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Effects of ocean acidification on different life history stages of northern abalone (Haliotis kamtschatkana) Crim, Ryan Nathanial 2010

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   EFFECTS OF OCEAN ACIDIFICATION ON DIFFERENT LIFE HISTORY STAGES OF NORTHERN ABALONE (HALIOTIS KAMTSCHATKANA)   by   Ryan Nathanial Crim   B.Sc., Western Washington University, 2008    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   The Faculty of Graduate Studies   (Zoology)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   October 2010    © Ryan Nathanial Crim, 2010    ii Abstract  Anthropogenic atmospheric CO2 levels are rapidly increasing; however, much of this CO2 (ca. 30%) dissolves into the surface ocean (upper 200 m) where it reacts with seawater and disrupts both ocean pH and carbonate chemistry, a process termed ocean acidification. Average pH of the surface ocean has already decreased by 0.1 units since the beginning of the Industrial Revolution and is expected to drop another 0.2 to 0.4 units by the end of this century. Of primary concern is the potential for ocean acidification to dramatically disrupt biological processes, especially biogenic calcification. Different life history stages may also be affected in different ways. Furthermore, interactions between ocean acidification and other environmental perturbations are often non-additive and thus result in non-predictive outcomes. Here, I investigate the effects of ocean acidification on different life history stages of an endangered abalone, Haliotis kamtschatkana. I reared larvae and adults under elevated CO2 conditions (800 and 1800 ppm), representing levels expected by the end of this century and beyond. Adults were also reared under two temperatures (9 and 12°C) to investigate interactions between CO2 and temperature. Larval survival, shell size and shell morphology were negatively affected by elevated CO2. At 1800 ppm, almost all larvae completely lacked a shell. Adults seem more tolerant of elevated CO2. Survival, growth and feeding rates were unaffected by elevated CO2, at either temperature. Early life history stages may be more sensitive due to differences in calcification processes. Near future levels of ocean acidification may dramatically impair early development of H. kamtschatkana but later life history stages may be more tolerant. Since H. kamtschatkana population growth is thought to be currently limited by successful fertilization, decreases in larval survival may have severe  iii consequences for the recovery of this endangered species. Efforts to mitigate the dramatic population decline of H. kamtschatkana will need to consider the potential repercussions of ocean acidification.                                          iv Preface  The work outlined in this M.Sc. thesis was primarily designed, carried out, analyzed, and written by myself, Ryan Crim, though the manuscripts in Chapters 2 and 3 were coauthored. All research complied with the University of British Columbia Animal Care Committee (Animal Care Certificate A05-1495).  The research outlined in Chapter 2 was performed primarily by myself. J. Sunday assisted with the technical aspects of larval rearing and other laboratory assistance during the experiment. I was responsible for the design and setup of the CO2 manipulation system as well as data collection and analysis. I wrote the initial draft of the manuscript with subsequent revisions by C.D.G. Harley and J. Sunday. C.D.G. Harley contributed to the conceptual design of this project. A version of this chapter has been submitted for publication.  The research detailed in Chapter 3 was carried out by primarily by myself. E. Tang assisted with some laboratory work and was responsible for collecting pH, salinity, and temperature data. I was responsible for the experimental design and setup as well as most of the data collection, analysis, and initial draft of the chapter. C.D.G. Harley contributed to the initial experimental design and chapter revisions.              v Table of Contents  Abstract ............................................................................................... ii Preface ................................................................................................ iv Table of Contents ..................................................................................v List of Tables....................................................................................... vi List of Figures .................................................................................... vii Acknowledgements............................................................................. ix Chapter 1: Introduction ........................................................................1 1.1   Climate Change and Carbonate Chemistry...........................................................1 1.2   Biological Implications of Climate Change..........................................................4 1.2.1   Effects on Different Life History Stages........................................................6 1.2.2   Multiple Climatic Stressors ...........................................................................7 1.3   Abalone Biology..................................................................................................8 1.4   Haliotis kamtschatkana Population Decline .......................................................10 1.5   Research Objectives...........................................................................................12 Chapter 2: Effects of Elevated CO2 on Growth and Survival of Larval Abalone (Haliotis kamtschatkana).................................................... 16 2.1   Introduction .......................................................................................................16 2.2   Materials and Methods.......................................................................................18 2.2.1   Animal Collecting and Rearing ...................................................................18 2.2.2   Carbonate Chemistry...................................................................................20 2.2.3   Data Analysis..............................................................................................21 2.3   Results...............................................................................................................22 2.4   Discussion .........................................................................................................23 2.4.1   Conclusions ................................................................................................27 Chapter 3: Effects of Elevated CO2 and Temperature on Growth and Feeding of Adult Abalone (Haliotis kamtschatkana) ........................ 37 3.1   Introduction .......................................................................................................37 3.2   Materials and Methods.......................................................................................39 3.2.1   Experimental Setup.....................................................................................39 3.2.2   Carbonate Chemistry...................................................................................41 3.2.3   Data Analysis..............................................................................................41 3.3   Results...............................................................................................................42 3.4   Discussion .........................................................................................................43 Chapter 4: General Conclusions ......................................................... 60 4.1   Significance of Overall Results ..........................................................................60 4.1.1   Summary ....................................................................................................60 4.1.2   Vulnerability to Ocean Acidification at Different Life History Stages .........61 4.2   Priorities for Future Research.............................................................................63 4.3   Conclusion.........................................................................................................66 References .......................................................................................... 67  vi List of Tables  Table 2.1   Carbonate chemistry of experimental seawater during the course of the experiment. pH error refers to deviation among replicates. Temperature, salinity and alkalinity error refers to deviation over time. Data are presented as means (± SD). Significance levels calculated using one-way ANOVAs, small letters denote significant differences based on post-hoc Tukey HSD comparisons .........................29 Table 3.1   Mean temperature (°C), salinity (psu) and pHNBS throughout the course of the experiment. Data are means (standard error). Small letters denote significant differences assessed with multiple comparison Tukey HSD tests .............................48 Table 3.2   Two-way ANOVA results of growth rates (calculated from length, width, area and wet weight), feeding rate and survival........................................................49 Table 3.3   MANOVA results of final dry weights (tissue and shell) .............................50   vii List of Figures  Figure 1.1   Worldwide distribution of abalones (Gastropoda: Haliotidae). Known distributions (thick orange lines), scarce populations (thin orange lines). Haliotis kamtschatkana comprises two subspecies: H. kamtschatkana kamtschatkana (black dots) and H. kamtschatkana assimilis (white dots). This thesis refers only to H. kamtschatkana kamtschatkana. Adapted from Geiger et al. 2000.............................14 Figure 1.2   Biphasic life cycle of Haliotis kamtschatkana ............................................15 Figure 2.1   Schematic of experimental design outlining CO2 manipulation setup. MFC = Mass Flow Controller ..............................................................................................30 Figure 2.2   Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Scale bar = 100 µm.........................31 Figure 2.3   Daily mean pHNBS during the experiment (circles: 400 ppm CO2; diamonds: 800 ppm CO2; triangles: 1800 ppm CO2). Error bars are SD (n = 8).........................32 Figure 2.4   Mean percent survival of Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Small letters that differ denote significant differences assessed by one-way ANOVA and post-hoc Tukey HSD comparison. Error bars are SEM (n = 8) ...............................................33 Figure 2.5   Mean percent of Haliotis kamtschatkana larvae that developed normal shell morphology after exposure to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Small letters that differ denote significant differences assessed by a non-parametric Kruskal Wallis and post-hoc Tukey HSD comparison. Error bars are SEM (n = 8).......................................................................................................34 Figure 2.6   Mean final shell length (µm) of larval Haliotis kamtschatkana exposed to elevated CO2 levels (400 ppm [ambient] and 800 ppm) for 8 days. Data are for “normal” shells only; there were not enough normal shells to quantify size in the highest CO2 treatment. * indicates significant difference. Error bars are SEM (n = 8). ................................................................................................................................35 Figure 2.7   Mean percent settlement of Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Settlement calculated from total initial number of larvae (A) and surviving larvae (B). Percent settlement calculated from surviving larvae. No significant differences were observed across