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Acidification, deoxygenation, and warming are escalating changes in coastal waters throughout the world ocean, with potentially severe consequences for marine life and ocean-based economies. To examine the influence of these oceanographic changes on a key biological process, we measured the effects of current and expected future conditions in the California Current Large Marine Ecosystem on the fertilization success of the red abalone (Haliotis rufescens). Laboratory experiments were used to assess abalone fertilization success during simultaneous exposure to various levels of seawater pH (gradient from 7.95 to 7.2), dissolved oxygen (DO) ($60 and 180 mm. kg SW) and temperature (9, 13, and 18 C). Fertilization success declined continuously with decreasing pH but dropped precipitously below a threshold near pH 7.55 in cool (9 C—upwelling) to average (13 C) seawater temperatures. Variation in DO had a negligible effect on fertilization. In contrast, warmer waters (18 C) often associated with El Nino Southern Oscillation conditions in central California acted antagonistically with decreasing pH, largely reducing the strong negative influence below the pH threshold. Experimental approaches that examine the interactive effects of multiple environmental drivers and also strive to characterize the functional response of organisms along gradients in environmental change are becoming increasingly important in advancing our understanding of the real-world consequences of changing ocean conditions.
Effects of current and future coastal upwelling conditions on the
fertilization success of the red abalone (Haliotis rufescens)
Charles A. Boch
*, Steven Y. Litvin
, Fiorenza Micheli
, Giulio De Leo
, Emil A. Aalto
Christopher Lovera
, C. Brock Woodson
, Stephen Monismith
, and James P. Barry
Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA
Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, USA
College of Engineering, University of Georgia, Athens, GA 30602, USA
Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305, USA
*Corresponding author: tel: 831-775-1849; fax: 831-775-1620; e-mail:
Boch, C. A., Litvin, S. Y., Micheli, F., De Leo, G., Aalto, E. A., Lovera, C., Woodson, C. B., Monismith, S., and Barry, J. P. 2017. Effects of current and
future coastal upwelling conditions on the fertilization success of the red abalone (Haliotis rufescens). – ICES Journal of Marine Science,
Received 30 August 2016; revised 10 January 2017; accepted 29 January 2017.
Acidification, deoxygenation, and warming are escalating changes in coastal waters throughout the world ocean, with potentially severe con-
sequences for marine life and ocean-based economies. To examine the influence of these oceanographic changes on a key biological process,
we measured the effects of current and expected future conditions in the California Current Large Marine Ecosystem on the fertilization suc-
cess of the red abalone (Haliotis rufescens). Laboratory experiments were used to assess abalone fertilization success during simultaneous ex-
posure to various levels of seawater pH (gradient from 7.95 to 7.2), dissolved oxygen (DO) (60 and 180 mm
kg SW) and temperature (9, 13,
and 18 C). Fertilization success declined continuously with decreasing pH but dropped precipitously below a threshold near pH 7.55 in cool
(9 C—upwelling) to average (13 C) seawater temperatures. Variation in DO had a negligible effect on fertilization. In contrast, warmer
waters (18 C) often associated with El Ni~
no Southern Oscillation conditions in central California acted antagonistically with decreasing pH,
largely reducing the strong negative influence below the pH threshold. Experimental approaches that examine the interactive effects of mul-
tiple environmental drivers and also strive to characterize the functional response of organisms along gradients in environmental change are
becoming increasingly important in advancing our understanding of the real-world consequences of changing ocean conditions.
Keywords: climate change, fertilization, Haliotis rufescens, hypoxia, multiple drivers, ocean acidification, ocean warming, upwelling.
Fossil fuel CO
emissions are driving massive and rapid changes
in global temperature and ocean chemistry (Bakun, 1990;
Caldeira and Wickett, 2003;Sabine et al., 2004;Solomon et al.,
2007;Chan et al., 2008). These global scale impacts are leading to
a cascade of changes in ocean stratification, transport, convection,
and other key processes at both regional and local scales. In the
California Current Large Marine Ecosystem (CCLME), shoaling
of the oxygen minimum zone (Stramma et al., 2010) has pro-
moted a reduction in the pH and dissolved oxygen (DO) content
of upwelled waters that are advected into nearshore habitats
(Chan et al. 2008;Feely et al., 2008;Bograd et al., 2008;Connolly
et al., 2010;Keeling et al., 2010;Deutsch et al., 2011;Booth et al.
