Biology lab experiment report ( due in 20 hours)

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nsf_project_description_2013_final.pdf

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RESULTS FROM PRIOR NSF SUPPORT

First time primary investigator, no prior NSF support.

PROJECT DESCRIPTION

RUI: OCEAN ACIDIFICATION: EFFECTS OF CONSTANT AND VARIABLE CO2-INDUCED OCEAN ACIDIFICATION ON FISH PHYSIOLOGY AND BEHAVIOR

STATEMENT OF THE PROBLEM

Recent investigation of ocean acidification (OA) impacts on larval and juvenile fishes has indicated a wide range of sublethal effects, including commonly reported impacts on behavior (Dixson et al. 2010; Munday et al. 2010) and otolith formation (Checkley et al. 2009; Bignami et al. 2013a). Behavioral and otolith effects have both been linked to a basic physiological response of fishes to increased environmental pCO2: the sustained increase in blood bicarbonate ion concentration ([HCO3

-]; Checkley et al. 2009; Esbaugh et al. 2012; Nilsson et al. 2012). At this point in time, investigations of OA effects on fishes have begun to provide a foundation of understanding that will help guide future research. However, nearly all studies have relied upon constant acidification treatments throughout the duration of experimental exposure. This does not accurately simulate naturally variable pCO2 that many species experience on a diurnal, episodic, and seasonal timescale (Hofmann et al. 2011; Frieder et al. 2012). The biological impact of naturally variable OA could be greatly different than what is now understood based upon studies using constant OA (Shaw et al. 2013). For example, OA-induced changes in blood chemistry may undergo similar diurnal cycles in response to variable pCO2, possibly reducing or eliminating effects on fish behavior or otolith growth. The characteristics of such a physiological response to variable pCO2 have not been described in the literature and could greatly affect the current understanding of OA impacts on fishes.

The overarching goal of this proposed research is to characterize the physiological response of fishes to variable-pCO2 OA and determine if such variability mitigates or eliminates the previously observed behavioral and otolith growth impacts of OA. This proposed research will be transformative to the field of OA research on fishes: it will address one of the most important knowledge gaps facing the field and produce data that could fundamentally change the current understanding of OA effects on fishes. The results of this research will guide the direction for future OA research, improve the ability to understand OA impacts on fishes in general, and ultimately influence future strategies to mitigate the impacts of OA on fish populations. Additionally, a description of the physiological response of fishes to variable pCO2 will provide the broader scientific community with information that is relevant to research on the physiology, biology, or ecology of fishes and their communities in present-day variable-pCO2 habitats.

BACKGROUND

Early literature on the effects of elevated pCO2 on marine fishes reported few impacts on adults and juveniles until treatments reached extreme levels (often 50,000 µatm pCO2 or higher; reviewed by (Ishimatsu et al. 2008). Comparatively, anthropogenic OA is projected to reach nearly 1000 µatm pCO2 by the year 2100 according to the Representative Concentration Pathway 8.5 model (RCP 8.5), corresponding to a 0.3 unit decline in average ocean pH (IPCC 2013). Concern over the susceptibility of the early life stages of fishes to OA has led to many recent studies that have re-focused attention on this taxonomic group under projected anthropogenic OA conditions. To date, these studies have reported mostly sublethal effects including changes in metabolism (Miller et al. 2012), growth, survival (Baumann et al. 2011), reproductive output (Miller et al. 2013), neural receptor function (Nilsson et al. 2012; Hamilton et al. 2013), otolith formation (Checkley et al. 2009; Bignami et al. 2013a), and behavioral characteristics (Dixson et al. 2010; Munday et al. 2010; Hamilton et al. 2013, among others). These

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effects are not always negative and typically vary among species and regions, making it difficult to draw general conclusions about the potential impact that future OA will have on fishes.

Two consistently reported impacts of OA on fishes are altered otolith formation and behavioral changes. Otoliths are paired, calcium carbonate, inner ear stones which contribute to auditory and vestibular sensation in teleost fishes. Calcium carbonate is passively deposited in annual and daily increments, with widths that are proportional to fish somatic growth, therefore they are often used as a proxy for age and growth by fisheries biologists and ecologists (Campana 2005). Enhanced otolith growth has been commonly observed under OA conditions of 800 µatm pCO2 and above (Checkley et al. 2009; Munday et al. 2011b; Bignami et al. 2013a; 2013b, among others), although a lack of effect on otolith growth (Munday et al. 2011a; Frommel et al. 2013) and one report of reduced otolith growth (Maneja 2012), have been reported over a range of pCO2 treatments (600 to 3200 µatm). Changes in otolith formation have implications for the sensory function of otoliths (Bignami et al. 2013a) as well as the interpretation of otolith growth history data, due to a decoupling of the relationship between otolith increment width and somatic growth (Bignami et al. 2013b). Both implications are relevant under future OA conditions or in present day acidified environments such as fjords (Thomsen et al. 2010) or upwelling zones (Feely et al. 2008).

