BEHAVIORAL AND MORPHOLOGICAL EFFECTS OF IN VITRO LARVAL REARING ON ADULT HONEY BEES, APIS MELLIFERA L. By ASHLEY NICOLE MORTENSEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Ashley Nicole Mortensen To my loving family that has supported me through all of life’s inspirations ACKNOWLEDGMENTS I am grateful to Daniel Schmehl and Hudson Tomé for their guidance troubleshooting the in vitro rearing protocol; Cameron Jack and Ping Li Dai for assistance in rearing many honey bee larvae; Brandi Simmons, Emily Helton, and Branden Stanford for ordering and preparing project supplies; Logan Cutts and Liana Teigan for managing healthy honey bee colonies; Vince Alderman for his diligent construction of project supplies; Tomas Bustamonte, Branden Stanford, and Jon Novak for assistance measuring and entering data; Edzan Van Santan for statistical consulting, and my advisory committee, Jamie Ellis, Christine Miller, Andrea Lucky, and Emily Miller-Cushon, for their support and guidance. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 RATIONALE AND SIGNIFICANCE ........................................................................ 11 2 A HONEY BEE COLONY’S NATURAL BROOD SURVIVAL RATE PREDICTS ITS IN VITRO LARVAL SURVIVAL RATE .............................................................. 15 Introduction ............................................................................................................. 15 Materials and Methods............................................................................................ 16 Brood Isolation ................................................................................................. 17 Colony Rearing Assay ...................................................................................... 17 In vitro Rearing Assay ...................................................................................... 18 Statistical Analysis ............................................................................................ 19 Results .................................................................................................................... 19 Discussion .............................................................................................................. 20 3 COMPARATIVE MORPHOLOGY OF ADULT HONEY BEES REARED IN VITRO OR IN THEIR PARENT HIVE ..................................................................... 28 Introduction ............................................................................................................. 28 Materials and Methods............................................................................................ 30 Dry Bee Weight ................................................................................................ 31 External Morphology ........................................................................................ 32 Statistical Analysis ............................................................................................ 32 Results .................................................................................................................... 33 Discussion .............................................................................................................. 33 4 EFFECTS OF ARTIFICIAL REARING ENVIRONMENT ON THE BEHAVIOR OF ADULT HONEY BEES ...................................................................................... 39 Introduction ............................................................................................................. 39 Materials and Methods............................................................................................ 43 Queen Recognition ........................................................................................... 45 Brood Rearing .................................................................................................. 47 Trophallaxis ...................................................................................................... 48 Sucrose Responsiveness ................................................................................. 49 5 Statistical Analysis ............................................................................................ 50 Results .................................................................................................................... 51 Queen Recognition ........................................................................................... 51 Brood Rearing .................................................................................................. 51 Trophallaxis ...................................................................................................... 51 Sucrose Responsiveness ................................................................................. 52 Discussion .............................................................................................................. 52 5 FIELD-BASED BEHAVIORAL OBSERVATIONS OF ADULT HONEY BEES REARED IN VITRO ................................................................................................ 67 Introduction ............................................................................................................. 67 Materials and Methods............................................................................................ 70 Results .................................................................................................................... 73 Discussion .............................................................................................................. 74 6 CONCLUSION ........................................................................................................ 84 LIST OF REFERENCES ............................................................................................... 90 BIOGRAPHICAL SKETCH .......................................................................................... 103 6 LIST OF TABLES Table page 1-1 Comparison of the environmental conditions experienced by in vitro- and colony-reared honey bees. ................................................................................. 14 2-1 Means comparisons of the rearing environment by time point interaction effect observed for survival percentage. ............................................................. 25 3-1 Summary of the results of each quantitative morphometric trait measured. ....... 38 4-1 Summary of the distribution function, link function, and random effects for each response group/variable by social behavior. .............................................. 62 4-2 Summary of parameters tested to assess the effect of developmental environment (parental colony or in vitro) on adult worker bee behavior. ............ 66 5-1 Categories used when recording adult honey bee behavior during observational periods. ........................................................................................ 79 5-2 Age at which honey bees were participating in each behavior category, shown by rearing environment. ........................................................................... 82 5-3 Comparisons of the mean age at which each behavior category was performed by honey bees that were reared by their parental colony or in vitro. 83 6-1 Summary of parameters tested to assess the effects of developmental environment (parental colony or in vitro) on adult worker honey bees. ............... 88 7 LIST OF FIGURES Figure page 2-1 The average percent survival by rearing environment (parental colony or in vitro) and time point (day 11 or at adult emergence) for developing honey bees.. .................................................................................................................. 24 2-2 Predictive relationship between day 11 survival percentages and adult emergence survival percentages by rearing environment.. ................................ 26 2-3 Predictive relationship between the day 11 survival percentages in the parental hive and the adult emergence survival percentages of larvae grafted from those parental hives and reared in vitro...................................................... 27 3-1 External features measured for morphological comparisons between adult honey bees that were reared in vitro or in their parental colony.. ....................... 37 3-2 External morphological characteristics used to categorize individuals as worker honey bees, queens, or queen/worker intercastes.. ............................... 37 4-1 Images of the queen recognition assay.. ............................................................ 57 4-2 Composite image of the petri dish cages in round two of the brood rearing assay.. ................................................................................................................ 58 4-3 Images of the trophallaxis assay.. ...................................................................... 59 4-4 Images of