DOI: 10.5061/dryad.m0cfxpp99
title: Within- and transgenerational stress legacy effects of ocean acidification on red abalone (Haliotis rufescens) growth and survival

description: Understanding the mechanisms by which individual organisms respond and
 populations adapt to global climate change is a critical challenge. The
 role of plasticity and acclimation, within and across generations, may be
 essential given the pace of change. We investigated plasticity across
 generations and life stages in response to ocean acidification (OA), which
 poses a growing threat to both wild populations and the sustainable
 aquaculture of shellfish. Most studies of OA on shellfish focus on acute
 effects, and less is known regarding the longer-term carryover effects
 that may manifest within or across generations. We assessed these
 longer-term effects in red abalone (Haliotis rufescens) using a
 multi-generational split-brood experiment. We spawned adults raised in
 ambient conditions to create offspring that we then exposed to
 high pCO2 (1,180 μatm; simulating OA) or low pCO2 (450 μatm;
 control or ambient conditions) during the first three months of life. We
 then allowed these animals to reach maturity in ambient common garden
 conditions for four years before returning the adults into high or
 low pCO2 treatments for 11 months and measuring growth and
 reproductive potential. Early-life exposure to OA in the F1 generation
 decreased adult growth rate, even after 5 years, especially when abalone
 were re-exposed to OA as adults. Adult, but not early-life exposure, to OA
 negatively impacted fecundity. We then exposed the F2 offspring to high or
 low pCO2 treatments for the first three months of life in a fully
 factorial, split-brood design. We found negative transgenerational effects
 of parental OA exposure on survival and growth of F2 offspring, in
 addition to significant direct effects of OA on F2 survival. These results
 show that the negative impacts of OA can last within and across
 generations, but that buffering against OA conditions at critical
 life-history windows can mitigate these effects.
description: We ran a fully crossed factorial, split-brood experiment with a
 low pCO<sub>2</sub> (mean condition: 450 matm,
 pH<sub>T</sub> 8.00, Waragonite = 2.08) treatment representing
 non-upwelling, current ocean conditions and a
 high pCO<sub>2</sub> (mean condition: 1,180
 matm, pH<sub>T</sub> 7.65, W<sub>aragonite</sub> =
 0.91) treatment representing strong upwelling pH values observed along the
 California coast, as well as near-future persistent values predicted to
 become increasingly common with ocean acidification. We created F1
 offspring from brood stock (F0) described below and carried out F1 and F2
 exposure treatments during the parents’ (F1) larval and post-settlement
 phases (first three months of life; F1 early-life exposure; detailed in
 Swezey et al. 2020), the parents’ (F1) mature reproductive phase (for 14
 months total prior to spawning; F1 adult exposure), and again during the
 offsprings’ (F2) larval and post-settlement phases (first three months; F2
 early-life exposure) (Fig. 1). We performed experiments in a flow-through
 system used in previous OA manipulation experiments allowing us to
 precisely control water temperature (held at 14°C) and chemistry during
 the early-life (larval, post-larval, and juvenile phases) and adult
 exposures.
description: # Within- and transgenerational stress legacy effects of ocean
 acidification on red abalone (Haliotis rufescens) growth and survival
 Data derived from
 experimental measurements during multiple stages of the experiment (F1
 adult through F2 early life) analyzed in the categories outlined below.
 Each category has its own R notebook and corresponding data: * F1 adult
 performance (growth in terms of length and weight) * F1 adult fecundity
 (spawning success; gonad tissue thickness measured via ultrasound) * F2
 performance (survival at 10, 28, and 97 DPF; size at 10 and 97 DPF;
 Inorganic/Shell mass at 97 DPF) * Data documenting seawater chemistry
 (temperature and pH/pCO2) throughout these phases of the experiment also
 included. ## Description of the data and file structure **F1 Adult
 performance** Length and Weight measurements analyzed for F1 adult
 abalone. Data taken in Fall of 2019, Fall of 2020, and Spring of 2021.