OA-induced behavioral effects on fishes range from disruption of olfactory homing ability (Munday et al. 2009), auditory behavior (Simpson et al. 2011), and behavioral lateralization (Domenici et al. 2012), to altered learning ability, risk assessment (Ferrari et al. 2012a; 2012b), and increased anxiety (Hamilton et al. 2013). It has also been demonstrated that the altered behavior of juvenile fishes raised under OA conditions can produce up to 9-fold higher predation mortality (Munday et al. 2010) and alter fish habitat preference (Devine & Munday 2012) in the wild. Under future OA conditions, such effects could ultimately influence important ecological processes such as recruitment of juvenile fishes, post- settlement ecological interactions, and population dynamics. The occurrence of behavioral effects in multiple study species, across trophic levels (Dixson et al. 2010; Munday et al. 2012), and between geographic regions (i.e., temperate Forsgren et al. 2013; Hamilton et al. 2013, as well as tropical) implies that these effects may be a pervasive impact of OA on fishes throughout the ocean.

It has been proposed that the otolith and behavioral effects of OA have a common driving mechanism, tied to the basic physiological response of fishes to elevated environmental pCO2. When exposed to elevated pCO2, teleost fishes experience a corresponding decline in blood pH, which is compensated for via the retention and active uptake of bicarbonate ions (Claiborne & Heisler 1986; Esbaugh et al. 2012). Full compensation of blood pH is achieved in as little as 2 to 4 hrs (Esbaugh et al. 2012) and is maintained for the duration of elevated pCO2 exposure (Claiborne & Heisler 1986; Michaelidis et al. 2007; Esbaugh et al. 2012). Increased extracellular bicarbonate ion concentration ([HCO3

-]) likely produces an increased aragonite saturation state in the endolymph fluid that surrounds otoliths, therefore contributing to accelerated calcification (Checkley et al. 2009; Bignami et al. 2013a). Additionally, as HCO3

- is retained in the blood, negatively charged chloride ions (Cl-) are excreted from the blood to balance electric charge (Claiborne & Heisler 1986). However, this process also alters the electrochemical gradient across neuron cell membranes. In adult vertebrates, GABAA receptors are the main neuronal inhibitory mechanism, which hyperpolarize the neuron by allowing the flow of Cl- from extracellular to intracellular space (Bormann et al. 1987; Hamilton et al. 2013). Nilsson et al. (2012) suggest that under OA conditions, decreased extracellular [Cl-] results in a reversal of Cl- conductance (i.e. outflow from the neuron), and thus an excitatory depolarization of the neuron when GABAA is activated. Data supporting this mechanism was provided when administration of a GABAA receptor antagonist (gabazine) was shown to reverse OA-induced behavioral effects by preventing the activation of GABAA receptors (Nilsson et al. 2012). More recently, Hamilton et al. (2013) verified this mechanism with the use of both a GABAA agonist (muscimol) and a GABAA

antagonist (gabazine) in splitnose rockfish (Sebastes diplopora). Consistent with a reversed gradient for Cl- ions in OA exposed rockfish, Hamilton et al. (2013) observed an increase in OA-treatment fish anxiety that was potentiated by

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muscimol, whereas muscimol administration produced a decrease in anxiety in control rockfish, consistent with the proposed OA-induced changes in electrochemical gradient (Fig. 1).

