Factors Influencing Mountain Pine Beetle (Coleoptera: Curculionidae: Scolytinae) Dispersal by Flight and Subsequent Trade-off Between Beetle Flight and Reproduction by Asha Nishadini Wijerathna A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Ecology Department of Biological Sciences University of Alberta © Asha Nishadini Wijerathna, 2016 Abstract The mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae), has an obligatory dispersal phase before selection of a suitable host for brood production. Flight during the dispersal phase is affected by both beetle physiology and environmental factors. Mountain pine beetle utilizes energy obtained during larval development and adult feeding before emergence from the natal tree host to fuel flight. Energy expenditure on flight may have consequences for subsequent reproduction. Mountain pine beetles were flown on computer-linked flight mills to establish how beetles utilize energy during flight and to determine how environmental conditions affect flight capacity. Mountain pine beetles utilize both lipids and carbohydrates to fuel the flight. Neutral lipids are the major fuel for flight. Beetles held at lower temperatures were more likely to fly and flew for longer distances than beetles held at warmer temperatures. Beetles increase flight distance and duration with day length. Beetles fly against wind speeds of 0-2 m/s in a wind tunnel. The effect of flight on subsequent reproduction was tested by introducing beetles flown on flight mills and un-flown control beetles into bolts of either lodgepole, Pinus contorta Douglas. ex. Loud. var. latifolia), or jack, Pinus banksiana Dougl., pine to reproduce. There is a trade-off between flight and reproduction in the mountain pine beetle as control beetles that did not fly produced a significantly higher number of offspring than flown beetles. The pine host that offspring were reared in affected the number of offspring and their condition, as more offspring emerged from jack pine but higher quality offspring emerged from lodgepole pine. Offspring from flown parents have a slightly lower body condition compared to the offspring from control adults. The study reveals physiological and environmental factors that influence dispersal capacity of mountain pine beetle and a trade-off between flight and reproduction in mountain pine beetle. ii Preface This document presents three studies (Chapter 2, Chapter 3 and Chapter 4) intended for publication and is a collaborative work led by Dr. Maya Evenden of the University of Alberta. I was responsible for data collection and analysis, literature review and manuscript composition. Dr. Evenden was involved with concept formation and manuscript composition throughout the work. Dr. Heather Proctor provided manuscript edits. Additionally, Caroline M. Whitehouse of the University of Alberta collected data used in Chapter 4. All research presented in this thesis was conducted in accordance with all applicable laws and rules set forth by provincial and federal governments and the University of Alberta and all necessary permits were in-hand when the research was conducted. iii Acknowledgements I would like to express my gratitude to Dr. Maya Evenden whose support, ideas and guidance added considerably to my graduate experience. The input of time and ideas by Dr. Heather Proctor and Dr. Keith Tierney is also much appreciated. I extend great thanks to Devin Letourneau of Alberta Agriculture and Forestry for bolt collection and Corey Davis and Troy Locke of Molecular Biology Service Unit of University of Alberta for assisting with biochemical analysis. I also thank Evgeni Matveev, Chetna Saran and Jackson Lai for their laboratory work and Boyd Mori, Stephen Trevoy for helping me with cutting bolts. This thesis could not have been written without the help of Sarah McPike, Nathan Bird and all the wonderful people at the Center for Writers at the University of Alberta. Thank you Evenden lab members for your support. Thank you Dad and Akalanka for your encouragement and patience along the way. Funding for this project was provided by the TRIA-Network, Natural Science and Engineering Research Council, Canada. iv Table of Contents Chapter 1 : Introduction .................................................................................................................. 1 Objectives ................................................................................................................................... 7 LITERATURE CITED ............................................................................................................... 9 Chapter 2 : Energy use by the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) for dispersal by flight. ......................................................................................................................... 20 2.1 Introduction ......................................................................................................................... 20 2.2 Methods............................................................................................................................... 22 2.2.1 Biochemical Assay....................................................................................................... 22 2.2.1.1 Beetles ....................................................................................................................... 22 2.2.1.2 Beetle Flight Treatment ............................................................................................ 22 2.2.1.3 Biochemical Analysis ................................................................................................ 23 2.2.2 Flight Muscle Experiment............................................................................................ 26 2.2.2.1 Beetles ....................................................................................................................... 26 2.2.2.2 Beetle Flight .............................................................................................................. 26 2.2.2.3 Flight muscle dissections .......................................................................................... 26 2.2.3 Data Analysis ............................................................................................................... 27 2.3 Results ................................................................................................................................. 