2012;Walter et al. 2014). In addition, an increase in El Ni~
conditions in combination with global ocean warming has re-
sulted in recent increases in temperatures in coastal ecosystems of
the CCLME (Trenberth and Hoar, 1997;Lee and McPhaden,
2010;Cai et al., 2014). These growing changes in key ocean con-
ditions are predicted to directly affect the physiology of many
marine organisms, with potentially profound effects on the sus-
tainability of marine populations (Vaquer-Sunyer and Duarte,
2008;Portner and Farrell, 2008;Somero et al., 2016). To date,
CInternational Council for the Exploration of the Sea 2017. All rights reserved.
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ICES Journal of Marine Science (2017), doi:10.1093/icesjms/fsx017
however, most research has focused on the response of marine or-
ganisms to shifts in a single oceanographic parameter (reviewed
by Gattuso et al., 2015) and our understanding of biological re-
sponses to simultaneous changes in multiple ocean conditions is
poorly understood. Experimental approaches that assess the re-
sponse of species, assemblages, and marine communities to realis-
tic future environmental variation among multiple drivers (e.g.
Kroeker et al., 2013) are needed to refine our ability to predict the
future stability of ecosystem function and the sustainability of
ocean-based economies (Gattuso et al., 2015).
The persistence of natural populations depends on successful
reproduction, and much research has focused on the effects of
ocean acidification, hypoxia and temperature variation on fertil-
ization success and early development in marine species (re-
viewed by Byrne, 2011). Although several biological factors (e.g.
gamete concentration, sperm:egg ratios, gamete age; Babcock and
Keesing, 1999;Baker and Tyler, 2001;Huchette et al., 2004)or
changes in ocean conditions (e.g. pH; Kurihara and Shirayama,
2004;Havenhand et al, 2008) are known to affect fertilization
success, few studies have examined the effects of simultaneous ex-
posure to multiple environmental changes (e.g. Byrne et al,
2010). Thus, we are just beginning to address how simultaneous
exposure to multiple drivers may influence these key processes in
marine organisms, and importantly, whether non-linear re-
sponses and tipping points exist across chemo-physical drivers.
These questions are particularly relevant in systems where envir-
onmental conditions are highly variable in space and time, and
where this variability is predicted to increase under future climate
change scenarios, such as upwelling ecosystems (Bakun, 1990;
Sydeman et al., 2014;Bakun et al., 2015). Here, we addressed the
individual and interactive effects of current and expected future
pH, DO, and temperature on fertilization success using a near-
shore benthic invertebrate, the red abalone Haliotis rufescens,asa
model system. Red abalone (H. rufescens), naturally inhabit the
intertidal to a depth of ca. 30 m from Southern Oregon to
Central Baja California of the CCLME (Boolootian et al., 1962).
Abalone and other ecologically important and economically
valuable benthic invertebrates are unable to escape bottom hyp-
oxia because of their limited mobility. They are also are negatively
affected by high pCO
(low pH) that can interfere with shell de-
position and growth, and are impacted by high temperatures, dir-
ectly or indirectly, through the loss of their algal food resources
(Shepherd et al., 1998;Orr et al., 2005;Micheli et al., 2012;
Gazeau et al., 2013;Kim et al., 2013). The question of how differ-
ent combinations of drivers may independently or interactively
affect abalone and other species remains unanswered. However,
recent experiments indicate that exposure to even a limited range
of low pH and low DO may have deleterious effects on mortality
and growth of early stage of red abalone juveniles (Kim et al.,
2013). Therefore, to increase our understanding of the likely fu-
ture effects of climate-driven changes in ocean conditions for
marine species, it is crucial to explore the effects of simultaneous
exposure to key environmental variables on critical processes
such as fertilization.
Here we examined the effects of variation in seawater pH, DO,
and temperature on the fertilization success of red abalone H.
rufescens. These oceanographic parameters are highly variable in
upwelling affected areas of nearshore habitats in the CCLME, and
their range of variability is shifting in response to climate change
(Sydeman et al., 2014) and as such, future upwelling is expected
to be increasingly stressful for coastal species (Somero et al.,
2016). We employed a hybrid (regression/factorial) experimental
approach to assess abalone fertilization success across a gradient
of seawater pH (regression, 40þpH levels), combined with a fac-
torial approach for DO (two levels: 6 and 2 mg/l DO) and tem-
perature (three levels: 9, 13, and 18C). This novel hybrid design
allowed us to characterize the functional response of fertilization
over a large range of pH, under multiple co-varying conditions,
and assess potential thresholds to changes in pH beyond current
day variation.