 Difference in length and weight from Fall 2019 to Fall 2020 used as growth
 metric for analyses. Files: F1_Weights_Lengths_12.19.23.csv Code:
 ORA_F1_performance_8.17.23.Rmd 1. Number of variables: 26 2. Number of
 cases/rows: 120 1. Variable list (*denotes used or described in analyses
 presented in manuscript): *Family - Maternal identity of the F1 abalone
 (individuals C, D, E as outlined in Swezey et al. 2020) *Juv_Treatment -
 (Juvenile Treatment) Treatment the F1 abalone experienced during the first
 three months of life. *Adult_Treatment - Treatment the F1 abalone
 experienced as adults during the second exposure. Code - A short hand way
 to indicate the combination of juvenile and adult treatment each abalone
 experienced (W = High-High, X = High-Low, Y = Low-High, Z = Low-Low).
 Juv_Adult_Code - An abbreviated indication of the combined juvenile and
 adult treatment each abalone experienced (Hi = High pCO2, Lo = Low pCO2).
 *Tag - The individual identifying tag number demarcating each individual
 abalone throughout the experiment. *Bucket_2019 - The bucket the adult F1
 abalone were housed in during the 11 month adult exposure portion of the
 experiment (Nov 2019 - Oct 2020). Each bucket contained 5-7 adult abalone.
 ID - A unique numerical identification for each individual abalone
 independent of their tag number. Length_11_19 - The length of the abalone
 as measured along the longest axis of the shell in November of 2019 (mm).
 Weight_11_19 - The weight of the abalone measured via balance in November
 of 2019 (g). Notes_11_19 - Notes taken during the measuring of adult
 abalone in November of 2019. Length_10_20 - The length of the abalone as
 measured along the longest axis of the shell in October 2020 (mm).
 Weight_10_20 - The weight of the abalone measured via balance in October
 of 2020 (g). Notes_10_20 - Notes taken during the measuring of adult
 abalone in October of 2020. Length_4_21 - The length of the abalone as
 measured along the longest axis of the shell in April of 2021 (mm).
 Weight_4_21 - The weight of the abalone measured via balance in April of
 2021 (g). 4_21_Notes - Notes taken during the measuring of adult abalone
 in April of 2021. *Length_Change_2019_2020 - The length of each abalone
 measured in November of 2019 subtracted from their length as measured in
 October 2020 (mm). *Weight_Change_2019_2020 - The weight of each abalone
 measured in November of 2019 subtracted from their weight as measured in
 October 2020 (g). Length_Change_2020_2021 - The length of each abalone
 measured in October 2020 subtracted from their length as measured in April
 2021 (mm). Weight_Change_2020_2021 - The weight of each abalone measured
 in October 2020 subtracted from their weight as measured in April 2021
 (g). Length_Change_2019_2021 - The length of each abalone measured in
 November 2019 subtracted from their length as measured in April 2021 (mm).
 Weight_Change_2019_2021 - The weight of each abalone measured in November
 2019 subtracted from their weight as measured in April 2021 (g).
 Weight_Length_2020 - The ratio of Weight to Length (Weight(g)/Length(mm))
 as measured in October of 2020. Weight_Length_2019 - The ratio of Weight
 to Length (Weight(g)/Length(mm)) as measured in November 2019. WL_Change -
 Change in Weight/Length Ratio from 2019 to 2020 (Weight/Legth Ratio 2020 -
 Weight/Length Ratio 2019) 3. Missing data codes: None 4. Specialized
 formats or abbreviations used: High = Stressful high pCO2 (lower pH)
 treatment Low = Control or ambient low pCO2 (higher pH) treatment **F1
 Fecundity**\ Spawning success (binary response) along with gonadal tissue
 measurements via ultrasound documented during spawning event in Spring of
 2021. Files: GonadSexSpawn_12.18.23.csv Code: ORA_F1_fecundity_8.17.23.Rmd
 1. Number of variables: 14 2. Number of cases/rows: 143 3. Variable list
 (*denotes used or described in analyses presented in manuscript): *Family
 - Maternal identity of the F1 abalone (individuals C, D, E as outlined in
 Swezey et al. 2020) *Juv_Treatment - (Juvenile Treatment) Treatment the F1
 abalone experienced during the first three months of life.