CO2 treatments in both A and B. Error bars are SEM (n = 8) ..........36 Figure 3.1   Mesocosm schematic outlining the self-filtration system of experimental aquaria. MFC = Mass Flow Controller.....................................................................51 Figure 3.2   Experimental design outlining CO2 manipulation. MFC = Mass Flow Controller ................................................................................................................52 Figure 3.3   Relationship between initial shell length (mm) and growth rate (mm d-1) of Haliotis kamtschatkana............................................................................................53  viii Figure 3.4   Mean monthly temperature (A and B), pH (C and D) and salinity (E and F) of experimental mesocosms during experiment (circles: 400 ppm CO2; diamonds: 800 ppm CO2; triangles: 1800 ppm CO2) at 9°C (A, C, and E) and 12°C (B, D, and F). Error bars are SEM (n = 4) ......................................................................................54 Figure 3.5   Effects of temperature and CO2 on abalone growth rates calculated from width (A), length (B) during course of experiment. Error bars are SEM (n = 4) .......55 Figure 3.6   Effects of temperature and CO2 on abalone growth rates calculated from area (A), wet weight (B) during course of experiment. Error bars are SEM (n = 4)..........56 Figure 3.7   Final dry weights [soft tissue (A) and shell (B)] of abalone exposed to various temperature (9 and 12°C) and CO2 concentrations (400 ppm, 800 ppm and 1800 ppm). Error bars are SEM (n = 4)....................................................................57 Figure 3.8   Effects of temperature and CO2 on feeding rates of abalone. Error bars are SEM (n = 4).............................................................................................................58 Figure 3.9   Effects of temperature and CO2 on survival of abalone. Error bars are SEM (n = 4) .....................................................................................................................59  ix Acknowledgements  First and foremost, I would like to thank my advisor Dr. Chris Harley for his support over the past two years. I am especially appreciative of his willingness to allow me to pursue research interests beyond the scope of my M.Sc. thesis. Chris’ passion and enthusiasm for marine science was inspiring and contagious and I am grateful to have had the opportunity to absorb his knowledge of Pacific Northwest marine life. I would also like to thank the members of my graduate research committee, Dr. Colin Brauner and Dr. Mike Hart for their intellectual contributions and continued support throughout the development of this project.  Many thanks to Gerald Singh, Jennifer Jorve, Rebecca Gooding, Rebecca Kordas and the rest of the Harley Lab group for their intellectual, technical and emotional support. I would also like to thank other members of the Abalone Research Team, Dr. Louis Gosselin and Dr. Elizabeth Boulding and graduate students Christine Hansen and Kaitlyn Read for their intellectual support.  I would also like to thank the numerous undergraduate volunteers who helped with laboratory work. This project would not have been done without the many technical contributions from John Richards at the Bamfield Huu-Ay-Aht Community Abalone Project (BHCAP) as well as the University of British Columbia shop staff Bruce Gillespie, Don Brandys and Vincent Grant. I would like to thank BHCAP for providing embryos with which experiments were carried out and Bamfield Marine Science Centre (BMSC) for providing research space and equipment. Many other individuals contributed their thoughts and / or assisted me in the laboratory and for such I am grateful to B. Love, R. Strathmann, R. Emlet, L. Gosselin, D. Levitan, K. Reuter, K. Lotterhos, A. Lambert, J.  x Maclean, R. Godfrey, C. Thompson, J. Nelson, S. Nyrose. A. O’Connell, R. Wynne, C. Clark.  Last and certainly not least I would like to acknowledge my parents who unwittingly planted the seed which led to my interest in marine biology by first introducing me to ocean life. My parents, to whom I am truly grateful for their endless love and support, have always pushed me to succeed and urged me to pursue my own interests and for that I am extremely thankful. Funding was provided by a UBC graduate student fellowship and Colville Tribal Education Assistance Program Higher Education Grant (to RC) and a Natural Sciences and Engineering Research Council Strategic Projects Grant and a Natural Sciences and Engineering Research Council Discovery Grant (to CH).                     1 Chapter 1 Introduction  1.1 CLIMATE CHANGE AND CARBONATE CHEMISTRY   Since the industrial revolution, humans have emitted 488 billion metric tons of carbon dioxide into the atmosphere, mostly from the burning of fossil fuels, deforestation and cement production (Canadell et al. 2007). The rate at which CO2 is being emitted is also increasing at a rate of about 1.03% each year and in 2007, 8365 million tons of anthropogenic CO2 were emitted into the atmosphere (Boden et al. 2010). These emissions have resulted in the increase of average global atmospheric CO2 concentrations from 280 ppm (pre-industrial) to the current 390 ppm CO2. The Intergovernmental Panel on Climate Change (IPCC 2007) has projected atmospheric CO2 concentrations may reach levels as high as 540 to 970 ppm by the year 2100.  In the last several decades, this increase in atmospheric CO2 concentrations has received much attention with regards to climate change. Of primary concern is the role of CO2 in contributing to the greenhouse effect. Atmospheric CO2 in the troposphere reflects radiative energy from the earth, ultimately causing the planet to warm. Average global temperatures have already increased 0.76°C (as of 2005; IPCC 2007) and are expected to increase another 2.4 to 6.4°C (relative to levels observed between 1980 – 1999; SRES scenario A1F1, IPCC 2007) by the end of this century. The last two decades have seen average global surface warming of ca. 0.2°C decade-1. A warming earth has  2 severe consequences for the biosphere that are only beginning to be understood. However, much of the CO2 humans have emitted into the atmosphere in the last several centuries has not stayed there. Approximately 28% of anthropogenic atmospheric CO2 has dissolved into the oceans (Canadell et al. 2007) where it causes major shifts in surface ocean carbonate chemistry, a process termed “ocean acidification” (see Caldeira and Wickett 2003).  Ocean acidification is commonly referred to as “the other CO2 problem” in reference to global climate change, because both ocean acidification and global climate change primarily result from increasing anthropogenic atmospheric CO2. As CO2 dissolves in seawater, it reacts with H2O molecules according to the following equation:  CO2 + H2O  ↔  H2CO3  ↔  H+ + HCO3-  ↔  2H+ + CO32-  The reaction equilibrium is dependant on a number of physiochemical characteristics of the water including but not limited to temperature, salinity, and total alkalinity. Contemporary CO2-induced ocean acidification is driving a decrease in both the pH and carbonate ion concentration [CO32-] in the surface ocean (upper 200 m of water column). Since the industrial revolution, average global surface ocean pH has declined by 0.1 units; because the pH scale is logarithmic, this corresponds to ca. 30% increase in [H+] (Royal Society 2005). Therefore, humans have already substantially changed the chemistry of the world’s surface ocean. As atmospheric CO2 levels continue to rise, nearing 1000 ppm by the year 2100, average global surface ocean pH is expected to drop another 0.2 to 0.3 units (Feely et al. 2009). Within the next several centuries, surface  3 ocean pH may drop by as much as 0.77 units, to pH levels not observed in the ocean during the last 300 million years (Caldeira and Wickett 2003).  Carbonate chemistry in the open ocean is well studied and fairly easy to calculate with just a few measured environmental parameters (see Lee et al. 2006). Coastal oceans, however, are much more dynamic in terms of physiochemical fluxes due to terrestrial influences and high benthic biomass of coastal shelves. In these systems, carbonate chemistry is much harder to predict and the lack of data regarding carbonate chemistry in coastal marine ecosystems exacerbates this difficulty. Some recent investigations regarding coastal carbonate chemistry have revealed some dramatic surprises. Feely and colleagues (2008) found low pH seawater associated with upwelling at the ocean surface along the North Pacific coast of the US. These waters had pH values as low as 7.6 and were undersaturated with respect to aragonitic CaCO3. A long term, high-resolution data set of pH in a highly mixed low intertidal tide pool on Tatoosh Island off the coast of Washington State shows dramatic diurnal, daily, and annual fluxes of pH (Wootton et al. 2008). Within a day, pH typically fluctuated ±0.24 units, but was sometimes as extreme as ±0.6 units. Substantial variation between days and years showed pH fluxes as great as ca. 1.0 unit. Coastal ocean pH flux can be attributed to biological processes such as daytime photosynthesis and nighttime respiration and physical processes such as oceanic upwelling of high CO2, low pH seawater from the deep ocean, variable sunlight, temperature and alkalinity, as well as increasing atmospheric CO2 levels.     4 1.2     BIOLOGICAL IMPLICATIONS OF OCEAN ACIDIFICATION  The effects of ocean acidification on marine biota are only beginning to be understood. However, research in this area is progressing rapidly. Of primary concern is the potential for ocean acidification to impair the performance of marine calcifiers by disrupting biogenic calcification processes. However, ocean acidification may negatively affect marine organisms in many other ways (reviewed in Orr et al. 2005, Doney et al. 2008). Perhaps most prominent is the potential for decreasing pH to affect organismal physiology (Portner et al. 2004). Furthermore, decreasing pH has been shown to decrease the ability of the oceans to absorb low-frequency sound waves (Ilyina et al. 2010), which could impact marine mammal bioacoustics, and disrupt chemo-sensory signals in larval fish important in locating suitable adult habitat. More subtle effects of ocean acidification on marine organisms are steadily being discovered. The growing consensus is that ocean acidification will likely have profound effects on ocean ecosystems and these effects will likely manifest within this century (Guinotte and Fabry 2008, Doney et al. 2009, Kleypas and Yates 2009).  Changes in ocean pH will likely have severe consequences on marine organisms, but perhaps more important ecologically is the reduced availability of [CO32-]. Biogenic calcification relies on the availability of [CO32-] to produce calcium carbonate (CaCO3) structures. Calcium carbonate is currently supersaturated in most of the world’s surface ocean, thus favoring its precipitation. However, the degree to which it is supersaturated is decreasing and many calcifying organisms have been reported to respond negatively to less supersaturation of CaCO3 (Wood et al. 2008, Martin and Gattuso 2009, Nienhuis et al. 2010). Furthermore, not all CaCO3 is created equal. Biogenic calcification relies on  5 multiple polymorphs of CaCO3 with differing solubility products. The two most common polymorphs are low-magnesium calcite and aragonite with aragonite being twice as soluble as calcite. Another common polymorph is high magnesium calcite, which is 30X more soluble than low magnesium calcite. Other even more soluble polymorphs include vaterite, which is so unstable it is rarely used by organisms, and amorphous calcium carbonate (ACC). ACC is often used as a precursor for larval shell formation in echinoderms and mollusks (Weiss 2002).  