The behavioral effects of OA can be induced via exposure to OA for a minimum period of 2 to 4 d (Munday et al. 2010; Nilsson et al. 2012), which implies that a sustained period of high [HCO3

-]/low [Cl-] may be required to elicit an effect (given the ability of fishes to acclimatize their blood chemistry within hours; Esbaugh et al. 2012). To date, exposure to constant OA treatments has been the status quo in all OA-related behavioral studies of fishes and in all physiological studies of fish blood chemistry response to elevated pCO2. However, constant ocean pH/pCO2 is not an accurate assumption for the natural environments in which most study species live (McElhany & Shallin Busch 2012; Shaw et al. 2013), such as coral reefs, seagrass meadows, and kelp forests. These habitats can experience large diurnal pH fluctuations of up to 0.7 units (Ohde & van Woesik 1999; Perez-Dominguez et al. 2006; Hofmann et al. 2011; Frieder et al. 2012). In particular, kelp forests off southern California have been shown to exhibit diurnal pH variability of up to 0.36 units, with an average of 0.11 units, largely driven by differences in nocturnal respiration (high pCO2) and daytime photosynthesis (low pCO2; Frieder et al. 2012). Put into context with projected future OA, a pH change of 0.30 units is equivalent to the total magnitude of change in average ocean pH projected to occur by year 2100 under RCP 8.5 (936 µatm atmospheric pCO2; IPCC 2013). Therefore, a diurnal swing of 0.30 pH units (equivalent to approximately 600 µatm pCO2) in kelp forest ecosystems could drive pCO2 levels 300 µatm above and below projected atmospheric values. For example, under RCP 6.0 projected conditions for the year 2100 (670 µatm pCO2; IPCC 2013), organisms in a kelp forest could experience pCO2 values ranging from 370 µatm during the day to 970 µatm at night. Likewise, if conditions meet the RCP 8.5 projections of 936 µatm pCO2 (IPCC 2013), values could range from 636 (i.e., day) to 1236 µatm (i.e., night). It has recently been suggested that variable environmental conditions could greatly influence the impact of OA on fishes, as diurnal cycling may shift pH/pCO2 above and below an organism’s “threshold” for impact (Bignami 2013c; Shaw et al. 2013). However, these arguments have been made in theory alone, and the empirical data

Figure 1. Model of ocean acidification-induced changes in GABAA receptor ionic gradients. Under normal conditions the concentration of Cl- ions is slightly higher in the cerebrospinal fluid (extracellular) relative to the cytoplasm (intracellular) and the equilibrium potential for Cl- (ECl) is near the resting membrane potential. When GABAA receptors open, Cl- flows into the neuron and counters depolarization, keeping the membrane potential more negative, and reducing neuronal activity. Ocean acidification (OA) induces hypercapnic acidosis in plasma, which fish counteract by excreting excess H+ and accumulating HCO3

-. This leads to a decrease in plasma [Cl-] (to maintain charge balance), thus leading to an alteration in ECl. In this condition, opening of GABAA receptors results in net Cl- movement out of the neuron, causing membrane depolarization and increasing excitation of neural pathways. Gabazine is an allosteric antagonist of GABAA receptors that prevents the channel from opening, whereas muscimol is a selective GABAA agonist that binds to the same site as GABA and increases channel opening (independent of presynaptic GABA release). Muscimol has opposite effects in control and OA-acclimated fish when the membrane potential is close to resting because ECl is altered. Figure and legend provided courtesy of T. Hamilton (Hamilton et al. 2013).

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necessary to address this important and potentially paradigm-shifting concept is currently lacking. This proposed research aims to produce the data necessary to address this crucial knowledge gap.

When blood pH changes more than what can be compensated for with non-bicarbonate blood buffering capacity, then retention/excretion of HCO3

- is necessary (Melzner et al. 2009). The result of exceeding this non-bicarbonate blood buffer capacity has been demonstrated in gulf toadfish (Opsanus beta), which increase blood [HCO3

-] when exposed to environmental pCO2 levels at or above 750 µatm (Esbaugh et al. 2012). Conversely, toadfish exhibit no significant change in blood chemistry at 560 µatm, suggesting that 560 µatm pCO2 does not induce a change in blood pH beyond the non-bicarbonate blood buffer capacity (Esbaugh et al. 2012). While some studies have indirectly addressed the consequences of removing OA conditions by evaluating the persistence of behavioral effects (which can last hours to days; Munday et al. 2010; Devine & Munday 2012; Hamilton et al. 2013), the influence of variable pCO2 on the blood chemistry of fishes is not known. However, if diurnal shifts in pCO2 cross the blood buffer threshold, then similar variability in blood [HCO3

-] can be expected because removal of high-pCO2 conditions will likely result in a shift from HCO3

- retention to excretion. Likewise, diurnal cycles in pCO2 may drive a similar diurnal variation in blood chemistry. Until the entire range of diurnal pCO2 variability occurs above the non-bicarbonate blood buffer capacity, daily pCO2 minimums could result in a “release” of the physiological need to retain HCO3

-. This would prevent the occurrence of the sustained 2-4 d exposure necessary to cause behavioral effects in fishes, and could effectively reduce or remove the basic physiological mechanism driving OA-induced behavioral and otolith effects in fishes. This has the potential to fundamentally alter the current understanding of projected OA impacts on fishes and is the primary justification for pursuing this proposed research.