 *Adult_Treatment - Treatment the F1 abalone experienced as adults during
 the second exposure. Treatment_Code - A short hand way to indicate the
 combination of juvenile and adult treatment each abalone experienced (W =
 High-High, X = High-Low, Y = Low-High, Z = Low-Low). *Tag_ID - The
 individual identifying tag number demarcating each individual abalone
 throughout the experiment. *Bucket - The bucket the adult F1 abalone were
 housed in during the 11 month adult exposure portion of the experiment
 (Nov 2019 - Oct 2020). Each bucket contained 5-7 adult abalone. Spawned -
 Denotes whether the abalone successfully spawned (produced sperm or eggs)
 during the spawning event in April of 2021 (responses are "yes"
 or "no"). *Spawned_Binom - Denotes whether the abalone
 successfully spawned (produced sperm or eggs) during the spawning event in
 April of 2021 (responses are binary 0 or 1). *Sex - The sex of the abalone
 as determined by their production of either sperm or eggs across spawning
 attempts (October 2020 and April 2021). Blank cells denote animals where
 sex was not able to be determined as they did not successfully spawn.
 Gonad_Score - A qualitative score given to abalone during ultrasound that
 captures the thickness/ripeness of the gonadal tissue (see Boles et al.
 2022 for full details). Gonad_mean_Winter - The thickness of the gonadal
 tissue as measured from still ultrasound images and Fiji software. Values
 are calculated from averaging three measurements across the thickest,
 medial portion of the gonad tissue (cm). Ultrasound images captured in
 January of 2021. *Gonad_mean_Spring - The thickness of the gonadal tissue
 as measured from still ultrasound images and Fiji software. Values are
 calculated from averaging three measurements across the thickest, medial
 portion of the gonad tissue (cm). Ultrasound images captured on the day of
 spawning, prior to spawning, in April of 2021. Length_4_21 - The length of
 the abalone as measured along the longest axis of the shell in April of
 2021 (mm). Gonad_Spring_Length - The gonad thickness measured in April
 2021 divided by the length of the animal in April of 2021 to see if animal
 size impacted gonadal thickness. 4. Missing data codes: Sex - Blank cells
 denote animals where sex was not able to be determined as they did not
 successfully spawn. Gonad Score - Blank cells denote animals where we were
 unable to get a clear ultrasound image and therefore did not get to
 accurately score. Gonad Mean (Winter and Spring) - We were not able to
 ultrasound all abalone prior to there spawning. Missing values indicate
 individuals where we were unable to capture still images to analyze.
 Length - Blank cells are missing data from that timepoint. 5. Specialized
 formats or abbreviations used: High = Stressful high pCO2 (lower pH)
 treatment Low = Control or ambient low pCO2 (higher pH) treatment **F2
 performance** Survival was calculated from animal counts per settlement
 cup at 10, 28, and 97 DPF (days post-fertilization) and survival from
 10-28 and 28-97 DPF periods were analyzed. Growth (area of abalone shell)
 was measured via microscope photos at 10 DPF and 97 DPF and calculated in
 Fiji image processing software. Final dry weights were obtained using a
 microbalance. Foil boats were placed in muffle furnace and weighed
 individually empty (starting weight of foil boat), then weighed with dried
 animals (taken at 97 DPF) (total mass), then after boats placed into
 muffle furnace and all organic matter burned off (shell/inorganic mass),
 this final weight subtracted from the dried weight gives organic mass and
 finally organic/inorganic mass ratio can be calculated from there. Files:
 ORA_finalCounts_6.16.22.csv; Ash_weight_abalone_12.19.23.csv;
 10dpf_Size.csv; F2_GrowthData_Density_8.23.22.csv Code:
 ORA_F2_performance_8.17.23.Rmd ORA_finalCounts_6.16.22.csv 1. Number of
 variables: 15 2. Number of cases/rows: 192 3. Variable list (*denotes used
 in analyses presented in manuscript): Treatment_Code - A short hand way to
 indicate the combination of juvenile and adult treatment each F1 parent
 abalone experienced (W = High-High, X = High-Low, Y = Low-High, Z =
 Low-Low). Rep - The replicate number of each cup used to house the baby F2
 abalone during the exposure period. Each treatment combination (F1 early
 life, F1 adult, F2 early life) had 24 replicate cups (8 treatment
 combinations, 192 cups total). *ID - The unique identifier for each cup
 combining treatment code and replicate. *Current_Treatment - The treatment
 applied to each cup housing F2 offspring abalone for the three month
 exposure period. *Juv_Treatment - (Juvenile Treatment) Treatment the F1
 parental abalone experienced during the first three months of life.
