INTERACTIVE EFFECTS OF OCEAN ACIDIFICATION AND MULTIPLE STRESSORS ON PHYSIOLOGY OF MARINE BIVALVES by Omera Bashir Matoo A dissertation submitted to the faculty of The University of North Carolina at Charlotte in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Charlotte 2013 Approved by: ______________________________ Dr. Inna M. Sokolova ______________________________ Dr. Matthew W. Parrow ______________________________ Dr. Mark G. Clemens ______________________________ Dr. Stanley S. Schneider ______________________________ Dr. Andy Bobyarchick ii ©2013 Omera Bashir Matoo ALL RIGHTS RESERVED iii ABSTRACT OMERA BASHIR MATOO.Interactive effects of ocean acidification and multiple stressors on the physiology of marine bivalves (Under direction of Dr. INNA M. SOKOLOVA) The continuing increase of carbon dioxide (CO2) levels in the atmosphere leads to increase in sea-surface temperature and causes ocean acidification altering seawater carbonate chemistry. Estuarine and shallow coastal areas, which are hotspots for biological productivity, are especially prone to these changes, because of low buffering capacity of brackish waters, biological CO2 production, and large fluctuations of temperature and salinity in these habitats. These additional stressors may exacerbate the acidification trend and significantly affect the physiology of marine calcifiers. Bivalves are a key group of marine calcifiers that serve as ecosystem engineers and key foundation species in estuarine and coastal environments. However, the interactive effects of elevated CO2 and other stressors, including elevated temperature and reduced salinity, are not yet fully understood in bivalves and require further investigation. This study focused on the physiological responses in two ecologically and economically important bivalve species - the eastern oyster (Crassostrea virginica) and hard shell clam (Mercenaria mercenaria). Juveniles and adults were exposed to environmentally relevant salinities at either ~32 or ~16 with different PCO2 levels (~400, 800 and 1500 µatm) and temperature (22°C and 27°C), as predicted by future global climate change scenarios, for 11-21 weeks (in juveniles) and 15 weeks (in adults). Survival, metabolism and calcification were iv assessed. Elevated PCO2 alone and in combination with either reduced salinity or elevated temperature led to reduced survival and growth, and altered the shell mechanical properties (microhardness and fracture resistance) in juvenile and adult bivalves. Tissue energy reserves (lipid and glycogen) were reduced in juveniles under elevated PCO2 and low salinity. Standard energy metabolism (SMR) increased under the conditions of elevated PCO2 and temperature indicating higher costs of basal maintenance. In adult bivalves, elevated temperature led to the depletion of tissue energy reserves in both species indicating energy deficiency, and resulted in higher mortality in oysters but not in clams. In adult clams and oysters, elevated PCO2 alone had a small effect on the metabolism. No persistent oxidative stress (measured by total antioxidant capacity as well as oxidative markers of proteins and lipids) was seen in the bivalves after prolonged exposure to elevated temperature and PCO2. The results of this study indicate that the interactions between common abiotic stressors (salinity and temperature) and elevated PCO2 are complex, non-linear, and species-specific. Our study shows that elevated temperature and reduced salinity are predominant stressors that affect survival, metabolism, and biomineralization response of the studied bivalves. Furthermore, these stressors modulate the responses of these bivalves to ocean acidification while elevated CO2 levels have a modest impact in the absence of these stressors. v DEDICATION To the courage of all those who survive in the land without post offices. vi ACKNOWLEDGMENTS The author would like to express her appreciation and gratitude to all those who made this dissertation possible: Her advisors, Dr. Inna M. Sokolova for making this work possible from idea to publication, Dr. Mark G. Clemens for advice on experimental design, Dr. Matthew W. Parrow, Dr. Stanley S. Schneider and Dr. Andy Bobyarchick for academic support; To Dr. Elia Beniash and Dr.Gary H. Dickinson for productive collaboration on ideas, experimental support and publication of this study; To the Department of Biology, UNCC for the opportunity for study; To colleagues Dr. Anna V. Ivanina, Dr. Rita Bagwe, Ilya Kurochkin, Ryan Rutledge and Chelsea Hawkins for support and making a great learning atomosphere in laboratory possible; To Valerie Williams, Ashley Dickinson, Claus Ullstad, Allen Baheri and many undergraduate reserachers for technical support in the laboratory; To friends