An increasing number of studies are reporting negative effects of ocean acidification on calcification by a wide variety of marine organisms from microbes (coccolithophores: Riebesell et al. 2000, Langer et al. 2006, Feng et al. 2008, De Bodt 2010; foraminifera: Kuroyanagi et al. 2009) to corals (Reynaud et al. 2003), echinoderms (Dupont et al. 2010), and mollusks (Gazeau et al. 2007). The negative impacts observed and predicted by specific case studies have been corroborated by a comprehensive meta- analysis incorporating all studies using realistic near future ocean acidification scenarios finding overall negative effects of ocean acidification on calcification, growth, survival and reproductive processes across all taxa (Kroeker et al. accepted). In general, responses vary across major taxonomical groups. However, high levels of inter- and intra-specific variability of organismal responses to ocean acidification are emerging (Ries et al. 2009, Langer et al. 2006, Langer et al. 2009), making broad extrapolations from the species to the community level very difficult, even within major clades.     6 1.2.2     EFFECTS ON DIFFERENT LIFE HISTORY STAGES  Organismal responses to CO2 can vary dramatically with ontogeny. Many different life history processes may be affected in different ways by ocean acidification (i.e. fertilization, cell division, morphogenesis, metamorphosis, post-larval development; see Kurihara 2008). While a majority of CO2 perturbation studies investigate effects only at the adult level, earlier life history stages may be more vulnerable to ocean acidification. Fertilization dynamics of broadcast spawning marine invertebrates are likely to be affected by ocean acidification as CO2 decreases sperm motility (Havenhand 2008) which has been shown to decrease fertilization efficiency in several species (Havenhand 2008, Reuter et al. in press, Parker et al. 2009, Kurihara 2004). Furthermore, at high sperm concentrations, eggs become more at risk of polyspermy (more than one sperm fertilizing one egg resulting in egg death) (Desrosiers et al. 1996, Reuter et al. 2010). However, fertilization efficiency was unaffected in the oyster, Crassostrea gigas (Havenhand 2009) suggesting fertilization dynamics of some species may be fairly robust in the face of acidification. After fertilization, embryonic development and hatching success was unaffected in several crustacean species at ecologically relevant CO2 levels (Kurihara and Ishimatsu 2008, Mayor et al. 2007); however, crustaceans in general tend to be more tolerant of ocean acidification conditions (see Kroeker et al. in press). Marine invertebrate larval development may be particularly sensitive to ocean acidification stress. In the laboratory, near 100% mortality of the brittle star, Ophiothrix fragilis, was observed after exposure to minor pH decreases (Dupont et al. 2008). In other taxa, exposure to elevated CO2 has  7 resulted in decreased calcification (Comeau et al. 2010), abnormal shell morphology (Dupont et al. 2008, Kurihara et al. 2009), delayed development (Kurihara et al. 2009, Ellis et al. 2009, Talmage and Gobler 2009), and decreased growth (Talmage and Gobler 2009).  1.2.3     MULTIPLE CLIMATIC STRESSORS  Ocean acidification is not the only current environmental change the ocean is experiencing. Anthropogenic emissions of greenhouse gases are also increasing sea surface temperature as well as UV radiation. Disruptions in climate patterns are affecting ocean salinity, dissolved oxygen, storm frequency and intensity, oceanic circulation and upwelling (Harley et al. 2006, Narayan et al. 2010). Many of these potential physical stressors will simultaneously impact marine biota. Therefore understanding how these different environmental stressors will interact to affect marine organisms is crucial to understanding community level impacts of global climate change.  Predicting the consequences of multiple environmental stressors is difficult. Organismal responses to multiple stressors can either be additive, antagonistic or synergistic. A recent meta-analysis found that 77% of studies on multiple stressors resulted in either antagonistic or synergistic responses (Darling and Cote 2008) suggesting multiple stressors will likely result in non-predictive outcomes. It remains unclear how organisms and ecological communities will respond to ocean acidification combined with other environmental stressors. Many published studies are finding non- additive biological effects associated with ocean acidification plus another environmental stress (Reynaud et al. 2003, Russell et al. 2009).  8  1.3     ABALONE BIOLOGY  Abalone represent an ancient lineage of marine gastropod mollusks (Vetigastropoda, Haliotidae) originating most likely in the Indo-Pacific (Geiger 2000). The earliest fossil specimens of haliotids date back to the late cretaceous period, 99.6 to 65.5 million years ago (Geiger and Groves 1999). They are morphologically unique as the only gastropods (extant or extinct) with a highly flattened shell, a row of tremata, and a high translation of the spire (Geiger and Groves 1999). Abalone have a world-wide distribution of at least 56 species (Geiger 2000) in tropical and temperate coastal marine environments (see Figure 1.1). Abalone are absent or severely underrepresented along the east coast of N. and S. America and the west coast of central and S. America. They appear restricted to latitudes between 50°S and 50°N, with the exception of Haliotis kamtschatkana which can be found as high as 60°N.  H. kamtschatkana is a cold water abalone native to NE Pacific coastal waters. Its range extends from Alaska to Baja California with two distinct subspecies. H. kamtschatkana kamtschatkana comprises the more northern population and H. kamtschatkana assimilis comprises the southern population; all further mention of H. kamtschatkana in this manuscript will refer to the former. Low temperature appears to limit its northerly distribution (Paul and Paul 1981) and there is some evidence that increasing temperatures as a result of anthropogenic climate change are limiting its southern distribution (Rogers-Bennett 2007).  H. kamtschatkana has a typical biphasic life cycle (Figure 1.2) consisting of a short lecithotrophic larval stage (ca. 10 d) before metamorphosing into an adult. All  9 haliotids are broadcast spawners, releasing their gametes directly into the water column where fertilization occurs. Fertilized embryos develop for ca. 19 h, at 10 – 13°C, before hatching as non-calcified ciliated trochophore larvae (Caldwell 1981). Collin and Voltzow (1998) clearly demonstrated that calcification of H. kamtschatkana is initiated during this stage prior to larval torsion. Such knowledge is critical, and often overlooked, in understanding how ocean acidification affects early larval development. Mollusk larval calcification begins with an initial deposition of amorphous calcium carbonate followed by aragonite (Weiss et al. 2002). As the larval shell develops, the larva transforms into a veliger, comprised of two fully retractable ciliated velar lobes protruding from the opercular opening of the shell. As a larva approaches metamorphic competency, a muscular foot develops between the velum and the operculum, along with several other morphological changes (i.e. eyespots develop). Larval behavior changes as the larva descends to the benthos in search of suitable juvenile habitat identified by chemical cues from, most notably, crustose coralline algae (CCA) and specifically gamma-amino- butyric acid naturally occurring both on the surface of CCA (Morse et al. 1980, Morse and Morse 1984) and associated with benthic diatoms and conspecific adult mucus (Roberts 2001). Upon metamorphosis, the velar lobes are shed and juvenile calcification begins.  Following metamorphosis, juveniles live a cryptic life on rocky substrate under boulders and in crevices. They live at greater depths than adults and migrate to more shallow depths as they mature. Early juvenile abalone feed mainly on benthic diatoms and as they mature, they begin feeding on macroalgae (Paul et al. 1977, Won et al. 2010). Gonadal maturation occurs after 2 to 3 years and shells have developed to ca. 50 mm  10 length. Individual fecundity is size-dependent. A single large, mature female can release 2.3 million eggs in a single spawning event (Caldwell 1981) and individuals may live 20 (Paul and Paul 2000) to 50 years (Breen 1980). Spawning typically occurs in late spring as seawater warms with local populations aggregating to form large spawning clusters. Spawning individuals crawl to the highest possible points, on rocks and macroalgae and even stack on top of one-another (Stekoll and Shirley 1993). While most spawning occurs in late spring / early summer, some trickle spawning can occur throughout the summer.  1.4     HALIOTIS KAMTSCHATKANA POPULATION DECLINE  Like many other abalone species worldwide, Haliotis kamtschatkana has experienced dramatic population declines in recent decades. H. kamtschatkana was once abundant on Pacific Northwest shores and was an important cultural resource for many coastal indigenous populations (Jones 2000, Reese 2000) and also supported recreational fisheries as well as small commercial fisheries in Alaska and British Columbia (Sloan and Breen 1988). However, in the early 1990’s widespread population declines throughout the range of H. kamtschatkana forced governments to officially close all legal harvesting of this species. Abalone population abundances prior to the large-scale eradication of the sea otter (Enhydra lutris), a major predator of abalone (Watson 2000), are unknown. It is likely that decreased predation pressure associated with E. lutris eradication contributed to abalone population growth, effectively over-estimating the scale of the current abalone population decline.  Despite a full closure of the H. kamtschatkana fishery in both Canada and the US (with the exception of a small recreational skin diving fishery in Alaska), populations  11 have continued to decline (Rothaus et al. 2008). In 1999, the species was listed as “threatened” according to the Committee on the Status of Endangered Wildlife in Canada (COSEWIC 2009) and recently re-listed as “endangered” in 2009. H. kamtschatkana is listed as a “species of concern” by the National Marine Fisheries Service (NMFS) in the US and “endangered” by the International Union for Conservation of Nature and Natural Resources (McDougal et al. 2006). The primary reason for H. kamtschatkana population declines is illegal harvesting. Abalone poaching continues unabated because of its high economic value and the difficulty in enforcing regulations due to the remoteness of its habitat. An estimated 80 to 90% of illegal collection of H. kamtschatkana occurs undetected by authorities (COSEWIC 2009), despite harsh convictions of offenders. H. kamtschatkana is especially prized as a cold-water abalone with market values as high as $50 lb-1 CAN (pers. comm. J. Richards BHCAP). The threats to abalone populations are not restricted to poaching. Many current populations are believed to exist in densities too low to support successful recruitment (Rothaus et al. 2008). Because abalone are broadcast spawners, releasing gametes directly in the water column, fertilization is dependent on sufficiently high densities of adult spawners. When adult spawning densities drop below a certain threshold value, fertilization is inhibited, a condition known as the Allee effect (Allee et al. 1949). Berec et al. (2007) estimate a minimum H. kamtschatkana population density of 0.15 abalone m-2 is needed for successful reproduction, though aggregating behaviours during mating and very high individual egg production may mitigate any Allee effect in nature. Current population densities are already lower than this in many locations such as the San Juan  12 Islands (Rothaus et al. 2008), and in other survey sites, declining densities are quickly approaching this Allee threshold (COSEWIC 2009). Other threats to the recovery of H. kamtschatkana include E. lutris predation, habitat loss or degradation, and climate change (COSEWIC 2009). There is some evidence suggesting the southern distribution of H. kamtschatkana is already being restricted by increasing temperatures (Rogers-Bennett 2007).  