Based on previous studies, we hypothesized that decreasing pH
may have a negative effect on fertilization (Kroeker et al., 2010;
Gazeau et al., 2013). However, potential interactions with DO
and temperature were difficult to predict because of the limited
range or the number of pH levels that have been previously exam-
ined (Byrne et al., 2010). While decreasing pH may reduce fertil-
ization success, an additional driver may not further affect
fertilization or the independent effects of a secondary driver may
dominate, thus decoupling fertilization success from the effects of
pH (dominant stressor model; Halpern et al., 2008). In a more
complex process, a secondary driver may interact with pH, mod-
ifying the decline in fertilization rates along the pH gradient by
ameliorating or exacerbating negative impacts (antagonistic and
synergistic effects, respectively; Crain et al., 2008). Finally, the ef-
fects of an additional environmental driver may be non-linear
along a pH gradient.
Preparation of seawater treatments
Two days before each experiment, seawater sources were pro-
duced using the nitrogen, air, and CO
gas control system
described in Barry et al. (2008) and stored at 13C in gas-tight 10
l mylar bags (Calibrated Instruments, Inc., NY, USA) with one-
way Luer Lock stopcocks. The target pH (total scale) and DO
concentration of the seawater sources (Supplementary Table S1)
were adjusted in order to achieve the ranges utilized in the fertil-
ization experiments (pH 7.95–7.2 and DO 6 and 2 mg/l) while ac-
counting for the process of adding gametes (see ‘Assessment of
fertilization success’ section below). After 24 h, pH (n ¼3 each;
UV-1601 spectrophotometer, Shimadzu, Kyoto, Japan) and total
alkalinity (n ¼8; TA, TitroLine 7000 open cell, potentiometric ti-
tration system, SI Analytics, Germany) of each seawater source
were measured and used to estimate dissolved inorganic carbon
concentrations (DIC, at 13C and 33 psu). In addition, DO con-
centrations were verified using an Aanderaa 3830 optode (Xylem
Inc., NY, USA). Based on DIC and TA values (Supplementary
Table S1 and S2), mixtures of each seawater source needed to
achieve the desired range of pH and DO for a particular experi-
ment (see Supplementary material ‘Preparation of seawater sour-
ces and Determination of volumetric mixtures of seawater
sources to achieve target pH and DO’ section) were determined
using CO
SYS ( These
predetermined mixtures were then loaded into 50 ml gas-tight
glass syringes fitted with three-way Luer Locks (“experimental
syringes”; Tomopal, Japan). To remove any bubbles, 15 ml of sea-
water was extruded and the syringe re-sealed, retaining 35 ml of
treatment seawater. Syringes were then stored overnight in a tem-
perature controlled seawater baths appropriate for the given ex-
periment (8.5, 13, or 18.5C, see description of experiments
below). In preliminary studies, we determined that the change in
2C. A. Boch et al.
carbonate chemistry and DO within experimental syringes was
negligible over several days.
Abalone spawning, gamete concentration determination,
and gamete density adjustment
Abalone were obtained from American Abalone Farms,
Davenport, CA, USA. For each experiment, six males (mean
shell length ¼86.4 mm 64.0 SD) and six females (mean shell
length ¼94.5 mm 65.8 SD) were conditioned in two tanks with
flowing 13C seawater, 0:24 Light:Dark photoperiod, and fed
giant kelp, Macrocystis pyrifera,ad libitum for 2 weeks prior to the
day of experiment. On the day of each experiment, we separated
the male and female brooders into individual induction con-
tainers and used the tris-buffer, hydrogen peroxide, and tempera-
ture protocol to individually induce spawning (Morse et al.,
1977). We delayed the initiation of the protocol for males by
1.5h, relative to females, in order to synchronize spawning be-
tween sexes. Over the experiments, 95% of male and female aba-
lone spawned within a 1-h window.
Upon commencement of spawning, sperm and eggs from all
spawning individuals were pooled in separate 500 ml, autoclaved
glass beakers. Gamete collection was limited to 45 min after the first
animal spawned to reduce the potential effects of gamete age on
fertilization success. To determine initial density of the pooled
sperm stock, micrographs of sperm stained with Lugol’s solution
were taken on a hemocytometer (Bright-Line, PA, USA) with an
Axioscop compound microscope (10, Zeiss, Germany, Olympus
ZH71 camera attachment and CellSens software). ImageJ (https:// software was then used to automatically enu-
merate sperm (n ¼2 samples from pooled sperm stock, see
Supplementary material ‘Estimation of sperm density via image
analysis’ section and Supplementary Figures S1A–C). To estimate
initial egg density, the number of eggs in 50 ml(n¼4) subsamples
of the pooled egg stock were counted on an Olympus SZH10 dis-
secting microscope. From these estimates and accounting for the
process of adding gametes into seawater treatments (final volume
of 40 ml in each syringe, see ‘Assessment of fertilization success’
section below), sperm and egg stock densities were diluted with
control sea water to achieve a final concentration of sperm and
eggs in each syringe of 10
and 60 ml
, respectively (see
Supplementary material ‘Determination of experimental sperm
density and exposure time’ section). During and after density ad-
justments, gamete stocks were maintained at 13C.
