Tolerance and Physiological Response to Environmental Stress in Antarctic Arthropods
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MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Michael A. Elnitsky Candidate for the Degree: Doctor of Philosophy __________________________________________ Director Richard E. Lee, Jr. __________________________________________ Reader Alan B. Cady __________________________________________ Reader Jon P. Costanzo __________________________________________ Reader Kathleen A. Killian __________________________________________ Graduate School Representative Robert L. Schaefer ABSTRACT TOLERANCE AND PHYSIOLOGICAL RESPONSE TO ENVIRONMENTAL STRESS IN ANTARCTIC ARTHROPODS by Michael A. Elnitsky The Antarctic Peninsula is characterized by harsh and dynamic environmental conditions. Organisms inhabiting this environment may be challenged by extremes of low temperature, limited water availability, dramatic seasonal fluctuations of light availability and ultraviolet radiation, and high salinity. This dissertation describes three projects examining the tolerance and physiological responses to such environmental stress of two Antarctic arthropods, the midge Belgica antarctica and the collembolan Cryptopygus antarcticus. The first investigation examined the ability of B. antarctica larvae to resist inoculative freezing at subzero temperatures and instead dehydrate as a strategy for winter survival (i.e., cryoprotective dehydration). When cooled to subzero temperatures in the presence of ice, the body fluid melting point was depressed to near equilibrium with the ambient temperature, due to reductions of body water content and the accumulation of several osmolytes, suggesting larvae can undergo cryoprotective dehydration at subzero temperatures. Under more natural conditions, the use of cryoprotective dehydration versus freeze tolerance for winter survival appears to depend upon the moisture content of the surrounding soil. The purpose of the second study was to assess the tolerance and physiological response to desiccation of C. antarcticus under ecologically-relevant conditions. Slow dehydration at high relative humidities characteristic of the austral summer induced the accumulation of several organic osmolytes and increased the tolerance of water loss. A mild drought acclimation further increased the subsequent desiccation tolerance of C. antarcticus. The springtails were also susceptible to water loss at subzero temperatures and likely rely upon such dehydration as a key component for winter survival. As B. antarctica microhabitats may be periodically inundated with seawater, the final investigation examined the osmotic response and tolerance of larvae to hyperosmotic seawater exposure. The larvae displayed an impressive tolerance of the osmotic stress, as ~50% survived a 6-d submergence in pure seawater. Hyperosmotic stress induced the accumulation of organic osmolytes and resulted in a significant positive correlation between the rate of oxygen consumption and larval body water content. Finally, a brief seawater acclimation enhanced the subsequent tolerance of freezing and dehydration, but reduced the tolerance of heat shock. TOLERANCE AND PHYSIOLOGICAL RESPONSE TO ENVIRONMENTAL STRESS IN ANTARCTIC ARTHROPODS 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 Michael A. Elnitsky Miami University Oxford, Ohio 2008 TABLE OF CONTENTS Item Page Table of contents ii List of tables iv List of figures v Acknowledgements ix Chapter 1: General introduction 1 References 4 Chapter 2: Cryoprotective dehydration and the resistance to inoculative 6 freezing in the Antarctic midge, Belgica antarctica Summary 7 Introduction 8 Materials and methods 9 Results 12 Discussion 14 References 19 Table 22 Figure legends 23 Figures 24 Chapter 3: Desiccation tolerance and drought acclimation in Antarctic 28 collembolan Cryptopygus antarcticus Summary 29 Introduction 30 ii Materials and methods 31 Results 35 Discussion 37 References 43 Tables 47 Figure legends 49 Figures 50 Chapter 4: Osmoregulation and salinity tolerance in the Antarctic midge, 54 Belgica antarctica: seawater acclimation confers cross tolerance to freezing and dehydration Summary 55 Introduction 56 Materials and methods 57 Results 61 Discussion 63 References 70 Table 74 Figure legends 76 Figures 78 Chapter 5: Concluding remarks 83 iii LIST OF TABLES Table Page Chapter 2: 2.1 Table 1. Estimated osmotic contribution of initial osmolytes in the 22 hemolymph and osmolytes produced during slow cooling to -3oC in an environment at equilibrium with the vapor pressure of ice. Values are mean ± SEM. Chapter 3: 3.1 Table 1. The total body water content (N = 25-30) and osmolyte 47 concentrations (N = 6) of Cryptopygus antarcticus during drought acclimation at 4oC and 98.2 or 75.0% RH. Values are mean ± SEM. Within an osmolyte different letters denote significant differences between treatment groups (ANOVA; Bonferroni-Dunn test). 