1.5 RESEARCH OBJECTIVES  Here, I investigate the potential consequences of near future levels of ocean acidification on Haliotis kamtschatkana. One key aspect of this project is to compare different life history stages. Larval stages of marine invertebrates have long been thought to be more sensitive to environmental perturbations than their adult counterparts (Thorson 1950). Furthermore, larval calcification processes are expected to be more vulnerable to ocean acidification because of the high relative importance of more soluble polymorphs of calcium carbonate in larval shells and skeletons (ACC and aragonite; Weiss et al. 2002, Jardillier et al. 2008). Therefore, I expect that adult H. kamtschatkana will be more tolerant to elevated CO2 than larval H. kamtschatkana. A majority of ocean acidification research involves short-term laboratory experiments that aim to describe how organisms will respond to a future acidified ocean. However, to gain a more comprehensive understanding of the potential biological ramifications of ocean acidification, more long-term investigations need to be undertaken. In chapter 2, I accomplish this by assessing the impact of ocean acidification  13 on the entire larval duration of H. kamtschatkana, from the embryonic stage, four hours following fertilization, to metamorphic competency. I reared larvae under ambient (400 ppm) and elevated (800 and 1800 ppm) CO2 levels. I measured survival, settlement and shell size of all abalone and also characterized shell development (normal vs. abnormal). I expected survival, settlement, size and normal shell development to all be negatively affected by elevated CO2. Another key aspect of this project is to investigate the potential interaction between elevated CO2 and other climatic stressors (temperature). In nature, climate change and ocean acidification are both occurring for the same reason, high levels of anthropogenic CO2 emissions. However, both have the potential to affect biota in different ways. The difficulties involved with assessing biological impacts of multiple environmental stressors simultaneously limit our current understanding of the potential effects of ocean acidification. In chapter 3, I address this by rearing adult abalone under various CO2 and temperature treatments. This experiment was also carried out over a long time period (five months). I raised abalone under ambient (400 ppm) and elevated (800 and 1800 ppm) CO2 levels at both 9 and 12°C, in a full factorial design. Both of these temperatures are well within the natural temperature range experienced by H. kamtschatkana and are not considered stressful (Paul and Paul 1981). I measured growth (shell length, width, area, and wet weight) and feeding rates throughout the course of the experiment. I expected elevated CO2 to impair growth and feeding rates. I expected growth and feeding to increase at 12°C relative to 9°C. Lastly, I expected the effects of CO2 to be partially mitigated at higher temperature, due to increased organismal metabolic activity and decreased solubility of CO2 in seawater.  14 Figure 1.1 Worldwide distribution of abalones (Gastropoda: Haliotidae). Known distributions (thick orange lines), scarce populations (thin orange lines). Haliotis kamtschatkana comprises two subspecies: H. kamtschatkana kamtschatkana (black dots) and H. kamtschatkana assimilis (white dots). This thesis refers only to H. kamtschatkana kamtschatkana. Adapted from Geiger et al. 2000.  15 Figure 1.2 Biphasic life cycle of Haliotis kamtschatkana.          16 Chapter 2  Effects of Elevated CO2 on Growth and Survival of Larval Abalone (Haliotis kamtschatkana)   2.1      INTRODUCTION  Atmospheric CO2 levels are currently increasing at rates unprecedented in geological history (Sabine et al. 2004). Since the industrial revolution, atmospheric CO2 concentrations have increased from ca. 280 to 390 ppm. Much of the anthropogenic CO2 emitted into the atmosphere dissolves in the ocean where it reacts with H2O to form carbonic acid, which shifts the carbonate chemistry of the surface ocean and ultimately results in a decrease in both carbonate ion concentration [CO32-] and pH, a process termed “ocean acidification”. In the last several hundred years, the oceans have absorbed approximately 30% of anthropogenic CO2 which has corresponded to an average pH decrease of 0.1 units in the surface ocean (Caldeira and Wickett 2003, Orr et al. 2005). As atmospheric CO2 levels are expected to near 1000 ppm by the end of this century (IPCC 2007), pH may decrease another 0.5 units. Ecologically relevant CO2 perturbation experiments have revealed substantial adverse effects of ocean acidification for a wide range of marine organisms (reviewed in Orr et al. 2005, Fabry et al. 2008, and Doney et al. 2009). Calcifying organisms (those producing calcium carbonate shells or skeletons) are expected to be the most vulnerable  17 to ocean acidification due to increasing costs / decreasing rates of calcification and increasing rates of shell or skeletal dissolution as calcium carbonate saturation levels decrease (Fabry et al. 2008, Doney et al. 2009, Nienhuis et al. 2010). Early life history stages may be particularly sensitive to ocean acidification (Kurihara 2008), because these stages are often most sensitive to environmental conditions. Indeed, recent studies have observed negative effects of ocean acidification on many early developmental processes including fertilization, larval growth, larval duration, and settlement (see Kurihara, 2008; Dupont and Thorndyke 2009, Talmage and Gobler 2009, Reuter et al. 2010). Even minor changes in pH can fully impair larval survival in certain species (Dupont et al. 2008).  Impairment of survival, development, and growth at early life history stages may have particularly severe consequences for species where population persistence or recovery from disturbance hinge on larval recruitment. One such species is the northern abalone, Haliotis kamtschatkana (Class Gastropoda, Family Haliotidae), which is endemic to coastal NE Pacific waters. Like many abalone species worldwide, H. kamtschatkana has experienced severe population declines in recent decades. Annual surveys conducted along the British Columbia coast suggest many populations have declined by about 80% since 1978 (Hankewich and Lessard 2008). Population declines have persisted since the official closure of both the recreational and commercial fishery in Canada and the US (Rothaus et al. 2008), and a limited supply of recruiting larvae has been suggested as a potential reason for the lack of recovery (COSEWIC 2009). Owing to a scarcity of spawners and a lack of larval recruitment, current populations may not be viable in some areas (Rothaus et al. 2008). The pessimistic outlook for the species as a  18 whole has led to northern abalone being listed as endangered (McDougal et al. 2006, COSEWIC 2009). Ocean acidification is known to impair key life history stages and transitions, including larval development. To date, however, ocean acidification has not been investigated as a significant threat to northern abalone or any other endangered species. As calcifiers, abalone larvae may be sensitive to changing ocean chemistry. Abalone develop calcified shells at all post-embryonic stages of development, and secrete more soluble polymorphs of CaCO3 (amorphous CaCO3 and aragonite) during the larval stage (Weiss et al. 2002, Jardillier et al. 2008) suggesting H. kamtschatkana larvae may be especially vulnerable to the effects of ocean acidification. Here, I investigate the effects of CO2-induced acidification on survival, growth and settlement of larval abalone (Haliotis kamtschatkana). Using levels of CO2 expected for this species within the next century and beyond, I reared larvae in various CO2 treatments for the entire larval duration of ~eight days and investigated a response to CO2 in terms of shell growth rates, shell deformities, larval survival, and the proportion of larvae competent to metamorphose by day eight.  2.2      MATERIALS AND METHODS  2.2.1     ANIMAL COLLECTION AND REARING Fertilized embryos of Haliotis kamtschatkana were obtained from the Bamfield Huu-ay-aht Community Abalone Project (BHCAP) hatchery in June 2009. Mature adult abalone broodstock were collected by SCUBA at nearby Sanford Island in Barkley  19 Sound, British Columbia in April 2009 by BHCAP staff. Wild abalone broodstock were reared under ambient conditions until ready to spawn. Three males and three females were induced to spawn with H2O2 following methods outlined by Strathmann (1987). Gametes from all individuals were combined in a common container to initiate fertilization. This fertilization protocol is not ideal as it adds unnecessary within- treatment variance due to variation among half-sibling families in their response to the experimental treatments, making any observed treatment effect a conservative estimate. Embryos were transferred into experimental treatments within four hours of fertilization (eight cell stage).  Homogenous mixtures of dense cultures of fertilized embryos were inoculated into experimental culture containers. Initial larval density (1.52 ml-1) was estimated by counting the number of larvae of a known volume of four extra samples. Abalone larvae were reared in 400 mL plastic tri-pour beakers with nitex mesh bottoms (74 um) placed inside 600 mL glass beakers (n = 8 treatment-1). Beakers were then placed within Plexiglas environmental control chambers (4 beakers chamber-1). To maintain equilibrial aqueous pCO2, chambers were filled with appropriate CO2 gas and sealed shut (see figure 2.1). Each chamber was then placed inside a 12°C incubator. Larvae were reared until metamorphic competency (ca. 8 d) at three target pCO2 levels: 400 ppm (ambient control), 800 ppm and 1800 ppm CO2. CO2 levels were chosen to represent a level of atmospheric CO2 expected by the year 2100 (800 ppm; IPCC 2007, Feely et al. 2009) and near maximal levels expected by the year 2300 (Caldeira and Wickett 2003). Water changes were performed every 24 hours, and gas inside environmental control chambers was replaced every 12 hours.  20  To induce metamorphosis, larval cultures were inoculated with gamma- aminobutyric acid (GABA) at a final concentration of 1 uM on day eight. After 12 h for metamorphosis, all swimming larvae were gently filtered and rinsed into 20 mL of CaCO3 buffered formalin at a final concentration of 1%. Attached larvae were rinsed with 70% ethanol and subsequent freshwater to induce dislodgement from the culture container, then filtered and rinsed into 20 mL of fixing solution. All larvae were counted and photographed (Olympus CAMEDIA C-5050 ZOOM) under a compound inverted microscope. Both swimming and settled larvae counted on day eight were considered survivors, and percent survival was calculated in relation to the initial number of larvae for each culture. Shell structure was scored as either normal or abnormal; abnormal shells were either completely absent or highly deformed (jagged and unmeasureable; see figure 2.2c). Shell lengths were measured using ImageJ 1.42q, and only normal larval shells were measured. Percent settlement was calculated in two ways: as, ! #of larvaesettled #of survivinglarvae •100%  and,  ! #of larvaesettled #of initial larvae •100%    2.2.2     CARBONATE CHEMISTRY I manipulated CO2 in experimental seawater by mixing ambient air (400 ppm) and CO2 gas (3% CO2, balance air; PraxAir) with Smart-Trak® mass flow controllers (Sierra Instruments, Inc.) to desired CO2 concentrations. Actual CO2 gas concentrations were  21 verified with Qubit S151 CO2 Analyzer. Experimental gases were then bubbled into 20L polyethylene carboys filled with 0.5 µm filtered seawater and allowed to equilibrate for at least 24 h. Treatment and control water was allowed to adjust to incubation temperature for 12 hours within sealed glass jars (with minimal air) inside incubation chambers. I monitored pH of jars immediately before daily water changes with an Omega PHH-830 pH meter. To ensure no substantial change in pH occurred within experimental beakers, I also measured pH of each sample immediately after water changes. Salinity and temperature were monitored daily. Total alkalinity (AT) was measured via Gran titrations with an Accumet model 15 pH meter (Fisher Scientific). I then input pH, AT, salinity and temperature into CO2SYS software to calculate remaining carbonate chemistry parameters (Table 2.1): dissociation constants for carbonate [CO32-], bicarbonate [HCO3-] and dissolved carbon dioxide [CO2] determined by Mehrbach et al. (1973), refit by Dickson and Millero (1987) and KSO4 using Dickson (1990).  