 *Adult_Treatment - Treatment the F1 parentl abalone experienced as adults
 during the second exposure. DPF_10_Total - The number of abalone observed
 in each cup via dissecting microscope at 10 days post fertilization.
 DPF_28_Total - The number of abalone observed in each cup via dissecting
 microscope at 28 days post fertilization. DPF_97_Total - The number of
 abalone observed in each cup via dissecting microscope at 97 days post
 fertilization. *Surv_10_28 - The proportion of abalone that survived in
 each cup from day 10 to day 28 post fertilization. *Surv_28_97 - The
 proportion of abalone that survived in each cup from day 28 to day 97 post
 fertilization. Surv_10_97 - The proportion of abalone that survived in
 each cup from day 10 to day 97 post fertilization. DPF10_minus_28 - The
 number of surviving abalone in each cup from day 10 to day 28 post
 fertilization (subtract counts from day 10 from counts from day 28).
 DPF10_minus_97 - The number of surviving abalone in each cup from day 10
 to day 97 post fertilization (subtract counts from day 10 from counts from
 day 97). DPF28_minus_97 - The number of surviving abalone in each cup from
 day 28 to day 97 post fertilization (subtract counts from day 28 from
 counts from day 97). 4. Missing data codes: Empty cells in Survival and
 columns subtracting counts from each other (e.g., DPF10_minus_28) indicate
 that all animals had died (count = zero) at the earliest time point so
 there was no calculation to conduct (for example, if all animals were dead
 at 28 DPF there is no value for the proportion of survivors between 28 DPF
 and 97 DPF, cannot divide by zero). In some cases, there was an error in
 the counts and more animals were counted at a later time point than there
 had been previously (this happened between 10 and 28 DPF as abalone are
 hardest to count when they are young and very small and some still
 swimming and yet to settle, these were removed as outliers as survival
 rate cannot be above 100%). 5. Specialized formats or abbreviations used:
 High = Stressful high pCO2 (lower pH) treatment Low = Control or ambient
 low pCO2 (higher pH) treatment DPF = days post fertilization
 Ash_weight_abalone_12.19.23.csv 1. Number of variables: 21 2. Number of
 cases/rows: 96 3. Variable list (*denotes used in analyses presented in
 manuscript): Weigh_boat_num - The number assigned to the foil weigh boat
 used to measure dry and ash weight of abalone. Treatment - A shorthand
 that encompasses all treatments into one code, current treatment (Hi =
 High, Lo = Low), parental treatment (W = High-High, X = High-Low, Y =
 Low-High, Z = Low-Low) and the cup replicate (1-24). *Current_Trt - The
 treatment applied to each cup housing F2 offspring abalone for the three
 month exposure period. Trt_Code - A short hand way to indicate the
 combination of juvenile and adult treatment each F1 parent abalone
 experienced (W = High-High, X = High-Low, Y = Low-High, Z = Low-Low).
 *F1_juv_Trt - (Juvenile Treatment) Treatment the F1 parental abalone
 experienced during the first three months of life. *F1_adult_Trt -
 Treatment the F1 parental abalone experienced as adults during the second
 exposure. Cup_ID_Code - The replicate number of each cup used to house the
 baby F2 abalone during the exposure period. *Number_abalone - The number
 of abalone left in each cup after the three month experiment that were
 used in these measurements. Each weigh boat contains abalone originating
 from a single cup. Weigh_boat - The weight of the foil weigh boat used to
 measure the abalone after being ashed at 500°C for 3h and weighed using a
 microbalance (µg). Dry_abs_boat - The weight of the dried abalone (paced
 drying oven at 30°C for 24 h) plus the weight of the foil boat µg.