28 2.3.1 Biochemical analyses ................................................................................................... 28 2.3.2 Flight muscle dissections ............................................................................................. 30 2.4 Discussion ........................................................................................................................... 31 LITERATURE CITED ............................................................................................................. 41 Chapter 3 : Variation of flight capacity in mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) with environmental conditions. ................................................................................. 45 v 3.1 Introduction ......................................................................................................................... 45 3.2 Methods............................................................................................................................... 48 3.2.1 Beetles .......................................................................................................................... 48 3.2.2 Beetle Flight ................................................................................................................. 48 3.2.3 Temperature-flight bioassay ........................................................................................ 49 3.2.4 Light-flight bioassay .................................................................................................... 50 3.2.5 Wind speed-flight bioassay .......................................................................................... 51 3.2.6 Data Analysis ............................................................................................................... 51 3.3.1 Temperature flight bioassay ......................................................................................... 52 3.3.1.1 Beetle flight propensity ............................................................................................. 52 3.3.2 Light-flight bioassay .................................................................................................... 53 3.3.2.1 Beetle flight capacity ................................................................................................. 53 3.3.3 Wind speed-flight bioassay .......................................................................................... 55 3.3.3.1 Beetle flight propensity ............................................................................................. 55 3.3.3.2 Beetle flight capacity ................................................................................................. 55 3.4 Discussion ........................................................................................................................... 57 LITERATURE CITED ............................................................................................................. 73 Chapter 4 : The potential for a trade-off between flight and reproduction in the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) on two pine hosts. .............................................. 78 4.1 Introduction ......................................................................................................................... 78 4.2 Methods............................................................................................................................... 81 4.2 1 Beetles .......................................................................................................................... 81 4.2.2 Parental Beetle Flight Treatment ................................................................................. 81 4.2.3 Bolt Inoculation and Offspring Rearing ...................................................................... 82 vi 4.2.4 Fat Extraction and Offspring Condition ...................................................................... 82 4.2.4 Data Analyses .............................................................................................................. 83 4.3 Results ................................................................................................................................. 84 4.4 Discussion ........................................................................................................................... 88 LITERATURE CITED ............................................................................................................. 96 Chapter 5 : General conclusion ................................................................................................... 104 LITERATURE CITED ........................................................................................................... 107 Bibliography ............................................................................................................................... 109 Appendix I .................................................................................................................................. 128 Appendix II ................................................................................................................................. 129 vii List of Figures Figure 2.1: A) A mountain pine beetle on flight tether. B) A flight mill with tethered beetle attached by inserting the 2 cm portion of the aluminum tether. ................................................... 34 Figure 2.2: Successive steps used for extraction of proteins, soluble carbohydrates, glycogen, total lipids and triglycerides in a single individual mountain pine beetle. Reagents used for each extraction are given in parentheses. Method adapted from Foray et al. (2012). ........................... 35 Figure 2.3: The proportional energy budget of randomly selected flown and un-flown (control) female and male mountain pine beetles. Proteins, lipids and carbohydrates were extracted from each individual beetle sample using a colourimetric method. Each panel includes five individual representative samples (S1-S5) indicating protein, total lipids and total carbohydrates weight as a percentage of the total energy budget. Data were analyzed using generalized mixed effects model............................................................................................................................................. 