2.2.3     DATA ANALYSIS Two of the control beakers became contaminated with littorinid egg capsules (with developing embryos) and littorinid feces and were subsequently removed from analyses. Abalone survivorship was very low in both of these beakers. To assess differences in survivorship I used a one-way ANOVA with CO2 level as a fixed factor. Shell length was analyzed with a Students t-test, as only two CO2 levels were compared. Effects of CO2 on normal shell structure and larval settlement were analyzed with non-parametric Kruskal Wallis tests. I used Tukey HSD multiple comparisons tests to look at differences among treatments. Seawater pH was averaged over time for each replicate and analyzed for  22 differences among treatments with a one-way ANOVA and post-hoc Tukey HSD test. To analyze effects of CO2 on seawater carbonate chemistry (see Table 2.1), I used one-way ANOVAs and Tukey HSD tests. Carbonate chemistry data were log transformed when necessary to meet assumptions of normality and homoscedasticity. All analyses were done in JMP v8.0.2.  2.3 RESULTS  Bubbling seawater with CO2-enriched air resulted in pH changes of -0.22 and - 0.49 units at 800 and 1800 ppm, respectively. Daily average pH values were 8.30 (0.02 SD), 8.07 (0.01) and 7.81 (0.02) at 400, 800 and 1800 ppm CO2, respectively (Figure 2.3). Salinity (mean = 34.5), temperature (mean = 12.0) and AT (mean = 1973.13) showed very little fluctuation over time and there were no differences observed across treatments (P > 0.17 in all cases; table 2.1). Elevated CO2 negatively affected survival of larval abalone (ANOVA; F2,19 = 5.23,  P = 0.01; Figure 2.4). Larval mortality was 35% at control CO2 levels, and ca. 60% at both of the higher CO2 levels (800 and 1800 ppm CO2), with no difference in mortality between higher levels. Since initial larval densities were estimated by subsampling from a common pool, we reran the ANOVA multiple times after randomly varying the starting number of larvae in each beaker using the mean and variance of the four original counts. This analysis was robust to the error associated with estimated initial larval densities. Higher CO2 concentrations were associated with higher incidence of larval shell deformities (Kruskal Wallis; χ2 = 18.97, df = 2, P < 0.001; Figure 2.5). Almost all larvae  23 developed normal shells under control conditions (ca. 98%). At 800 ppm CO2, however, only ca. 60% of larvae developed normal shells. Almost all larvae (ca. 99%) reared at 1800 ppm CO2 either lacked a shell completely or developed highly abnormal shells. Larval shell size was also significantly reduced by ca. 5% at 800 ppm CO2 (t-test; t = 4.4099, df = 10, P = 0.0006; Figure 2.6). Larval size at 1800 ppm CO2 could not be assessed because sample sizes of non-deformed larvae were too small. Of those larvae that survived to the end of the experiment, there were no among- treatment differences in the proportion that settled by day 8 (ANOVA: F2,21 = 1.18, p = 0.33; Figure 2.7b). This trend was similar when calculated as a function of initial larval density (Kruskal Wallis; χ2 = 1.999, df = 2, P = 0.37; Figure 2.7a). However, because the settlement period had just begun, percentage settlement of the survivors was relatively low (mean percentage ± S.E.M; 27.0 ± 6.0, 34.3 ± 4.6, and 26.4 ± 1.5 for 400, 800, and 1800 ppm, respectively). At higher CO2 levels, larvae were able to settle with or without a shell.  2.4      DISCUSSION  The predicted ecological consequences of ocean acidification are widespread and dramatic (Fabry et al. 2008, Doney et al. 2009). Although changes in biodiversity, including extinctions, are expected as a general consequence of ocean acidification, this threat is rarely considered explicitly in the context of species conservation. Here, we provide the first direct experimental evidence that ocean acidification will negatively impact an endangered species (the northern abalone Haliotis kamtschatkana). Our results  24 suggest that H. kamtschatkana early development will be substantially impacted by ocean acidification via impaired shell development and increased larval mortality. Because H. kamtschatkana is presently thought to be limited by reproductive output and recruitment (Fisheries and Oceans Canada 2007, Rothaus et al. 2008), these effects are likely to scale up to the population level, creating greater limits on population growth. A negative relationship between larval survivorship and CO2 concentration have been observed for other mollusks. Ellis et al. (2009) observed a ca. 20% decrease in survival to metamorphosis in the direct developing gastropod Littorina obtusata exposed to pH decreases of 0.5 units. For bivalves, Talmage and Gobler (2009) found survival decreased substantially in the clam, Mercenaria mercenaria (-75%) and scallop, Argopecten irradians (-90%) but not in the oyster Crassostrea virginica when exposed to pH decreases of 0.5 to 0.6 units. However, Watson et al. (2009) observed high levels of mortality (43% and 72%) in the oyster Saccostrea glomerata exposed to pH decreases of 0.3 and 0.5, respectively. In our experiments, comparatively moderate reductions in pH (0.22 and 0.49 units) both resulted in a decrease in larval survival of 43% relative to controls. In addition to simple survivorship, calcification and development of the abalone larval shell appear to be particularly sensitive to changes in carbonate chemistry. At 800 ppm, those shells that developed without deformities were smaller than the controls, as seen in other mulluscan larvae given elevated CO2 conditions (Kurihara, et al. 2007, Kurihara 2008, Ellis et al. 2009, Talmage and Gobler 2009, Watson et al. 2009). Abnormal shell development of larvae exposed to elevated CO2 levels appears to be a common response among mollusks; however, we show that shell abnormalities in H.  25 kamtschatkana larvae can manifest with relatively minor increases in CO2 (40% of larvae developed abnormal shells at 800 ppm CO2). At 1800 ppm, virtually all larvae developed abnormal shells or lacked a shell completely. Similar results have been observed for bivalves where 91% of Crassostrea virginica larvae (Kurihara et al. 2007) and 99% of Mytilus galloprovincialis larvae (Kurihara et al. 2009) developed abnormal shells after exposure to ca. 2000 ppm CO2 for 48 hours post fertilization. Despite the complete or near-absence of a shell, malformed abalone larvae survived relatively well in our experiments (compare survival in the 1800 and 800 ppm CO2 treatments). In nature, however, naked and malformed larvae would likely be more susceptible to planktonic predation (Hickman 1999, 2001). In contrast to the effect on shell development, CO2 did not detectably affect settlement competency of the H. kamtschatkana larvae that survived to the end of the experiment. This is contrary to what has been observed in many other larvae (Kurihara 2008, Talmage and Gobler 2009), and may be explained by the developmental life history of this species. Dupont and Thorndyke (2009) hypothesized that developmental rates are slowed under elevated CO2 in some species due to changes in larval feeding rates, which should result in settlement-at-time differences in planktotrophic species but not in lecithotrophic (non-feeding) species such as H. kamtschatkana (also see Dupont et al. 2010). Although CO2 effects were not permitted to manifest over the full duration of the settlement period in our experiment, settlement of the first ca. 25% of larvae as measured here supports the hypothesis that CO2 effects on development rate in other species are manifest through feeding. Furthermore, almost all of the larvae that settled and metamorphosed at 1800 ppm developed abnormal shells or lacked a detectable shell  26 entirely, which would presumably make them highly susceptible to predators in the wild (Gosselin and Qian 1997).  Natural populations of H. kamtschatkana may experience periodic exposure to elevated CO2 levels near to those used in our study well before the end of this century. Coastal carbonate chemistry dynamics are quite variable and in NE Pacific waters are strongly influenced by upwelling (Feely et al. 2008) and seasonal and diurnal variations in photosynthesis and respiration (see Wootton et al. 2008). H. kamtschatkana spawns in late spring to early summer, during the peak of the upwelling period. The greater mortality rates found here at 800 ppm CO2 suggest that larvae are not predisposed to tolerating these elevated levels, and abalone larvae may suffer greater mortality in years when strong upwelling brings corrosive water to the surface. Increasing intensities of upwelling associated with anthropogenic climate change may enhance the delivery of CO2-enriched seawater to nearshore surface waters (Narayan et al. 2010) and further exacerbate effects on larval survival and shell morphology.  Low population densities and associated recruitment limitation are only one of many impediments to the successful recovery of H. kamtschatkana populations; other threats include illegal harvest, sea otter predation, climatic warming (particularly in their southern range; Rogers-Bennett 2007) and disease (McDougall et al. 2006). At the latitudinal scale, northern abalone may be caught between different aspects of anthropogenic climate change at opposite ends of their range. Abalone living near their northern range limits are already being exposed to seawater of relatively low pH (Byrne et al. 2010), which is expected to become undersaturated with respect to aragonite within this century (Feely et al. 2009). In the southern portion of the range, there is evidence  27 suggesting that increasing ocean temperatures are resulting in H. kamtschatkana population declines (Rogers-Bennett 2007). If the range limit of this species collapses from both directions, the overall impact of climate change may be greater than would be predicted from acidification or warming alone.  Ocean acidification is likely to affect H. kamtschatkana populations in other ways that we did not measure. By inoculating fertilized embryos into experimental treatments, we bypassed any effect of high CO2 / low pH on fertilization (see Reuter et al., 2010) and early cell division (Desrosiers et al. 1996, Kurihara and Shirayama 2004). It is also possible that high CO2 negatively affected the structural integrity of the shell, increasing vulnerability of larvae to predation. Since seawater pH can disrupt chemosensory signaling (Munday 2009), the ability of H. kamtschatkana larvae to detect proper settlement locations may be impaired by ocean acidification. The effects of ocean acidification on larvae may persist into late juvenile and adult stages, and such carry-over effects may manifest even if later developmental stages are more tolerant of ocean acidification stress (Harris et al. 1999; Pechenik 2006).  