 Ash_abs_boat - The weight of the ashed abalone (boat and dried abalone
 placed in a muffle furnace heated to 460°C for 4 h) and the weigh boat
 (µg). *Dry_abs - The weight of the dried abalone and the weigh boat minus
 the weight of the foil boat on its own (µg). *Ash_abs - The weight of the
 ashed abalone and the weigh boat minus the weight of the foil boat on its
 own (µg). This represents the shell or inorganic mass of the abalone.
 *Dry_minus_ash - The weight of the dried abalone minus the weight of the
 ashed abalone (µg). This represents the tissue or organic mass of the
 abalone. *Area_Total - The total are of shell as measured in Fiji added
 across all abalone left in a given cup (mm^2). Area_Avg - The total are of
 shell divided by the number of abalone in a cup to get the average shell
 area per abalone. *Shell_div_AreaTotal - The weight of the ashed abalone
 (Ash_abs) divided by the total area of shell in the cup (Area_Total) to
 get the shell mass per unit area (g/mm^2). *Tissue_div_AreaTotal - The
 weight of the dried organic mass or tissue (Dry_minus_ash) divided by the
 total area of shell in the cup (Area_Total) to get the tissue mass per
 unit area (g/mm^2). *Shell_div_Tissue - The ratio of shell mass to tissue
 mass as calculated by dividing Shell_div_AreaTotal by
 Tissue_div_AreaTotal. Dry_div_AreaTotal - The weight of the whole animal
 dried (Dry_abs; Shell and Tissue) divided by the total area of shell in
 the cup (Area_Total) to get mass per unit area (g/mm^2). Notes - Notes
 relevant to data collection or analysis. 4. Missing data codes: Some
 extreme outliers removed (see notes column). 5. Specialized formats or
 abbreviations used: None 10dpf_Size.csv 1. Number of variables: 6 2.
 Number of cases/rows: 94 3. Variable list (*denotes used in analyses
 presented in manuscript): *F2_Treatment - The treatment applied to each
 cup housing F2 offspring abalone for the three month exposure period.
 *Adult_Treatment - Treatment the F1 parental abalone experienced as adults
 during the second exposure. *Juv_Treatment - (Juvenile Treatment)
 Treatment the F1 parental abalone experienced during the first three
 months of life. Code_Treatment - A short hand way to indicate the
 combination of juvenile and adult treatment each F1 parent abalone
 experienced (W = High-High, X = High-Low, Y = Low-High, Z = Low-Low). Cup
 - A shorthand that encompasses all treatments into one code, current
 treatment (Hi = High, Lo = Low), parental treatment (W = High-High, X =
 High-Low, Y = Low-High, Z = Low-Low) and the cup replicate (1-24). *Area -
 The shell area of a subset of individuals at 10 DPF measured by
 photographing them under a microscope and using Fiji software tracing the
 outline of each individual abalone to calculate their area (mm^2). 4.