36 Figure 2.4: Protein content as a proportion of individual body weight, determined for female and male mountain pine beetle (n=20-32) in accordance with flown and control flight conditions. Mountain pine beetles were flown on flight mills for 23 h. The proteins were extracted from flown and un-flown (control) beetles using a Bradford assay. Protein content of beetles was compared with the known contents of bovine serum albumin as the standard. Data were analyzed using a generalized mixed effects model. Raw data are plotted. .................................................. 37 Figure 2.5: Carbohydrates as a proportion of individual body weight, determined on female and male mountain pine beetle (n=20-32) in accordance with flown and un-flown (control) flight conditions. Mountain pine beetles were flown on flight mills for 23 h. The glucose, trehalose and glycogen were extracted from flown and un-flown beetles using a Hot anthrone reaction. Glucose and glycogen contents of beetles were compared with the known contents of +D- glucose as the standard. Trehalose content of beetles was compared with the known contents of +D-trehalose as the standard. Data were analyzed using generalized mixed effects models for each carbohydrate. Raw data are plotted. ..................................................................................... 38 viii Figure 2.7: Mountain pine beetle flight duration in relation to flight muscle volume. Beetles were flown on flight mills for 23 h and the beetles were dissected to measure the flight muscle volume. Data were analyzed using a general mixed effects model. ............................................. 40 Figure 3.1: Experimental design of temperature flight bioassay. Mountain pine beetles flight capacities were tested at four different holding-flying temperature regimes. .............................. 61 Figure 3.2: Experimental design of light flight bioassay. Mountain pine beetles flight capacities were tested at two different light cycles (16L:8D and 18L:6D) with different day lengths. ........ 62 Figure 3.3: Experimental design of wind speed flight bioassay. Mountain pine beetles flight capacities were tested at four different wind speeds (0, 0.5, 1, 2 m /s). ....................................... 62 Figure 3.4: Diagrammatic view of the wind tunnel (0.1m X 0.05m X 0.05m) used for the wind speed bioassay. Beetles were tethered by attaching a beading wire to the pronotum. Tethers were attached to the roof of the wind tunnel so that beetles were separated by 5 cm and suspended 15 cm from the roof and 40 cm from the upwind end of the tunnel. Five beetles were tested at each trial and were placed facing upwind. ............................................................................................ 63 Figure 3.5: Mountain pine beetle flight duration (h) under the tested holding and flight temperature regimes. Male and female beetles were held separately at two holding temperatures: Cold (5°C) and Warm (24°C) for 23 hours before the initiation of the flight bioassay which was conducted under cold (20°C) or warm (24°C) conditions for 23 hours. Beetles from both holding temperatures were flown at the same time under a selected flying temperature. Females and males were flown separately on alternative days. Data were analyzed using a general mixed effects model. Raw data are plotted. ............................................................................................. 64 Figure 3.6: Mountain pine beetle flight distance (km) under the tested holding and flight temperature regimes. Male and female beetles were held separately at two holding temperatures: cold (5°C) and warm (24°C) for 23 hours before the initiation of the flight bioassay which was conducted under cold (20°C) or warm (24°C) conditions for 23 hours. Beetles from both holding temperatures were flown at the same time under a selected flying temperature. Females and ix males were flown separately on alternative days. Data were analyzed using a general mixed effects model. Raw data are plotted. ............................................................................................. 65 Figure 3.7: Mountain pine beetle flight speed (km/h) under the tested holding and flight temperature regimes. Male and female beetles were held separately at two holding temperatures: cold (5°C) and warm (24°C) for 23 hours before the initiation of the flight bioassay which was conducted under cold (20°C) or warm (24°C) conditions for 23 hours. Beetles from both holding temperatures were flown at the same time under a selected flying temperature. Females and males were flown separately on alternative days. Data were analyzed using a general mixed effects model. Raw data are plotted. ............................................................................................. 66 Figure 3.8: Mountain pine beetle flight duration (h) in the scotophase and photophase of the two tested light cycles: short (16L: 8D) and long (18L: 6D). Females and males were flown separately on alternate days. Data were analyzed using a general mixed effects model. Raw data are plotted...................................................................................................................................... 67 Figure 3.9: Mountain pine beetle flight distance (km) in the photophase and scotophase of the two tested light cycles: short (16L: 8D) and long (18L: 6D). Females and males were flown separately on alternate days. Data were analyzed using a general mixed effects model. Raw data are plotted...................................................................................................................................... 68 Figure 3.10: Mountain pine beetle flight velocity (km/h) in the photophase and scotophase of the two tested light cycles: short (16L: 8D) and long (18L: 6D). Females and males were flown separately on alternate days. Data were analyzed using a general mixed effects model. Raw data are plotted...................................................................................................................................... 69 Figure 3.11: Mountain pine beetle total flight duration (min) at the tested wind speeds during the 50 min wind tunnel bioassay. Male and female beetles, 3-5 days post emergence were held separately at 5°C prior to flight for 23 h. Beetles were suspended within the wind tunnel for 50 minutes. Beetles were attached by tethers to the roof of the wind tunnel facing upwind. Tested wind speeds were: 0 m/s, 0.5m/s, 1m/s, 2 m/s. Females and males were flown separately on x