2.4.1     CONCLUSIONS  We found survival, shell structure, and shell size of Haliotis kamtschatkana larvae to be negatively impacted by future CO2 concentrations. In the near future, H. kamtschatkana larvae may develop at the same rate as present day, but shell size and structure, as well as survival in the plankton, may ultimately lead to lower recruitment in this reproductively limited endangered species. As wild populations of H. kamtschatkana  28 continue to decline, it is important to recognize changing ocean chemistry as an emerging threat to this endangered species. Ocean acidification has been identified as a key challenge for global biodiversity conservation efforts (Sutherland et al. 2009). For species that have already suffered substantial population declines due to other factors, additional changes in seawater pH and carbonate chemistry may be particularly deleterious. The CO2-mediated decline in larval survival that we document here suggests that northern abalone, which are already endangered, will become progressively less likely to recover from their current low population size as ocean acidification progresses. Because it is unlikely that ocean acidification will be reversed before potentially detrimental conditions are reached, conservation of species like the northern abalone will depend upon maintenance of genetic variation, and mitigation of other factors that impact population sizes, such as harvest rates, habitat loss, invasive species encroachment, and other pollutants.  29 Table 2.1     Carbonate chemistry of experimental seawater during the course of the experiment. pH error refers to deviation among replicates. Temperature, salinity and alkalinity error refers to deviation over time. Data are presented as means (± SD). Significance levels calculated using one-way ANOVAs, small letters denote significant differences based on post-hoc Tukey HSD comparisons. *calculated with CO2SYS®                  Parameter 400 ppm (ambient) 800 ppm 1800 ppm P Temperature (°C) 12.0 (0.10) Salinity 34.42 (0.19) 34.57 (0.19) 34.62 (0.16) 0.1712 pHNBS 8.30 (0.02)a 8.07 (0.01)b 7.81 (0.02)c <0.0001 AT (µmol kg-1 SW) 1978.08 (56.04) 1969.64 (60.00) 1971.67 (44.44) 0.9777 pCO2 (µatm)* 224.0 (12.9)a 404.4 (22.54)b 795.3 (52.75)c <0.0001 HCO31- (µmol kg-1 SW)* 1560.0 (15.91)a 1695.1 (11.77)b 1812.1 (9.06)c <0.0001 CO3 (µmol kg-1 SW)* 163.1 (6.21)a 107.1 (4.58)b 62.3 (3.53)c <0.0001 CO2 (µmol kg-1 SW)* 9.2 (0.53)a 16.6 (0.93)b 32.7 (2.17)c <0.0001 ΩCalcite* 3.90 (0.15)a 2.56 (0.11)b 1.49 (0.08)c <0.0001 ΩAragonite* 2.49 (0.09)a 1.63 (0.07)b 0.95 (0.05)c <0.0001  30  Figure 2.1 Schematic of experimental design outlining CO2 manipulation setup. MFC = Mass Flow Controller.  31      Figure 2.2 Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Scale bar = 100 µm.                               32   Figure 2.3 Daily mean pHNBS during the experiment (circles: 400 ppm CO2; diamonds: 800 ppm CO2; triangles: 1800 ppm CO2). Error bars are SD (n = 8).  33  Figure 2.4 Mean percent survival of Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Small letters that differ denote significant differences assessed by one-way ANOVA and post-hoc Tukey HSD comparison. Error bars are SEM (n = 8). a b b  34 Figure 2.5 Mean percent of Haliotis kamtschatkana larvae that developed normal shell morphology after exposure to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Small letters that differ denote significant differences assessed by a non-parametric Kruskal Wallis and post-hoc Tukey HSD comparison. Error bars are SEM (n = 8). a c b  35 Figure 2.6 Mean final shell length (µm) of larval Haliotis kamtschatkana exposed to elevated CO2 levels (400 ppm [ambient] and 800 ppm) for 8 days. Data are for “normal” shells only; there were not enough normal shells to quantify size in the highest CO2 treatment. * indicates significant difference. Error bars are SEM (n = 8). *  36 Figure 2.7 Mean percent settlement of Haliotis kamtschatkana larvae exposed to elevated CO2 levels (400 ppm [ambient], 800 ppm and 1800 ppm) for 8 days. Settlement calculated from total initial number of larvae (A) and surviving larvae (B). Percent settlement calculated from surviving larvae. No significant differences were observed across CO2 treatments in both A and B. Error bars are SEM (n = 8).  37 Chapter 3  Effects of Elevated CO2 and Temperature on Growth and Feeding of Adult Abalone (Haliotis kamtschatkana)   3.1  INTRODUCTION  Since the industrial revolution, humans have emitted 488 billion metric tons of carbon dioxide (CO2) into the atmosphere (Canadell et al. 2007). However, only about 43% of these emissions have remained in the atmosphere; terrestrial sinks have absorbed about 29% with the remaining 28% being taken up by the oceans (Canadell et al. 2007). The absorption of 142 billion metric tons of CO2 (on top of natural sources of atmospheric CO2) in the ocean has initiated changes in the carbonate chemistry of the surface ocean long thought impossible. As CO2 dissolves in the surface ocean, it quickly goes through a series of chemical reactions ultimately resulting in an increase in hydrogen ions [H+] and decrease in carbonate ions [CO22-]. These simultaneous changes are collectively referred to as “ocean acidification” (Caldeira and Wickett 2003). Average global surface ocean pH has already dropped by 0.1 units reflecting a 30% increase in H+ (Royal Society 2005). As the surface ocean continues to take up more anthropogenic CO2, ocean pH is expected to drop by 0.2 to 0.4 units by the year 2100 (Feely et al. 2009) and by as much as 0.77 units within the next several centuries  38 (Caldeira and Wickett 2003). However, much less is known about coastal carbonate chemistry dynamics, which are much more variable (Wootton et al. 2008) and highly influenced by terrestrial inputs (Borges and Gypens 2010, Feely et al. in press). The biological implications of ocean acidification are complex and only beginning to be understood (see Doney et al. 2008). While calcifying organisms may be more severely affected due to their requirement for readily available carbonate ions (Kroeker et al. In press), all organisms may be susceptible to physiological stress associated with changing pH (Portner et al. 2004). Additional effects of ocean acidification may emerge due to disruptions associated with ecological interactions (Wootton et al. 2008). Furthermore, there may be substantial inter- and intra-specific variability in response to ocean acidification (Langer et al. 2006, Langer et al. 2009, Ries et al. 2009, Kroeker et al. In press). To better understand the effects of ocean acidification, it is crucial that CO2 perturbation studies consider a wide variety of species with an emphasis on ecologically and economically important species rather than on model organisms. Also, since multiple climatic changes are happening simultaneously (i.e. temperature, pH, UV radiation, hypoxia, etc.) and recent research is revealing significant interactions among these variables on biological processes (Gooding et al. 2009, Parker et al. 2009, O’Donnell et al. 2009), it is important to consider how other climatic variables will interact with CO2 to get a more comprehensive view of the biological consequences of ocean acidification. Haliotis kamtschatkana (Gastropoda: Haliotidae) is a high-latitude abalone endemic to coastal NE Pacific waters. Once historically important as a cultural resource and harvested recreationally and commercially (in Alaska), this species is now  39 endangered (COSEWIC, IUCN). Populations have declined precipitously in recent decades primarily due to overharvesting, despite complete fishery closures in the early 1990’s in the US and Canada (Rothaus et al. 2008). Many overharvested populations are now believed to be at densities too low to support successful reproduction in this broadcast spawner (Rothaus et al. 2008). As H. kamtschatkana populations continue to decline it is critical to determine how anthropogenic induced ocean acidification will affect its biology and potential for recovery. In this study, I exposed adult abalone to elevated CO2 (800 ppm and 1800 ppm) and temperatures (9 and 12ºC) and measured growth and feeding rates over the course of five months. I expected growth rates to be negatively affected by CO2. Both temperatures are well within the normal range of temperatures for this species, so I expected growth would be positively influenced by temperature. I also predicted that increased metabolic activity at 12ºC might partially mitigate the effects of elevated CO2 on calcification. As CO2 has been observed to decrease metabolic activity, I expected feeding rates to decrease with increasing CO2. I also expected feeding rates would be greater at the higher temperature and a stronger effect of CO2 would be observed at the lower temperature.  3.2      MATERIALS AND METHODS  3.2.1     EXPERIMENTAL SETUP I obtained mature Haliotis kamtschatkana individuals (45 – 57.5 mm shell length) from the Bamfield Huu-ay-uht Community Abalone Project (BHCAP) hatchery in September 2008. Abalone were transferred to the University of British Columbia and  40 distributed among 24 experimental aquaria with re-circulating natural seawater. Each aquarium (246-l) was equipped with its own filtration system (sock filter - protein skimmer - biological filter - UV sterilizer), and chiller (for temperature regulation) (Figure 3.1). Abalone were fed bull kelp (Nereocystis leutkeana) ad libitum and allowed to acclimatize to experimental tanks at 9°C and a salinity of 32 psu for ca. three months. Once acclimatized, abalone were subjected to full factorial combinations of three CO2 levels (400 ppm, 800 ppm and 1800 ppm) and two temperatures (9°C and 12ºC) (n = 4) for five months (140 d) (see Figure 3.2). Tanks were assigned treatment levels in a stratified block design, so that each treatment was represented in different parts of the room (i.e. closest to door or windows). Five individually marked abalone were placed inside each tank within a clear plastic container with many small holes along the sides. Small pumps (Maxi-Jet 900) were placed inside each abalone container to ensure sufficient flow so that respiration was not impeded.  Abalone were fed Nereocystis leutkeana ad libitum for the entire duration of the experiment. As this kelp species exhibits annual growth cycles, dying back in winter months, I collected large quantities of drift N. leutkeana during the fall months of 2008. I subsequently dried the blades and stored them with silica beads until needed (within 4 months). Dried N. leutkeana was rehydrated in seawater before being fed to abalone. Feeding rates were measured during 3-day periods of each week for the entire duration of the experiment. Wet weight of rehydrated kelp was recorded before being fed to abalone. Wet weight of remaining kelp was again weighed 3 days later. The difference was recorded as the amount of kelp eaten during the 3-day period.  41  Shell length (SL), shell width (SW), and wet weight (WW) were measured each week during the course of the experiment. I measured SL and SW of each individual with calipers. WW of each individual was determined after dabbing each animal with a paper towel twice and weighing with a scale to the nearest 0.01 g. Shell shape was reasonably approximated as an ellipse, and shell area (SA) was therefore calculated as, SA = π • (SL/2) • (SW/2). Growth rates for each individual were then calculated based on SL, SW, SA, and WW as the slope of the size metric over time. Slopes were calculated in Microsoft Excel (v. 11.5.8). Only individuals with more than four discrete measurements were included in the analyses. The use of slopes helped overcome noise associated with measurement error at any particular sampling date. Upon terminating the experiment, abalone were dissected and dried (70°C) for 72 hours and dry weights of both tissue and shell were recorded.  