To address the target research area of predicting the consequences of OA on ecosystem health and function, it is necessary to gain a better understanding of the physiological mechanisms of individual organisms. Until the response to natural OA variability is better understood for fishes, it may not be possible to answer the question of To what extent will OA affect an organism’s performance? The proposed research will improve the ability to answer this question by providing definitive insight into the response of fishes to variable OA, influencing the interpretation of past data, and guiding research in the future. If variable pCO2 has an effect on the response of fishes to OA, then it will be necessary to determine if the results of past research are relevant to OA in the natural environment. More importantly, an improved understanding of the response to variable-pCO2 OA will greatly impact the direction and interpretation of future OA research, resulting in data that are more representative of the natural environment. This will create an opportunity to formulate more confident scientific conclusions, that will be necessary to consider and apply during the development of OA mitigation strategies or management efforts.

The potential impact of the proposed research also applies to disciplines other than OA. For example, ocean geochemistry is affected by marine fish because they contribute a significant portion of the oceanic carbonate budget (3-15 %) via precipitation of carbonate as a byproduct of maintaining osmotic balance (Wilson et al. 2009). This contribution greatly influences the oceanic carbon cycle and Wilson et al. (2009) predicted that it will be exaggerated under future high-pCO2 conditions. However, conclusions that are rooted in the current understanding of physiological mechanisms under constant- pCO2 exposure may need to be reinterpreted with consideration for how present day or future variability influences these processes. There is also a need to understand the physiological and behavioral response of fishes to variable pCO2 because fish that inhabit high-pCO2 environments such as upwelling zones (Feely et al. 2008) and fjords (Thomsen et al. 2010) may already exhibit the effects of dramatic natural variability in pCO2. For example, rockfish (Sebastes spp.) and many other species that inhabit coastal waters naturally experience pCO2 levels that exceed 1000 µatm (Feely et al. 2008). In an experimental setting, similar constant-pCO2 treatments (1125 µatm pCO2) produced increased anxiety and altered the sheltering behavior of juvenile rockfish (Hamilton et al. 2013). Although Hamilton et al. (2013) presented this study from an OA perspective, the authors also made the important distinction that these effects could

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be experienced during present day upwelling events and could ultimately influence juvenile mortality, community ecological interactions, and population dynamics. Such behavioral effects may also occur in other species and could be currently impacting fish population dynamics in regions with great temporal or spatial pCO2 variability. Therefore, it is critical to gain a better understanding of how fishes respond to these environmental factors regardless of projected OA scenarios, especially when working with ecologically and economically important species.

STUDY SPECIES

The study species proposed for this research is the kelp rockfish (Sebastes atrovirens). Sebastes spp. are native predatory fish in temperate Eastern Pacific waters and are a popular sport fishing and commercial fishery target, with many populations experiencing decline in recent decades (Love et al. 1998; Lea et al. 1999). The kelp rockfish grows to a maximum size of approximately 40 cm over a maximum lifespan of 25 years (Lea et al. 1999). Mating occurs between February and June, with viviparous females releasing planktonic larvae that settle into kelp forests after a 1-2 month pelagic larval duration (Nelson 2001). Late-stage larvae and juveniles recruit to the kelp canopy before migrating to midwater and demersal zones of kelp forests as they mature (Schiel & Foster 1985; Nelson 2001). Trials with adult fish will utilize this species, but depending on availability of recruits, trials with juveniles may utilize another species from the genus Sebastes.

HYPOTHESES, GOALS, AND OBJECTIVES

The physiological response of blood chemistry to variable OA could differ in a number of ways from the currently understood sustained increase in blood [HCO3

-] due to constant high-pCO2. For simplicity, the three potential scenarios addressed here will be referred to as Scenario A, B, and C. In Scenario A, pCO2 is diurnally elevated above and decreased below the blood buffer capacity. In response, blood [HCO3

-] reflects this change and undergoes full compensation and “recovery” with each increase and decrease in pCO2 (Fig. 2). Scenario A does not result in a sustained increase in [HCO3

