipaluria urich Copyright 2009. Elsevier B.V. Chapter 2 reproduced with permission from Collin, M.A., Camama, E., Swanson, B.O., Edgerly. J.S., Hayashi, C.Y. 2009. Comparison of embiopteran silks reveals tensile and structural similarities across taxa. Copyright 2009. American Chemical Society. Chapter 3 reproduced with permission from Collin, M.A., Mita, K., Sehnal, F., Hayashi, C. Y. Molecular evolution of lepidopteran silk proteins: insights from the ghost moth, Hepialus californicus. Copyright 2010. The Authors. v ABSTRACT OF THE DISSERTATION Characterization and Evolutionary Analyses of Silk Fibroins From Two Insect Orders: Embioptera and Lepidoptera by Matthew Aric Collin Doctor of Philosophy, Graduate Program in Genetics, Genomics and Bioniformatics University of California, Riverside, June 2010 Dr. Cheryl Y. Hayashi, Chairperson Silk production has independently evolved in several insect lineages, such as Hymenoptera (bees, ants and wasps), Siphonaptera (fleas), and Archeognatha (bristletails and silverfish). Primarily composed of proteins, silks are used for functions, such as protection, prey capture, dispersal, and reproduction. Because of the ecological significance of silks, natural selection can act on silk fibers and thus the underlying silk proteins (fibroins). Lepidoptera (moths and butterflies) and Embioptera (webspinners) are two insect orders that utilize silk mostly for protection. Characterizing fibroins from distantly related insect lineages may reveal convergent evolutionary patterns and provide insight into the functional elements of their fibroins. Within a lineage, conserved characteristics may yield a better understanding of silk evolution and fiber formation. I obtained fibroin sequence data through silk gland cDNA libraries of Lepidoptera and Embioptera. This was accomplished through construction of species-specific silk vi gland libraries for the lepidopteran Hepialus californicus (ghost moth), and five embiopteran species: Antipaluria urichi, Archembia n. sp., parthenogenic Haploembia solieri, Oligotoma nigra, and Saussurembia davisi. After screening the libraries, transcripts for fibroins were identified for each species. Hepialus californicus heavy chain fibroin and light chain fibroin were compared to other lepidopteran and trichopteran heavy and light chain fibroins. Analyses revealed that both heavy and light chain fibroins have historically experienced stabilizing selection and light chain fibroin has potential as a phylogenetic marker. For the embiopteran fibroins, many similar features were found to be shared across species, such as glycine/serine rich, repetitive motifs, and a short, non-repetitive carboxyl-terminal region. Conservation of these elements suggests that these regions are important for fiber formation and mechanical properties. Furthermore, the conserved carboxyl-terminal regions imply that embiopteran fibroins have undergone stabilizing selection. Additional characterizations were performed on embiopteran silks to determine amino acid composition, secondary structural conformations, and mechanical properties. These features were found to have a high degree of similarity across taxa, suggesting that embiopteran silk has remained largely unchanged since it arose over 100 million years ago. vii Table of Contents Introduction………………………………………………………………………….... 1 References……………………………………………………………………… 11 Chapter 1…..………………………………………………………………………….. 15 Abstract………………………………………………………………………… 16 Introduction…………………………………………………………………….. 17 Materials and Methods…………………………………………………………. 20 Results and Discussion………………………………………………………… 24 References……………………………………………………………………… 38 Chapter 2…..………………………………………………………………………….. 43 Abstract………………………………………………………………………… 44 Introduction……………………………………………………………………. 45 Materials and Methods………………………………………………………… 47 Results and Discussion………………………………………………………… 51 References……………………………………………………………………… 68 Supplementary Figures………………………………………………………… 74 viii Chapter 3……..………………………………………………………………………. 76 Abstract……………………………………………………………………….. 77 Introduction…………………………………………………………………… 78 Materials and Methods………………………………………………………... 80 Results and Discussion………………………………………………………... 84 References…………………………………………………………………….. 100 Supplementary Table………………………………………………………….. 104 Chapter 4……………………………………………………………………………... 105 Abstract……………………………………………………………………….. 106 Introduction…………………………………………………………………… 107 Materials and Methods………………………………………………………... 109 Results and Discussion………………………………………………………... 113 References…………………………………………………………………….. 132 Summary……………………………………………………………………………... 138 ix List of Figures Fig. 1.1. Photographs of Antipaluria urichi (Clothodidae) and silk in the field……….. 18 Fig. 1.2. SEM images of Antipaluria tarsus………………………………………........ 25 Fig. 1.3. Three representative stress-strain curves of Antipaluria silk tensile tests that depict the maximum, median, and minimum true stress values…...... 28 Fig. 1.4. Antipaluria fibroin partial-length cDNA sequence and translation (Accession # FJ361212)……………………………………………………....... 30 Fig. 1.5. Predicted amino acid composition of the translated Antipaluria fibroin cDNA in comparison to the empirically determined composition of Antipaluria spun silk………………………………………………………........ 31 Fig. 1.6. Northern blot analysis of the Antipaluria fibroin transcript………………...... 35 Fig. 2.1. Phylogenetic relationships of species in this study based on Szumik et al. (2008)……………………………………………………………. 48 Fig. 2.2. Representative stress-strain curves for silk fibers spun by Haploembia (dark blue), Oligotoma (purple), Antipaluria (red), Archembia (black), Austalembia (green) and Saussurembia (cyan)……………………………….. 53 Fig. 2.3. Box plots comparing tensile properties across taxa………………………….. 55 Fig. 2.4. Amide III region from Saussurembia original FTIR spectrogram (blue) with second derivative (red) above and component bands obtained through curve-fitting analysis underneath at 1235 cm-1 (orange), 1263 cm-1 (green), and 1277 cm-1(purple)……………………………………... 60 Fig. 2.5. Secondary structural component percentages from FTIR amide curve fitting analysis………………………………………………………………….. 62 Supplemental Fig. 2.1. Complete original FTIR spectrograms of embiopteran silks spun by Haploembia (green), Saussurembia (black), Antipaluria (red), and Archembia (blue)………………………………………. 74 x Supplemental Fig. 2.2. Amide III regions of (a) Haploembia, (b) Antipaluria, and (c) Archembia