3.2.2     CARBONATE CHEMISTRY I manipulated carbonate chemistry by mixing compressed CO2 (3% or 10% CO2, balance air; Praxair) with ambient air (air compressors: model). Precise CO2 levels were maintained by mass flow controllers (Sierra Instruments Inc.) and bubbled directly into experimental tanks at a flow rate of 800 ml min-1. I monitored seawater pH (YSI 556- MPS) and temperature (mercury thermometer) daily and salinity (refractometer) weekly.  3.2.3     DATA ANALYSIS Growth rates for each individual were averaged for each tank. Growth and feeding rates were analyzed as a 2 X 3 full factorial two-way ANOVA with CO2 and  42 temperature as fixed factors and tank location as a block. One tank (treatment: 9ºC / 800 ppm CO2) was removed from all analyses because pH levels were not different from the control and we assumed that seawater pCO2 had not equilibrated. No significant effect of block or initial abalone size (see figure 3.3) was observed in any analysis (P > 0.25) and thus was removed and the analyses were rerun. Final dry weights (tissue and shell) were analyzed using a MANOVA with temperature and CO2 as fixed factors. Temperature, pH and salinity were analyzed with two-way ANOVAs with temperature and CO2 and fixed factors. I used post-hoc Tukey Honestly Significant Differences (HSD) multiple comparisons tests to assess differences in pH across CO2 treatments. All analyses were conducted in JMP8.0.2.  3.3 RESULTS  The bubbling of seawater with CO2 resulted in a pH changes of -0.03 at 800 ppm and -0.14 at 1800 ppm CO2 at 9°C (see table 1; Figure 3.4). At 12°C, pH changes of -0.04 at 780 ppm and -0.12 at 1800 ppm CO2 were obtained. Both temperature and salinity (ca. 32.5 psu) were stable throughout the duration of the experiment (Table 3.1). Growth rates were highly variable both within and among tanks. Growth was unaffected by CO2, while temperature had a positive effect (Figure 3.5, 3.6; table 3.2); however, this trend was only significant when growth was calculated from shell width and area (see table 3.2). Shell addition in the 12°C treatment outpaced shell addition in the 9°C treatment by 1.42 mm2 d-1. Furthermore, neither temperature nor CO2 had a significant effect on final dry weights (tissue or shell) (Figure 3.7). Temperature had a  43 positive effect on feeding rates but CO2 had no impact (see table 3.2; Figure 3.8). At 9°C, each abalone ate ca. 0.2 g kelp d-1. This increased to 0.55 g kelp d-1 at 12°C. Mortality was slightly higher at 12°C, but this difference was not significant (p = 0.72; Figure 3.9). A posteriori power analyses (G*Power) confirmed I had sufficient power to detect temperature effects (> 90%); however, power was relatively low to detect CO2 and temperature-CO2 interaction effects (< 80%). Although some individuals exhibited some substantial gonadal swelling, I did not detect any patterns among treatments. I also did not find any differences between males and females regarding growth rates or survival (data not shown).  3.4 DISCUSSION  Overall, I found abalone growth and survival to be robust to increases in CO2 and associated seawater pH. Growth rates, feeding rates and mortality were all unaffected by the pH levels used in this study, at both 9 and 12°C. Abalone at this stage of development appear to have a high tolerance for long-term exposure to decreased pH levels. This is surprising given the relatively high importance of aragonite in abalone calcification (Lin and Meyers 2004). Aragonite is more soluble than calcite and thus organisms relying on aragonite precipitation are expected to be more vulnerable to ocean acidification (Morse et al. 2007). However, it is important to note that the minimal pH change observed across CO2 treatments suggests that seawater pCO2 may not have been fully equilibrated to target levels; however, I was unable to measure the necessary carbonate chemistry parameters to verify this possibility.  44 Several other mollusks appear to be fairly tolerant of moderate changes in seawater pH. Harris et al. (1999) found abalone (H. rubra and H. laevigata) feeding rates and survival were unaffected by pH levels as low as 7.16. However, at lower pH levels, growth and survival were substantially impaired by low pH. Furthermore, O2 consumption wasn’t affected until pH 6.72 for H. laevigata, below which O2 consumption decreased. For the bivalves, Mytilus edulis and Crassostrea gigas, while increased CO2 tended to decrease calcification rates linearly with sudden short-term exposure to high CO2 (Gazeau et al. 2007), Berge et al. (2006) found minimal changes in growth rates of M. edulis during long-term (44 d) exposure to CO2-induced pH shift to pH 7.4. On the extreme end, the cephalopod Sepia officinalis maintained growth rates and increased calcification when exposed to CO2 levels as high as 4000 and 6000 ppm (Gutowska 2008), possibly due to its high metabolic rate (Melzner et al. 2009). However, there appears to be substantial variation in susceptibility to ocean acidification among mollusks (see Ries et al. 2009) as well as other taxa, with certain species being more vulnerable (i.e. Limacina helicina, Comeau et al. 2008). H. kamtschatkana calcification may be sufficiently slow that it is not the growth-limiting step; perhaps slow-growing organisms will be less impacted by ocean acidification than faster growing species. Biological responses to high CO2 are highly variable among species; however, the physiological mechanisms responsible for differences in susceptibility to ocean acidification are still in the process of being understood. Melzner et al. (2009) describe possible mechanisms for this variation that may be related to animal physiology as well as localized environmental conditions. H. kamtschatkana lives in shallow subtidal habitats along the NE Pacific coast that experience naturally large pH fluxes from  45 upwelling and biological processes diurnally and seasonally. Wootton et al. (2008) reported large seawater pH fluxes diurnally (0.24 units) as well as daily and seasonally in a highly mixed tidepool off the Washington coast. Furthermore, local upwelling events have the potential to dramatically reduce seawater pH in surface waters along the west coast of N. America (Feely et al. 2008). As a result, H. kamtschatkana may normally experience pH fluxes greater than levels I used in this study and therefore may have developed compensatory mechanisms for dealing with highly variable pH.  I observed a strong positive effect of temperature on feeding rates of abalone. This is opposite what Lloyd and Bates (2008) reported from similar hatchery-reared H. kamtschatkana from BHCAP, where feeding rates increased as temperature dropped from 12 to 9°C, no matter what density the animals were reared in. However, their result is confounded by time and may be due to changes in nutritional quality of fresh kelp (N. leutkeana) toward the end of its growing season (late fall / early winter). Despite the fact that kelp consumption more than doubled at 12°C, relative to 9°C, minimal increase in growth was observed, suggesting a decrease of feed conversion ratios. Paul and Paul (1981) also found minimal temperature effects on growth of similar sized H. kamtschatkana between 8.5 and 11.5°C and hypothesized that differences in gonadal maturation may be responsible (Paul et al. 1977). Although I did not detect any noticeable patterns in gonadal swelling among treatments in my experiment, a more quantitative assessment of gonadal development may have identified differences that could explain the discrepancy between the feeding rates and growth rates I observed.  Abalone growth rates in my experiment are similar to published values. Paul and Paul (1981) reared similar sized abalone under various temperatures observing 0.03 to  46 0.05% d-1 increase in length at 8.5°C and 0.05 to 0.13% d-1 increase at 11.5°C, compared to 0.03 to 0.04% d-1  at 9°C and 0.05 to 0.07% d-1 increase in length at 12°C in my experiments. Lloyd and Bates (2008) saw slightly higher growth rates (0.08 mm d-1 SL) and feeding rates (1.45 g kelp abalone-1) of abalone reared at low density (227 abalone m2) and fed ad libitum. The lower growth and feeding rates I observed might be due to feeding the abalone dried and rehydrated kelp, the processing of which likely resulted in decreased nutrient levels compared to fresh kelp (Vilchis et al. 2005).  While I was unable to detect direct effects of decreased pH on H. kamtschatkana growth, survival and feeding rates, it is possible that low pH will affect abalone in ways I did not measure. Higher, more stressful temperatures associated with El Nino events and global climate change may inhibit the ability of abalone to cope with ocean acidification stress (O’Donnell et al. 2009). Other environmental perturbations (hypoxia, salinity, diseases, upwelling intensity) may also interact with ocean acidification to negatively impact abalone populations. Ontogenetic shifts in vulnerability to ocean acidification may also manifest (Waldbusser et al. 2009). My own research suggests larval development of H. kamtschatkana may be more impacted by low pH (see chapter 2). Ocean acidification may also impact behavior (Munday et al. 2009), susceptibility to infectious disease (Bibby et al. 2008), reproductive output (Kurihara et al. 2008) as well as indirect effects associated with ecological interactions (Wootton et al. 2008). Adult Haliotis kamtschatkana appear to be able to tolerate long-term exposure to moderate decreases in seawater pH at both 9 and 12°C. However, to fully understand the potential impact of ocean acidification on abalone growth and development, more work should be done over larger pH and temperature scales. Abalone may be more sensitive to  47 larger pH fluxes, which could naturally result from short-term upwelling events. Such extreme short-term periodic climatic events may even be more important in shaping ecological communities (Harley and Paine 2009).   48 Table 3.1     Mean temperature (°C), salinity (psu) and pHNBS throughout the course of the experiment. Data are means (standard error). Small letters denote significant differences assessed with multiple comparisons Tukey HSD tests.   9°C 12°C  400 ppm 800 ppm 1800 ppm 400 ppm 800 ppm 1800 ppm Temperature 9.06 (0.16)a 8.88 (0.04)a 9.16 (0.04)a 12.27 (0.06)b 12.05 (0.10)b 11.95 (0.13)b Salinity 32.8 (0.17) 32.5 (0.12) 32.9 (0.27) 32.9 (0.54) 32.9 (0.24) 32.5 (0.46) pHNBS 7.71 (0.01)a 7.68 (0.01)b 7.57 (0.01)c 7.76 (0.01)a 7.70 (0.01)b 7.64 (0.01)c                   49 Table 3.2     Two-way ANOVA results of growth rates (calculated from length, width, area and wet weight), feeding rate and survival.  Source df SS F P      Length Temp 1 0.00040323 3.0832 0.0971 CO2 2 0.00032388 1.2382 0.3148 Temp X CO2 2 0.00010566 0.4039 0.6739  Width Temp 1 0.00084190 9.2831 0.0073 CO2 2 0.00007583 0.4180 0.6649 Temp X CO2 2 0.00005917 0.3262 0.7261  Area Temp 1 107.79663 10.0695 0.0056 CO2 2 17.215480 0.8041 0.4638 Temp X CO2 2 6.94243 0.3144 0.7344  Wet Weight Temp 1 6.7372893 2.4674 0.1347 CO2 2 1.0759676 0.5346 0.5954 Temp X CO2 2 0.4207202 0.2718 0.7652  Feeding Rate Temp 1 6.1961778 36.5445 <0.0001 CO2 2 0.3756039 2.1076 0.3530 Temp X CO2 2 0.1397027 0.4120 0.6688  Survival Temp 1 50.00000 0.1335 0.7193 CO2 2 224.24242 0.2994 0.7451 Temp X CO2 2 453.33333 0.6052 0.5573          50 Table 3.3     MANOVA results of final dry weights (tissue and shell).         *numerator df, denominator df †Wilks’ Lambda approximate F                   Source df* F P     Overall Model 10,32 2.7614† 0.0141  Intercept 2,16 188.529 <0.0001 Temp 2,16 0.0645 0.6063 CO2 4,32 1.9181† 0.1314 Temp X CO2 4,32 2.1396† 0.0987   51 Figure 3.1 Mesocosm schematic outlining the self-filtration system of experimental aquaria. MFC = Mass Flow Controller.  