-] (or corresponding decrease in [Cl-]) and may therefore prevent the development of behavioral and otolith formation effects. In Scenario B, variable pCO2 diurnally crosses the blood buffer threshold as described in Scenario A, except the physiological result is [HCO3

-] oscillation that gradually increases average [HCO3

-] with more restricted diurnal shifts than in Scenario A. Scenario B does not provide full “recovery” of normal blood [HCO3

-] each day, and because it represents a sustained increase in average [HCO3

-], behavior and otolith formation may be impacted similarly to a constant elevated-pCO2 treatment. In Scenario C, the entire range of environmental pCO2 variability occurs above the blood

Figure 2. Diurnal variability of pCO2 (top panel) and response of blood [HCO3

-] (bottom panel). RCP 6.0 (top panel, blue curve) crosses a threshold for non-bicarbonate blood buffer capacity (black dashed line) each night (grey shaded areas), causing an increase in blood [HCO3

-]. The opposite occurs when pCO2 drops below the threshold during the day. Resulting [HCO3

-] could be variable with full diurnal recovery (Scenario A, dark blue dashed line) or an overall increase in [HCO3

-] and more limited diurnal variability (Scenario B, light blue dotted curve). RCP 8.5 (top panel, red line) consistently exceeds the buffer threshold, thus [HCO3

-] will remain elevated while undergoing some diurnal variation (Scenario C, dashed red curve). The well-described sustained response to constant elevated-pCO2 is displayed for comparison (black curve).

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buffer threshold. Therefore, recovery of normal blood chemistry never occurs despite diurnal variation in [HCO3

-], resulting in behavioral and otolith effects equivalent to a constant elevated-pCO2 treatment. In this proposed research, Scenarios A and B are represented by the RCP 6.0 variable-pH treatment, and Scenario C is represented by the RCP 8.5 variable-pH treatment (described below).

The overarching goal of the proposed research is to characterize the physiological response of fishes to variable-pCO2 and determine if such variability ultimately mitigates or eliminates previously observed impacts of OA. The specific objectives correspond to three subsets of alternative hypotheses that will be tested by exposing rockfish to constant-pCO2 and variable-pCO2 OA scenarios. The first two objectives focus on treatment impacts on adults, while the third extends the study to include the juvenile life stage. Juveniles are included because OA impacts could influence many processes that are of critical ecological importance during this stage (i.e., recruitment, sheltering, and habitat transition). Subsequent reference to “observed” effects refers to previously described OA effects on fishes, specifically increased anxiety in rockfish (Hamilton et al. 2013) and increased otolith growth in other species (Checkley et al. 2009; Bignami et al. 2013a).

Objective 1: To measure and compare the changes in blood chemistry of adult kelp rockfish in response to constant-pCO2 and diurnally-variable-pCO2 OA scenarios that reflect RCP 6.0 and 8.5 projections for the year 2100.

HA1a: Diurnal variability of environmental pCO2 under RCP 6.0 conditions will cause adult kelp rockfish to exhibit full diurnal compensation and recovery of blood [HCO3

-] (i.e., Scenario A).

HA1b: Diurnal variability of environmental pCO2 under RCP 6.0 conditions will cause adult kelp rockfish to exhibit a diurnal increase and decrease of blood [HCO3

-], but without full recovery of normal blood chemistry during periods of low-pCO2 (i.e., Scenario B).

HA1c: Diurnal variability of environmental pCO2 under RCP 8.5 OA conditions will cause an increase in average blood [HCO3

-] with variability but no diurnal recovery (i.e., Scenario C).

Objective 2: To determine and compare the behavioral and otolith growth effects of constant-pCO2 and diurnally-variable-pCO2 OA on adult rockfish, under RCP 6.0 and 8.5 projected scenarios for the year 2100.

HA2a: Diurnal variability of pCO2 under RCP 6.0 OA conditions will result in the reduction or absence of behavioral and otolith growth effects otherwise observed under constant-pCO2 RCP 6.0 OA conditions (i.e., Scenario A).

HA2b: Diurnal variability of pCO2 under RCP 8.5 conditions will produce similar effects compared to those observed under constant-pCO2 RCP 8.5 OA conditions (i.e., Scenario C).

Objective 3: To determine and compare the behavioral and otolith growth impacts on juvenile rockfish exposed to constant-pCO2 and diurnally-variable-pCO2 OA, under RCP 6.0 and 8.5 projected scenarios for the year 2100.