Sandra Goetze and Maria Mugica-Fernandez for discussion of ideas and the many fun trips together; To friends Kamalika,Vandana and Harpreet for their faith in me and keeping me focused in my goals; To her parents, Dr.Bashir Matoo and Shamshad Matoo, for their continued support, patience and guidance; And to her schoolteacher and friend Mr.Muzaffar for all his advice and love through the years. vii TABLE OF CONTENTS LIST OF FIGURES ix LIST OF TABLES x LIST OF ABBREVIATIONS xiii CHAPTER 1: INTRODUCTION 1 CHAPTER 2: ENVIRONMENTAL SALINITY MODULATES THE EFFECTS 28 OF ELEVATED CO2 LEVELS ON JUVENILE HARD-SHELL CLAMS, MERCENARIA MERCENARIA Introduction 29 Materials and Methods 32 Results 42 Discussion 49 Figures 56 Supplementary Materials 65 CHAPTER 3: INTERACTIVE EFFECTS OF SALINITY AND ELEVATED 70 CO2 ON JUVENILE EASTERN OYSTERS, CRASSOSTREA VIRGINICA Introduction 71 Materials and Methods 75 Results 90 Discussion 94 Conclusions 104 Figures 106 viii CHAPTER 4: INTERACTIVE EFFECTS OF ELEVATED TEMPERATURE 115 AND CO2 LEVELS ON METABOLISM AND OXIDATIVE STRESS IN TWO COMMON MARINE BIVALVES Introduction 116 Materials and Methods 120 Results 127 Discussion 130 Figures 137 CHAPTER 5: INTERACTIVE EFFECTS OF ELEVATED TEMPERATUE 141 AND CO2 ON THE ENERGY METABOLISM AND BIOMINERALIZATION OF MARINE BIVALVES CROSSOSTREA VIRGINICA AND MERCENARIA MERCENARIA Introduction 143 Materials and Methods 146 Results 156 Discussion 161 Figures 168 Supplementary Materials 175 CHAPTER 6: SUMMARY 181 REFERENCES 194 ix LIST OF TABLES TABLE 2.1: Summary of water chemistry parameters during experimental 63 exposures. TABLE 2.2: Effects of exposure PCO2, salinity and their interaction on shell 64 and physiological properties in juvenile Mercenaria mercenaria. TABLE 3.1: Summary of water chemistry parameters during experimental 112 exposures of juvenile eastern oysters, Crassostrea virginica. TABLE 3.2: ANOVA results of the effects of exposure salinity, PCO2 and their 113 interaction on shell and soft tissue mass, mechanical shell properties, enzyme activities and energy-related indices in juvenile Crassostrea virginica. TABLE 3.3: Summary of the effects of salinity and PCO2 levels on the studied 114 physiological and biomineralization traits in C. virginica juveniles. TABLE 4.1: Summary of water chemistry parameters during experimental 139 exposures. TABLE 4.2: ANOVA for the effects of temperature, CO2 levels and exposure 140 time on the studied physiological and biochemical traits of C. virginica. TABLE 4.3: ANOVA for the effects of temperature, CO2 levels and exposure 141 time on the studied physiological and biochemical traits of M.mercenaria. TABLE 5.1: Summary of water chemistry parameters during experimental 173 exposures. TABLE 5.2: Activity, activation energy and Arrhenius breakpoint temperature 174 (ABT) for carbonic anhydrase (CA) in different tissues of clams and oysters. x LIST OF FIGURES FIGURE 1.1: Gobal annual emissions of anthropogenic CO2 and other 2 greenhouse gases in 1970-2004. FIGURE 1.2: Global averages of surface warming for SRES scenarios using 3 Atmosphere-Ocean General Circulation Models (AOGCMs). FIGURE 1.3: Ecosystem impacts associated with global average temperature 4 change. FIGURE 1.4: CO2 emissions and concentration for a range of SRES (Special 5 Report on Emission Scenarios) scenarios. FIGURE 1.5: Schematic representation of ocean acidification and CaCO3 7 counter pump. 2- FIGURE 1.6: Time series of surface-ocean PCO2, pHT and [CO3 ], as well as for 11 the atmospheric mole fraction of CO2 at Mauna Loa (Black), ocean time- series stations ALOHA (green), BATS (red), and ESTOC (blue). FIGURE 1.7: Carbonate parameters changes in the Kennebec Estuary, Maine. 15 FIGURE 1.8: (a).World aquaculture production by composition and types of 20 different farmed species. (b).Production of major mollusk species from aquaculture in 2010. FIGURE 1.9: Schematic of the shell formation (excluding the hemocyte-related 21 calcification mechanisms) of a mollusk. FIGURE 2.1: Mortality and standard metabolic rate (SMR) in juvenile clam 56 (Mercenaria mercenaria) exposed to different PCO2 and salinity treatments. FIGURE 2.2: Changes in M. mercenaria shell and tissue mass in response to 57 salinity, PCO2 and aragonite saturation (ΩArg). FIGURE 2.3: Mechanical properties of the shells of M. mercenaria exposed to 58 different PCO2 and salinity levels. FIGURE 2.4: SEM micrographs of the exterior of M. mercenaria shells after 16 59 weeks exposure to experimental conditions. FIGURE 2.5: SEM micrographs of the interior of M. mercenaria shells after 16 60 weeks exposure to experimental conditions. xi FIGURE 2.6: SEM micrographs of the interior of the hinge region of 61 M. mercenaria shells after 16 weeks exposure to experimental