 Missing data codes: None. 5. Specialized formats or abbreviations used: Hi
 = High = Stressful high pCO2 (lower pH) treatment Lo = Low = Control or
 ambient low pCO2 (higher pH) treatment DPF = days post fertilization
 F2_GrowthData_Density_8.23.22.csv 1. Number of variables: 10 2. Number of
 cases/rows: 101 3. Variable list (*denotes used in analyses presented in
 manuscript): *Current_Trt - The treatment applied to each cup housing F2
 offspring abalone for the three month exposure period. Code - A short hand
 way to indicate the combination of juvenile and adult treatment each F1
 parent abalone experienced (W = High-High, X = High-Low, Y = Low-High, Z =
 Low-Low). *Juv_Trt - (Juvenile Treatment) Treatment the F1 parental
 abalone experienced during the first three months of life. *Adult_Trt -
 Treatment the F1 parental abalone experienced as adults during the second
 exposure. Cup - A shorthand that encompasses the parental treatments into
 one code, parental treatment (W = High-High, X = High-Low, Y = Low-High, Z
 = Low-Low) and the cup replicate (1-24). Cup_ID - A shorthand that
 encompasses all treatments into one code, current treatment (High, Low),
 parental treatment (W = High-High, X = High-Low, Y = Low-High, Z =
 Low-Low) and the cup replicate (1-24). Avg_Area - The estimated area per
 individual abalone (mm^2) calculated from the total area summed across
 each abalone in the image and divided by the number of abalone in each
 image (Ab_Num_97dpf). The area for each individual was measured by
 photographing them under a microscope and using Fiji software tracing the
 outline of each individual abalone to calculate their area (mm^2) at 97
 DPF. Ab_Num_97dpf - The number of abalone in each cup and photographed at
 97 DPF. Ab_Num_28dpf - The number of abalone in each cup and counted at 28
 DPF. Notes - Notes relevant to data collection or analysis. 4. Missing
 data codes: None 5. Specialized formats or abbreviations used: High =
 Stressful high pCO2 (lower pH) treatment Low = Control or ambient low pCO2
 (higher pH) treatment DPF = days post fertilization **Water chemistry:**
 File: ORA_Chemistry Data_12.18.23.xlsx Tab 1: ORA_F1_Phase 1 Water
 chemistry measured weekly throughout the adult F1 exposure period
 (November 2019 - October 2020). 1. Number of variables: 8 2. Number of
 cases/rows: 1018 3. Variable list: Date - The date a measurement was
 collected. Slope_percent - A value that verifies the probe is properly
 calibrated. Values between 85-100 are considered in acceptable range (see
 additional notes below). Slope_mV - A value that verifies the probe is
 properly calibrated. These values represent the offset from the slope.
 Source - Which treatment the water was collected from, high or low.
 Bucket_Sump - Where the water was collected from (a bucket housing abalone
 in the experimental system identified by number, incoming water from the
 flow-through seawater system prior to entering the experimental system,
 and sumps that held the treated water for both the high and low
 treatments). pH - The pH of the water sample. Temperature - The
 temperature of the water sample (°C). mV - Millivolts of the sample (mV).
 4. Missing data codes: Empty cells represent NA or that data were not
 collected from that source/date. (See additional notes for the
 Slope_percent and Slope_mV columns). 5. Specialized formats or
 abbreviations used: mV = millivolt; 0 mV = 7 on the pH scale Slope percent
 and Slope mV = These values are recorded after calibrating the pH probe as
 a way to compare measurements across time and ensure proper functioning of
 the pH probe. These two values are recorded once and apply to all values
 collected from the pH probe in a given session/date. They are independent
 from the observations recorded in that row, they are mapped to a given
 date. High = Stressful high pCO2 (lower pH) treatment Low = Control or
 ambient low pCO2 (higher pH) treatment Tab 2: ORA_F1_Phase 2 Water
 chemistry measured weekly throughout the adult F1 exposure period
 (February 2021 - April 2021). Water samples were collected from their
 source and placed into falcon tubes for measurements in the lab. 1. Number
 of variables: 8 2. Number of cases/rows: 333 3. Variable list: Date - The
 date a measurement was collected. Slope_percent - A value that verifies
 the probe is properly calibrated. Values between 85-100 are considered in
 acceptable range (see additional notes below). Slope_mV - A value that
 verifies the probe is properly calibrated. These values represent the
 offset from the slope. Source - Which treatment the water was collected
 from, high or low. Bucket_Sump - Where the water was collected from (a
 bucket housing abalone in the experimental system identified by number,
 incoming water from the flow-through seawater system prior to entering the
 experimental system, and sumps that held the treated water for both the
 high and low treatments). pH - The pH of the water sample. Temperature -
 The temperature of the water sample (°C). mV - Millivolts of the sample
 (mV). 4. Missing data codes: Empty cells represent NA or that data were
