Clemson University TigerPrints All Dissertations Dissertations 8-2019 Self-Repair and Self-Cleaning of the Lepidopteran Proboscis Suellen Floyd Pometto Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Pometto, Suellen Floyd, "Self-Repair and Self-Cleaning of the Lepidopteran Proboscis" (2019). All Dissertations. 2452. https://tigerprints.clemson.edu/all_dissertations/2452 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. SELF-REPAIR AND SELF-CLEANING OF THE LEPIDOPTERAN PROBOSCIS A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy ENTOMOLOGY by Suellen Floyd Pometto August 2019 Accepted by: Dr. Peter H. Adler, Major Advisor and Committee Co-Chair Dr. Eric Benson, Committee Co-Chair Dr. Richard Blob Dr. Patrick Gerard i ABSTRACT The proboscis of butterflies and moths is a key innovation contributing to the high diversity of the order Lepidoptera. In addition to taking nectar from angiosperm sources, many species take up fluids from overripe or sound fruit, plant sap, animal dung, and moist soil. The proboscis is assembled after eclosion of the adult from the pupa by linking together two elongate galeae to form one tube with a single food canal. How do lepidopterans maintain the integrity and function of the proboscis while foraging from various substrates? The research questions included whether lepidopteran species are capable of total self- repair, how widespread the capability of self-repair is within the order, and whether the repaired proboscis is functional. I demonstrated the capability of butterfly and moth species from families throughout the Apoditrysia to completely repair a totally separated proboscis. Generally, the repaired proboscis was capable of taking up fluid. Because self-repair is a process that has been modeled for nature-inspired designs of microfluidic devices, further questions involve the mechanisms used in repair. I found that labial palps, coiling, and wetting were not required for repair for Danaus plexippus and Vanessa cardui (Nymphalidae). Wing movements facilitated repair for V. cardui but not for D. plexippus. Self-cleaning has applications in bioinspirational research. How is the proboscis kept clean and free of debris during foraging? I found bacterial inhibition by the proboscises of V. cardui and Agraulis vanillae (Nymphalidae) on poured plates inoculated with Bacillus sutbtilis and Pseudomonas aeruginosa. The adaptive value of self-repair and self-cleaning of the proboscis goes beyond continued uptake of fluids. Lepidopterans with separated galeae would be excluded from foraging from narrow flower corollas. Additionally, the ability to coil the proboscis would optimize the aerodynamics of flight by reducing drag. Structural or chemical features of the proboscis that resist bacterial contamination would help protect from infection. ii DEDICATION I dedicate this work to my husband, Tony, with gratitude for his trust in God’s providence and goodness, and for his enduring support and encouragement through both phases of my career. Tony was dedicated and supportive in every way of our decision for me to stay home with our children throughout their formative years, and afterwards, he enthusiastically supported my efforts to pursue graduate studies. iii ACKNOWLEDGMENTS I offer deep gratitude to my committee, Dr. Adler, Dr. Benson, Dr. Blob and Dr. Gerard, for their instruction, guidance and efforts throughout my doctoral program. I greatly benefited from the breadth of their experience and the perspectives offered from their various disciplines. I especially thank Dr. Peter H. Adler for inspiring and facilitating my research and study in every way. Without his recognition of personal interest, I would not have been able to pursue graduate studies in Entomology. I model my teaching and research on his excellent example. I thank Dr. Konstantin G. Kornev for his close collaboration throughout this project and for funding this research through a joint grant with Dr. Adler. I thank Dr. Charles “Eddie” Beard for daily help with every aspect of the research including collection and rearing of specimens, solving computer issues, guiding statistical work, and providing extra hands whenever needed. Thank you to Dr. Brian Scholtens, College of Charleston, for his professional assistance with moth identification. Thank you to John Abercrombie, Senior Lecturer in Biological Sciences at Clemson University, for advice on planning the bacterial study, training in methodology, provision of supplies and lab space, and preparation of the inocula. Thank you to Justin Scott, Flow Cytometry Research