3.2 Table 2. Osmolyte concentrations (N = 6) of Cryptopygus antarcticus 48 during exposure at -3.0oC in an environment at equilibrium with the vapor pressure of ice. Values are mean ± SEM. Within an osmolyte different letters denote significant differences between days of exposure (ANOVA; Bonferroni-Dunn test). Chapter 4: 4.1 Table 1. Estimated osmotic contribution of initial osmolytes in the 74 hemolymph and osmolytes produced during hyperosmotic seawater exposure. Values are mean ± SEM. Within an osmolyte different letters represent significant differences between days of exposure (ANOVA; Bonferroni-Dunn test). iv LIST OF FIGURES Figure Page Chapter 2: 2.1 Fig. 1. Seasonal changes in temperature at a representative larval 24 Belgica antarctica microhabitat site on Torgersen Island, near Palmer Station, Antarctica (64o46’ S, 64o04’ W). Microhabitat temperatures were measured in 2005-2006 using single-channel temperature loggers (HOBO Water Temp Pro, Onset Computer, Pocasset, MA, USA). The dashed line represents the equilibrium freezing point of the body fluids of fully hydrated, control larvae. 2.2 Fig. 2. Changes in (A) body water content (N = 15) and (B) body fluid 25 melting point (N = 6) of larval Belgica antarctica during slow cooling to -3oC in an environment at equilibrium with the vapor pressure of ice. Different letters indicate significant differences between values (ANOVA, Bonferroni-Dunn test, P<0.05). Values are mean ± SEM. 2.3 Fig. 3. Body water content (WC) of individual Belgica antarctica 26 larvae (N = 30) during slow cooling to -3oC in contact with substrates -1 of varying moisture content: 0.80, 1.10, and 1.40 g H2O · g dry soil. Triangles denote WC of individuals at day 0, circles the WC of frost exposed individuals (day 16). Dashes denote the mean WC of individuals at day 0 and 16, separated into ‘high’ (frozen) and ‘low’ (dehydrated) WC groups. v 2.4 Fig. 4. Percentage of Belgica antarctica larvae frozen, as detected by 27 the maintenance of ‘high’ body water content, during cooling to -3oC in contact with substrates of varying moisture content. Different letters indicate significant differences between values (ANOVA, Bonferroni-Dunn test, P<0.05). Values are mean ± SEM of three groups of 10 individuals. Chapter 3: 3.1 Fig. 1. (A) Changes in total body water content of Cryptopygus 50 antarcticus during desiccation exposure within various relative humidity (RH) environments at 4oC. Values are the mean ± SEM of 25-30 individuals. (B) Percent survival as a function of total body water content of C. antarcticus during desiccation in various constant relative humidity environments. Values are the mean ± SEM of five groups of 10 individuals. 3.2 Fig. 2. Water loss rate of Cryptopygus antarcticus at 4oC and 0% RH. 51 A linear regression line was fitted to the points (y = -0.208x – 0.00650, R2 = 0.998), where the slope of the regression represents the water loss rate in percent of total body water per hour. Values are mean ± SEM of 25-30 individuals. 3.3 Fig. 3. Survival of Cryptopygus antarcticus desiccated for 5 d at either 52 96.0% RH (A) or 93.0% RH (B) at 4oC. Collembola were previously acclimated at 100% RH (control) or drought acclimated at 98.2 or 75.0% RH prior to assessment of desiccation tolerance. Values are mean ± SEM of five groups of 10 individuals. Asterisks denote a significant difference relative to the control treatment (Student’s t- test). vi 3.4 Fig. 4. Changes in (A) body water content (N = 15-20 individuals) and 53 (B) osmotic pressure of the body fluids (N = 6) of Cryptopygus antarcticus during slow cooling to -3.0oC in an environment at equilibrium with the vapor pressure of ice. Values are the mean ± SEM. Chapter 4: 4.1 Fig.1. (A) Survival (N = five groups of 10 larvae), (B) water content 78 (N = 25-30), and (C) hemolymph osmolality (N = 6) of Belgica antarctica larvae exposed to a various concentrations of seawater or freshwater (~0 mOsm kg-1). Values are mean ± 1 SEM. 4.2 Fig. 2. Effect of acclimation to seawater (~1000 mOsm kg-1) and 79 resultant changes of body water content on the rate of oxygen consumption of Belgica antarctica larvae (y = -0.853 + 0.602x; R2 = 0.822; P<0.001). 4.3 Fig. 3. The effect of acclimation to seawater on the freeze tolerance of 80 Belgica antarctica larvae. Larvae were acclimated to either seawater (~1000 mOsm kg-1) or freshwater (~0 mOsm kg-1) for 3 d prior to assessment of freeze tolerance. A third group of larvae (rehydrated) were acclimated to seawater for 3 d followed by rehydration for 24 h in freshwater. Larvae were frozen in groups of 10 in ~100 µL of freshwater for 6 h. Values are mean ± 1 SEM of five groups of 10 larvae. Asterisks denote a significant difference relative to the freshwater (control) treatment (ANOVA, Dunnett’s test, P<0.05). vii 4.4 Fig. 4. The effect of seawater acclimation on the desiccation tolerance 81 of Belgica antarctica larvae at 98.2 (A) or 75.0% RH (B) and 4oC. Larvae were acclimated to either seawater (~1000 mOsm kg-1) or freshwater (~0 mOsm kg-1) for 3 d prior to assessment of desiccation tolerance. Rehydrated larvae were acclimated to seawater for 3 d and then allowed to rehydrate for 24 h in freshwater prior to desiccation. Values are mean ± 1 SEM of five groups of 10 larvae.