 not collected from that source/date. (See additional notes for the
 Slope_percent and Slope_mV columns). 5. Specialized formats or
 abbreviations used: mV = millivolt; 0 mV = 7 on the pH scale Slope percent
 and Slope mV = These values are recorded after calibrating the pH probe as
 a way to compare measurements across time and ensure proper functioning of
 the pH probe. These two values are recorded once and apply to all values
 collected from the pH probe in a given session/date. They are independent
 from the observations recorded in that row, they are mapped to a given
 date. High = Stressful high pCO2 (lower pH) treatment Low = Control or
 ambient low pCO2 (higher pH) treatment Tab 3: ORA_F2_Larval Bucket_probe
 Water chemistry measured in all larval buckets twice throughout the larval
 exposure period (0-7 DPF). Water samples were collected from their source
 and placed into falcon tubes for measurements in the lab. 1. Number of
 variables: 8 2. Number of cases/rows: 50 3. Variable list: Date - The date
 a measurement was collected. Slope_percent - A value that verifies the
 probe is properly calibrated. Values between 85-100 are considered in
 acceptable range (see additional notes below). Slope_mV - A value that
 verifies the probe is properly calibrated. These values represent the
 offset from the slope. Source - Which treatment the water was collected
 from, high, low, or incoming (water from the flow-through seawater system
 prior to entering our experimental system). Bucket_Sump - Where the water
 was collected from (a bucket housing larval abalone in the experimental
 system identified by letter or sumps that held the treated water for both
 the high and low treatments). pH - The pH of the water sample. Temperature
 - The temperature of the water sample (°C). mV - Millivolts of the sample
 (mV). 4. Missing data codes: Empty cells represent NA or that data were
 not collected from that source/date. (See additional notes for the
 Slope_percent and Slope_mV columns). 5. Specialized formats or
 abbreviations used: mV = millivolt; 0 mV = 7 on the pH scale Slope percent
 and Slope mV = These values are recorded after calibrating the pH probe as
 a way to compare measurements across time and ensure proper functioning of
 the pH probe. These two values are recorded once and apply to all values
 collected from the pH probe in a given session/date. They are independent
 from the observations recorded in that row, they are mapped to a given
 date. High = Stressful high pCO2 (lower pH) treatment Low = Control or
 ambient low pCO2 (higher pH) treatment Tab 4: ORA_F2_in situ Water
 chemistry measured in all larval buckets twice throughout the larval
 exposure period (0-7 DPF). Water samples were collected from their source
 and placed into falcon tubes for measurements in the lab (pH) as well as
 measured in situ within the system (Insitu pH) and the pH reading from our
 sensors built into the experimental system were also recorded. 1. Number
 of variables: 9 2. Number of cases/rows: 81 3. Variable list: Date - The
 date a measurement was collected. Salinity - Salinity of the seawater
 (ppt) measured once per observation session on incoming seawater prior to
 entering the experimental system. Source - Which treatment the water was
 collected from, high, low, or incoming water prior to entering the
 experimental system. Bucket_Sump - Where the water was collected from
 (sumps that held the treated water for both the high and low treatments,
 empty cells signify that the measurements were collected from incoming
 seawater, see above). Temp - The temperature of the water measured within
 the sump or in the flow of incoming water (°C). pH - The pH of the water
 as measured by pH probe (see Tabs 1-3). Insitu pH - The pH of the water as
 measured within the experimental system. Wall pH - The pH reading from our
 sensors built into the experimental system. 4. Missing data codes: Empty
 cells represent NA or that data were not collected from that source/date.
 In the Salinity column, these values are recorded once on incoming water
 once and apply to all values collected in a given session/date. They are
 independent from the observations recorded in that row, they are mapped to
 a given date). 5. Specialized formats or abbreviations used: High =
 Stressful high pCO2 (lower pH) treatment Low = Control or ambient low pCO2
 (higher pH) treatment ## Code/Software We performed all analyses using R
 version 3.5.1 and the lme4 package for model construction (Bates et al.
 2015). We assessed the significance of fixed effects in our models by
 analysis of deviance, Type II Wald chi-square tests using the ANOVA
 function from the car package (Fox & Weisberg 2019). For models with
 scaled parameters, we used a Kenward-Roger approximation of degrees of
 freedom and used these to assess the significance of fixed effects using F
 scores (Venables & Ripley 2002, Kenward & Roger 2009). We explored
 any potential pair-wise differences using a Bonferroni correction and the
 “contrasts” function in the emmeans package (Searle et al. 1980).