MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Yuta Kawarasaki Candidate for the Degree: Doctor of Philosophy _____________________________________________ Director Richard E. Lee, Jr. _____________________________________________ Reader Jon P. Costanzo _____________________________________________ Reader Kathleen A. Killian _____________________________________________ Reader Paul J. Schaeffer _____________________________________________ Graduate School Representative A. John Bailer ABSTRACT SURVIVAL AND PHYSIOLOGICAL RESPONSES TO SUBZERO TEMPERATURES IN THE ANTARCTIC MIDGE, BELGICA ANTARCTICA: TO FREEZE OR NOT TO FREEZE by Yuta Kawarasaki Low-temperature survival of the Antarctic midge, Belgica antarctica, is promoted by alternative strategies of freeze tolerance and freeze avoidance. Larvae are freeze tolerant year round. Yet, provided they avoid inoculative freezing, they could remain unfrozen by either supercooling (during acute exposure) or cryoprotective dehydration (during prolonged exposure). The first two projects in this dissertation compared each of these two strategies of freeze- avoidance to that of freeze-tolerance. The purpose of the first project was to compare the induction of the rapid cold-hardening (RCH) response in frozen and supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater cryoprotection in frozen versus supercooled larvae. Since the primary difference between these two groups is cellular dehydration, and dehydration without chilling significantly increased larval cold tolerance, it was speculated that cellular dehydration caused by freeze concentration promoted the rapid development of cryoprotection in frozen larvae. The second investigation examined the alternative overwintering strategies of freezing and cryoprotective dehydration. Freezing had little effect on larval body water content and hemolymph osmolality. In contrast, cryoprotective dehydration resulted in a progressive loss of body water, causing a four-fold increase in hemolymph osmolality. Also, freezing and cryoprotective dehydration produced distinctly different patterns of glycogen breakdown. However, post-recovery levels of glycogen were similar in these two groups as were total lipids. In summary, freezing and cryoprotective dehydration were both effective in promoting winter survival of larvae, with only minor differences in energetic costs. The use of cryoprotective dehydration as an overwintering strategy in B. antarctica is constrained by inoculative freezing from environmental ice. Thus, the last project investigated the effect of different microhabitat substrates on larval overwintering by freezing or cryoprotective dehydration. When tested at ecologically-relevant hydration levels, all types of substrate created environmental conditions that were too moist to permit avoidance of freezing by inoculation. Consequently, it is likely that most larvae would likely be forced to overwinter in a frozen state. Yet, dehydrated larvae were found in the field. Therefore, spatial and temporal variations in microhabitat conditions can expose larvae to dehydration stress and potentially allow them to overwinter by cryoprotective dehydration. SURVIVAL AND PHYSIOLOGICAL RESPONSES TO SUBZERO TEMPERATURES IN THE ANTARCTIC MIDGE, BELGICA ANTARCTICA: TO FREEZE OR NOT TO FREEZE A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology by Yuta Kawarasaki Miami University Oxford, Ohio 2013 Dissertation director: Richard E. Lee, Jr. TABLE OF CONTENTS Item Page Table of contents ii List of tables iv List of figures v Acknowledgements x Chapter 1: General introduction 1 References 4 Chapter 2: The protective effect of rapid cold-hardening develops more 6 quickly in frozen versus supercooled larvae of the Antarctic midge, Belgica antarctica Summary 7 Introduction 8 Materials and methods 9 Results 13 Discussion 16 References 21 Tables 28 Figure legends 30 Figures 32 Chapter 3: Alternative overwintering strategies in larvae of the Antarctic 38 midge, Belgica antarctica: freezing versus cryoprotective dehydration Summary 39 ii Introduction 40 Materials and methods 42 Results 46 Discussion 49 References 56 Tables 62 Figure legends 64 Figures 65 Chapter 4: Moist hibernacula promote inoculative freezing and limit the 70 potential for cryoprotective dehydration in the Antarctic midge, Belgica antarctica Summary 71 Introduction 72 Materials and methods 74 Results 76 Discussion 79 References 83 Tables 86 Figure legends 87 Figures 88 Chapter 5: Concluding remarks 91 References 94 iii LIST OF TABLES Item Page Chapter 2: 2.1 Table 1. The effect of RCH and cold acclimation on glucose and trehalose 28 levels in larvae of B. antarctica. 