HA3a: Diurnal variability of pCO2 under RCP 6.0 OA conditions will result in the reduction or absence of behavioral and otolith growth effects otherwise observed under constant-pCO2 RCP 6.0 OA conditions (i.e., Scenario A).

HA3b: Diurnal variability of pCO2 under RCP 8.5 conditions will produce similar effects compared to those observed under constant-pCO2 RCP 8.5 OA conditions (i.e., Scenario C)

EXPERIMENTAL TIMELINE

The proposed research will take place over a 3-yr period, split into a development phase and subsequent experimental phases (Table 1). The development phase will involve construction and testing of a seawater system, OA treatment system, and laboratory equipment. The development phase will last approximately 10 months, during the 2014-2015 academic year. The subsequent experimental phases will

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occur throughout the remaining 26 months. Most research will be conducted during the summer months, when classes are not in session, although preliminary work and some experiments may be conducted during the academic year where time allows.

During the first summer, Objective 1 will be addressed using experiments designed to evaluate the impact of constant vs variable OA on blood chemistry. Experiments will last approximately 2 weeks each and will utilize adult rockfish. To accommodate four treatments per experiment (constant control and OA treatment, variable control and OA treatment), each experiment will be conducted at least once with control and RCP 6.0 treatments, and at least once with control and RCP 8.5 treatments. Objective 2 will be addressed during the second summer of research. These experiments will last at least three weeks, and will test the effects of constant vs variable OA on adult kelp rockfish behavior and otolith growth. To minimize the limitation of experimental duration due to mortality, no surgery or blood chemistry analyses will be attempted during these experiments. During the third summer of research, Objective 3 will be addressed by testing the impact of constant versus variable OA on juvenile rockfish behavior and otolith growth. These experiments will last at least three weeks and will not involve surgery or blood analysis.

Table 1. Objective overview & timeline with summarized treatments, hypotheses, samples sizes, & methods.

YEAR OBJECTIVE TREATMENTS ALTERNATIVE HYPOTHESES SAMPLE SIZE

METHODS

Development N/A N/A N/A Construction RAS, CO2 control system, & lab equipment

Present day constant & variable, Yr 2100 RCP 6.0 constant & variable

HA2a: Variable RCP 6.0 OA allows full [HCO3

-] recovery HA2b: Variable RCP 6.0 OA does not allow full [HCO3

-] recovery

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Objective 1: Adult rockfish blood chemistry (2 exp. minimum)

Present day constant & variable, Yr 2100 RCP 8.5 constant & variable

HA2c: Variable and constant RCP 8.5 OA do not allow full [HCO3

-] recovery

8 to 10 per treatment, per exper.

- Adult rockfish collected & acclimated to RAS - Fish surgically catheterized - Diurnal variation in pH vs. constant pH - Blood samples (high & low time resolution) for min. of 3 d - Blood pH & Total CO2 measured - Body size & weight compared pre/post experiment

Present day constant & variable, Yr 2100 RCP 6.0 constant & variable

HA3a: Behavior/otoliths will be impacted by constant but not variable RCP 6.0 OA

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Objective 2: Adult rockfish behavior & otoliths (2 exp. minimum)

Present day constant & variable, Yr 2100 RCP 8.5 constant & variable

H3b: Behavior/otoliths will be impacted by both constant and variable RCP 8.5 pH

8 to 10 per treatment, per exper.

- Adult rockfish collected & acclimated to RAS - Otoliths stained with Alizarin Red - Exposed to treatments; 3 wk minimum - Light/dark preference (anxiety) & shelter behavior assessed at 1 and 3 wk - Otoliths extracted for size, shape, increment analysis - Pre/post body length & weight compared

Present day constant & variable, Yr 2100 RCP 6.0 constant & variable

HA4a: Behavior/otoliths will be impacted by constant but not variable RCP 6.0 OA

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Objective 3: Juvenile rockfish behavior & otoliths (2 exp. minimum)

Present day constant & variable, Yr 2100 RCP 8.5 constant & variable

HA4b: Behavior/otoliths will be impacted by both constant and variable RCP 8.5 OA

15 to 25 per treatment, per exper.

- Rockfish recruits collected & acclimated - Otoliths stain (alizarin red) - Exposed to treatments; 3 wk minimum - Light/dark preference (anxiety) & shelter behavior assessed at 1 & 3 wk - Otoliths extracted for size, shape, increment analysis - Pre/post body length & weight compared

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METHODS

EXPERIMENTAL SEAWATER SYSTEM

All fish will be held and exposed to treatments in a …