52 Figure 3.2 Experimental design outlining CO2 manipulation. MFC = Mass Flow Controller.           53  Figure 3.3 Relationship between initial shell length (mm) and growth rate (mm d-1) of Haliotis kamtschatkana.  54 A F E D C B  Figure 3.4 Mean monthly temperature (A and B), pH (C and D) and salinity (E and F) of experimental mesocosms during experiment (circles: 400 ppm CO2; diamonds: 800 ppm CO2; triangles: 1800 ppm CO2) at 9°C (A, C, and E) and 12°C (B, D, and F). Error bars are SEM (n = 4)  55 A) Width B) Length Figure 3.5 Effects of temperature and CO2 on abalone growth rates calculated from width (A), length (B) during course of experiment. Error bars are SEM (n = 4).  56 B) Wet Weight A) Area Figure 3.6 Effects of temperature and CO2 on abalone growth rates calculated from area (A), wet weight (B) during course of experiment. Error bars are SEM (n = 4).  57 B) Shell A) Tissue Figure 3.7 Final dry weights [soft tissue (A) and shell (B)] of abalone exposed to various temperature (9 and 12°C) and CO2 concentrations (400 ppm, 800 ppm and 1800 ppm). Error bars are SEM (n = 4).  58 Figure 3.8 Effects of temperature and CO2 on feeding rates of abalone. Error bars are SEM (n = 4).  59 Figure 3.9 Effects of temperature and CO2 on survival of abalone. Error bars are SEM.           60 Chapter 4 General Conclusions  4.1 SIGNIFICANCE OF OVERALL RESULTS  4.1.1 SUMMARY In Chapter 2, I show evidence that ocean acidification has the potential to dramatically affect early development of H. kamtschatkana. I conducted careful laboratory experiments over the entire duration (note that most larvae had not reached settlement competency) of larval development from embryo (prior to the initiation of calcification) to juvenile metamorphosis. I found elevated CO2 levels negatively affected survival and shell development. Relatively few studies on ocean acidification rear larvae for their entire larval duration and many inoculate larvae into CO2 treatments after calcification has already begun. Increased duration of exposure to elevated CO2 likely has a stronger effect on organismal responses and for echinoderms and mollusks, calcification is believed to be initiated by the secretion of extremely soluble amorphous calcium carbonate. As a result, many studies are likely underestimating the impact of ocean acidification on larval development.  In Chapter 3, I show that long-term exposure of mature H. kamtschatkana to minor decreases in seawater pH does not impede growth and feeding rates. While the physiological mechanism for this tolerance is unclear, these results show that it is possible for an organism to cope with variable carbonate chemistry dynamics, despite its  61 large reliance on more soluble polymorphs of CaCO3 for calcification and low metabolic rates (see Melzner et al. 2009). However, larger fluctuations of seawater pH may induce negative effects on abalone growth and metabolism.  4.1.2     VULNERABILITY TO OCEAN ACIDIFICATION AT DIFFERENT LIFE HISTORY STAGES My results suggest that larval development of Haliotis kamtschatkana may be more sensitive to ocean acidification than their adult counterparts. Elevated CO2 negatively impacted survival, growth and calcification of abalone larvae, while no discernable impacts of CO2 were apparent on adult abalone. However, CO2-induced pH shifts were much larger in the larval experiment (-0.5 units at 1800 ppm CO2) than the adult experiment (-0.15 at 1800 ppm CO2), which may explain the difference between the two studies. For a species with a healthy population, such an effect may not result in substantial population-level effects, due to high levels of natural density-dependant mortality at larval and early juvenile stages. However, for H. kamtschatkana, this may have severe population-level consequences because this species already exists in unnaturally low densities where population growth is likely inhibited by reproductive efforts (Rothaus et al. 2008). Ocean acidification will likely have especially severe consequences for OA-sensitive species existing in depressed densities [e.g. other abalone (H. cracherodii, H. sorenseni, and H. currugata) and certain corals (Acropora palmata and A. cervicornis); listed as threatened, endangered or “species of concern” by the National Marine Fisheries Service (USA)].  62 There are very few published accounts of the potential effects of ocean acidification on multiple life history stages of marine organisms. In contrast to H. kamtschatkana, McDonald et al. (2009) found later stages of the barnacle (Amphibalanus amphitrite) were more sensitive to ocean acidification than earlier life history stages. This discrepancy could be due to major physiological differences in calcification processes of crustaceans, which in general tend to be more resilient (as compared to echinoderms and mollusks) to ocean acidification stress (Kroeker et al. In press). Certain calcifying species (e.g. corals) produce non-calcifying larvae and effects of ocean acidification on such organisms may not manifest until later stages of development (Albright et al. 2008). My results suggest that adult H. kamtschatkana may be able to cope with near- future ocean acidification stress but larval stages may be more vulnerable. Carbonate chemistry dynamics within the natural geographic range of H. kamtschatkana are extremely variable and substantially affected by ocean upwelling (Feely et al. 2008), biological processes (Wootton et al. 2008), and terrestrial inputs (Feely et al. 2010). Organisms existing in areas of such dynamic CO2 and pH fluxes are hypothesized to have some pre-adaptive qualities allowing them to cope with variable CO2 and pH stress (Melzner et al. 2009). The reduced survival and calcification of abalone larvae in our experiments suggest they lack physiological pre-adaptive qualities allowing them to deal with ocean acidification stress, despite the fact that reproduction occurs during the annual peak of upwelling.     63 4.2 PRIORITIES FOR FUTURE RESEARCH   While I was able to detect effects of ocean acidification on several specific biological processes, there are many other processes that may also be directly and indirectly vulnerable to ocean acidification stress. To get a more comprehensive picture of how abalone populations will respond to future ocean acidification, it is necessary to evaluate the following potentially sensitive processes.  Because H. kamtschatkana is a broadcast spawner, releasing gametes directly into the water column where fertilization takes place, it is imperative to evaluate the potential effects of ocean acidification on fertilization dynamics. Reuter et al. (In press) observed that both sperm limitation and risk of polyspermy increased at near-future levels of ocean acidification for the red sea urchin, Strongylocentrotus franciscanus. This essentially means that higher densities of spawning adults are required to achieve maximum fertilization success. If abalone fertilization responds in the same way, established minimum threshold densities (Berec et al. 2007) would need to be increased to account for effects of near-future levels of ocean acidification. While I did not detect an effect of CO2 on larval metamorphosis and settlement, this needs to be evaluated with a more careful experiment determining the effects of CO2 on larval settlement over time. My experiment only accounted for the first individuals competent to settle. In nature, settlement would likely proceed for several days if not more. It is possible that an effect of CO2 could have manifested if settlement was allowed to proceed for several days. It is also possible that CO2 could disrupt chemosensory signaling (Munday et al. 2009, Dixson et al. 2009) important for larval abalone for  64 detecting proper habitats in which to metamorphose. Such an effect would severely inhibit successful recruitment of juveniles to appropriate habitats and therefore impede population growth. Because recently settled juveniles are subject to high levels of natural mortality (Gosselin and Qian 1997), assessing the effects of CO2 on early juvenile growth and development should be a research priority. It is also possible that elevated CO2 may inhibit adult abalone fecundity (see Kurihara et al. 2008). H. kamtschatkana as well as many other haliotids are vulnerable to diseases that have the potential to severely reduce populations. Many of these diseases become more infectious as temperature increases and certain outbreaks in Southern California have reduced populations of H. cracherodii and H. sorenseni enough to grant them status of “threatened” or “endangered” (NMFS). Since elevated CO2 has been shown to suppress immune response in another mollusk (Bibby et al. 2008), it is critical to understand how H. kamtschatkana vulnerability to infectious disease will be affected by ocean acidification. H. kamtschatkana populations may be affected by ocean acidification in other indirect ways. Elevated CO2 has been shown to affect growth and chemical compositions in the kelps, Saccharina latissima and Nereocystis luetkeana (Swanson and Fox 2007), both of which are consumed by H. kamtschatkana. Connell and Russell (2010) found that elevated CO2 has the potential to initiate dramatic community shifts in kelp forests. Kelps are important for abalone as both a food source and habitat; elevated CO2-induced changes in community structure and chemical compositions of kelp may affect H. kamtschatkana populations. Furthermore, how other physical environmental  65 perturbations will interact with elevated CO2 levels to impact marine biota is largely unknown. The combination of multiple environmental factors will likely affect all the aforementioned biological processes and potentially in non-predictive (non-additive) ways. Understanding how elevated CO2 will interact with other environmental perturbations will be critical to fully understand how H. kamtschatkana populations will respond to near-future ocean acidification. Lastly, the potential for marine organisms to adapt to ocean acidification stress is unknown. The current rapid rate of ocean acidification, as compared to acidification events in geological history, may impair the ability of certain species (e.g. those with low population turnover rates or low genetic diversity) to successfully adapt to a high CO2 / low pH ocean. Sunday et al. (in prep) attempted to answer this question for the sea urchin, Strongylocentrotus franciscanus and the mussel, Mytilus trossulus. We found both species had some potential for adapting to near-future levels ocean acidification; however both species differed in their adaptability, with urchins being more adaptable than mussels, due to greater phenotypic variation in sea urchins. More research in this field is urgently needed to more thoroughly assess adaptation potentials across a wide variety of marine organisms. H. kamtschatkana biology is characterized by very long generation times (ca. 10 years), and individuals require several years to reach sexual maturity (McDougall et al. 2006); therefore, H. kamtschatkana may be less able to adapt than other species with shorter generation times.     66 4.3 CONCLUSION   Overall, my results are the first to provide evidence that ocean acidification has the potential to dramatically impair the recovery of an endangered species (COSEWIC 2009, McDougal et al. 2006). If other haliotids respond similarly, this has major implications for other abalone populations along the Pacific Coast of North America and elsewhere, many of which (e.g. H. cracherodii, H. sorenseni, and H. corrugata) are also threatened or endangered due to overharvest, diseases, and climate change. 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