2.2 Table 2. Effects of freezing, cryoprotective dehydration, and slow 29 dehydration on larval water content, hemolymph osmolality, and freeze tolerance in B. antarctica. Chapter 3: 3.1 Table 1. Effects of freezing and cryoprotective dehydration on 62 cryoprotectant levels, lipid content, and dry mass in larval B. antarctica during the 37-d experiment. 3.2 Table 2. Estimated osmotic contribution of original solutes in the 63 hemolymph due to loss of body water during cryoprotective dehydration. Chapter 4: 4.1 Table 1. Substrate characteristics of different microhabitats of 86 B. antarctica. iv LIST OF FIGURES Item Page Chapter 2: 2.1 Fig. 1. Temperature fluctuations during the late austral summer (February 32 3 − March, 31, 2011) at a representative microhabitat for larvae of Belgica antarctica on Humble Island, near Palmer Station, Antarctica (64°46 S, 64°04W). The broken line indicates the equilibrium melting point of fully hydrated larvae (~−0.7°C). 2.2 Fig. 2. Effects of RCH at different temperatures on (A) organismal 33 survival and (B) cell survival of larval midgut tissues in B. antarctica. RCH responses were induced by exposing larvae to various temperatures for 2 h, prior to testing their cold tolerance at −18°C for 24 h. The control group was directly exposed to −18°C for 24 h. Organismal survival was based on 47–51 larvae (± standard error of proportion). Cell survival was based on 4 or 5 replicates, with each replicate based on the mean of three groups of 100 cells (± s.e.m.). Values with different letters are significantly different within frozen groups. Asterisks (*) denote significant differences between frozen and supercooled groups (Bonferroni, family-wise P<0.05). 2.3 Fig. 3. Representative images of the effect of acclimation temperature on 34 cell viability in midgut (MG) tissues of B. antarctica larvae. Tissues were dissected from larvae that survived exposure to −18°C for 24 h. Green indicates living cells, whereas dead cells are stained red. Images of Malpighian tubules (MT) are also shown. v 2.4 Fig. 4. Effect of varying the duration of acclimation at −3°C on (A) 35 organismal and (B) cell survival of larval midgut tissues in B. antarctica. Prior to testing their cold tolerance at −18 or −21°C for 24 h, larvae were exposed to −3°C for various intervals. The control group was directly exposed to −18°C for 24 h. Organismal survival was based on 47–52 larvae (± standard error of proportion). Cell survival was based on 4 or 5 replicates, with each replicate based on the mean of three groups of 100 cells (± s.e.m.). Values with different letters are significantly different within frozen groups exposed to the same discriminating temperature. Asterisks (*) denote significant differences between frozen and supercooled groups. Crosses (†) denote significant differences between treatment and control groups (Bonferroni, family-wise P<0.05). 2.5 Fig. 5. Effect of varying the duration of exposure to −5°C on (A) 36 organismal survival and (B) cell survival of larval midgut in B. antarctica. Protective effects of RCH were tested by subsequently assessing larval cold tolerance at −18°C for 24 h. The control group was directly exposed to −18°C for 24 h. Organismal survival was based on 49–55 larvae (± standard error of proportion). Cell survival was based on 4 or 5 replicates, with each replicate based on the mean of three groups of 100 cells (± s.e.m). Values with different letters are significantly different (Bonferroni, family-wise P<0.05). 2.6 Fig. 6. Time course for the loss of protective effects generated by RCH. 37 Following RCH at −5°C for 2 h in the frozen state, larvae were allowed to thaw at 2°C for various intervals and survival was assessed after subsequent exposure to −18°C for 24 h. The control group was directly exposed to −18°C for 24 h. Organismal survival was based on 49–51 larvae (± standard error of proportion). Values with different letters are significantly different (Bonferroni, family-wise P<0.05). vi Chapter 3: 3.1 Fig. 1. Experimental protocol for the subzero exposure. Subsets of 65 samples were removed from the experiment on Days 14, 32, and 37 to assess larval body water content, hemolymph osmolality, cryoprotectant and glycogen levels, and total lipid content. 3.2 Fig. 2. Seasonal changes in temperature at a representative larval 66 microhabitat of B. antarctica on Humble Island, near Palmer Station, Antarctica (64°46 S, 64°04 W). Microhabitat temperatures were measured in 2011−2012 using a single-channel temperature logger. The broken line indicates the equilibrium melting point of winter-acclimatized larvae (−1.1°C; this study). 3.3 Fig. 3. Effects of freezing