Associate at the Clemson Light Imaging Center, for instruction in the use of ImageJ. Thank you to Dr. Anthony L. Pometto, III, professor in the Department of Food, Nutrition and Packaging Science at Clemson University, for guidance on the bacterial study and review of the manuscript. Thank you to Dr. Patricia Zungoli for hiring me as the instructor for ENT 2000 and for encouraging me to continue my graduate studies. iv LIST OF TABLES Table Page 3.1 Repeated measures ANOVA for rate of repair (change in percent galeal union/minute) in each of six species of Lepidoptera (controls: treatments): Danaus plexippus (11:11), Vanessa cardui (11:10), Papilio cresphontes (11:12), Phoebis sennae (5:6), Heliothis virescens (11:12) and Manduca sexta (7:10). Proboscises of controls were not separated (total n=56) and persisted at 100% union. ............................................. 54 3.2 Factors involved in proboscis repair for two species of nymphalid butterflies. ............................................................................ 58 4.1 The absorbance at 600 nm for each bacterial inoculum to determine the relative cell density. Inocula were prepared by adding 100 µL of each bacterial culture (or 175-200 µL for Bacillus subtilis) to 7 ml of warm, sterile tryptic soy agar liquid media. ........................................... 75 4.2 The proportions of inhibition area to proboscis area were compared for each species of butterfly and bacteria assayed. .................................................................................... 81 4.3 Comparison of the areas of inhibition associated with hemolymph samples taken before or after dissection of the proboscis from live specimens of Vanessa cardui and tested with Pseudomonas aeruginosa. If there was no inhibition, the area was reported as 0 mm2. ....................................................................................................... 81 A.1 Species of lepidopterans tested for ability to repair the proboscis. Specimens were collected at the Clemson University Insectary or South Carolina Botanical Gardens. ................................................................................................. 87 v LIST OF FIGURES Figure Page 2.1 Artwork by Allison Stoiser (used with permission) incorporates ecological interactions. A. Ants march along a Passiflora vine in the Peruvian Amazon as heliconian caterpillars consume the leaves (upper right). B. Clearwing butterflies of the tribe Ithomiini (Danainae, Nymphalidae) on Boraginaceae plants. The adult butterflies scratch the plant to release plant fluids, then use the proboscis to take up the fluids to obtain chemical protection. ......................................................6 2.2 Cross section of the proboscis of Manduca sexta, labeled to show the internal structures and the legulae (photograph by C.E.Beard). ....................................................20 3.1 Types of union of the galeae; schematic drawings in dorsal view on the left, photographs in lateral view from a single specimen of Danaus plexippus on the right. A. Fully united galeae; total length (Lt) equals the united portion of the galeae (Lu), Lu = Lt. B. Partially united galeae, Lu<Lt. C. Partially united galeae with a medial separation; the united portions are summed for Lu. D. Totally separated galeae, Lu = 0. .......................................................................................................46 3.2 Experimental setup for testing the effect of coiling on proboscis repair. The specimen’s wings and legs were restrained with two overlapping glassine sleeves held with a clothes pin. The butterfly was positioned on a Styrofoam stage in a groove ending in an open chamber. To prevent coiling, the proboscis was extended over a contiguous row of insect pins mounted on a plastic support. Two crossed insect pins prevented elevation of the head. The plastic support with row of pins and the two crossed pins were removed for controls and for the wetting experiments. ............................................................................51 vi List of Figures (Continued) Figure Page 3.3 Repair of the proboscis was followed as percent union of galeae (mean ± standard error), measured as U = (Lunited/Ltotal)*100 where Lunited is the united length of the proboscis and Ltotal is the total length of the proboscis, for six species of Lepidoptera after total galeal separation (controls: treatments): Danaus plexippus (11:11), Vanessa cardui (11:10), Papilio cresphontes (11:12), Phoebis sennae (5:6), Heliothis virescens (11:12)