Figure 1. General procedure during the fertilization experiments. A. Known concentrations of sperm (white cloud) are loaded after egg
injection (white flecks) into 50 ml gas-tight syringes (Tomopal, Japan) with specified pH, DO, and temperature levels. Seawater is kept water
tight in syringes with 3–way Luer Lock valve and connector. B. A final 1 ml of seawater is injected into the syringes to flush any gametes in
the valves. Then all the syringes are incubated for 600 seconds in the appropriate temperature tank. C. 20 ml out of the total 40 ml in each
syringe are loaded into a modified 50 ml syringe container to measure the pH (SentrON-Line 8100-100 ISFET probe connected by RS232 cable
to a logging computer), dissolved oxygen and temperature (NeoFox hyoxy probe, sensing patch and temperature probe connected via USB to
a logging computer). D. Sample micrograph showing unfertilized eggs (single cells indicated by red arrows) and 4-cell stage fertilized eggs
(remainder of the cells) after 600 seconds of treatment followed by 4-hours of incubation at non-stressful levels.
Effects of current and future coastal upwelling conditions 3
Assessment of fertilization success
To access fertilization success, sperm and eggs from the adjusted
density pooled gamete stocks were injected into 50 ml experimen-
tal syringes pre-loaded with volumetric mixtures of seawater
sources (see ‘Preparation of seawater treatments’ section above)
to achieve the range of pH and DO for a given experiment and
held at the appropriate temperature (8.5, 13, or 18.5C, see de-
scription of experiments below). A 2 ml of egg stock solution, fol-
lowed by 2 ml of sperm stock solution, were injected into the
experimental syringe using separate 5ml gas-tight syringes with
one-way Luer Locks (Figure 1a). Subsequently, 1 ml of control
seawater was injected from a separate 5 ml syringe to flush any
gametes remaining in the Luer Lock into each experimental syr-
inge (see Supplementary Tables S1 and S2 for final flush source),
bringing the final total volume to 40 ml. The order which experi-
mental syringes were inoculated with gametes was randomized
with the exception of controls (see descriptions of experiments
below), which were conducted at the start, finish and across regu-
lar intervals during each experiment.
After gamete injection, each experimental syringe was held in a
second temperature bath (Figure 1b, 9, 13, or 18C, see descrip-
tion of experiments below) and eggs were exposed to sperm for a
duration of 600 s (see Supplementary material ‘Determination of
experimental sperm density and exposure time’ section).
Subsequent to the exposure period, 20 ml of each experimental
syringe was expunged into a 50 ml falcon tube, with the bottom
removed and replaced with Nitex 90 mm mesh, then immersed re-
peatedly in control seawater (pH 7.95, DO 6.0 mg/l, 13C) to
wash away excess sperm and terminate the exposure period. Each
tube was held in bins filled with flowing control seawater for 4 h,
at a depth that maintained each tube approximately two-third
filled, to allow fertilized eggs to develop. Samples were then
washed into 20 ml glass scintillation vials and fixed with 500 mlof
10% formalin. For each sample, >100 cells were examined using
an Olympus SZ40 dissecting microscope to access proportional
fertilization success, with eggs reaching a two-cell stage or greater
counted as fertilized (Figure 1d).
Measurement of post-fertilization experimental
syringe pH and DO
The remaining 20 ml of sample was held in the sealed experimen-
tal syringe (<3h) at 13
C until pH and DO were measured.
A SentrON-Line 8100-100 ISFET probe (Sentron, NL) was used
to estimate pH (total scale) and a NeoFox spectrometer
(OceanOptics, FL, USA) system to measure DO and temperature
(Figure 1c, for details see supplementary material ‘Determination
of post fertilization experimental syringe pH and DO’ section).
Effects of ocean acidification and hypoxia on
fertilization rates
The range of current environmental variability along the central
California coast within the CCLME was used to determine the
range of treatments levels for pH, DO, and temperature over
which abalone fertilization success was measured. Records of
temperature, DO, and pH from a depth of 6 m off Hopkins
Marine Station were made during the spring upwelling period
(observations in April–June, 2013 season) (Figure 2), indicating
extremes during upwelling conditions of pH 7.5, DO 60 mmol/
kg SW (approximately 2.0 mg/l DO), and 9C. Although it is
difficult to predict the time scales and magnitude of changes in
the composition of upwelled waters in response to rising atmos-
pheric CO
(Feely et al., 2004,2008) we used a pH offset of -
0.3 pH (reference for global surface water pH change) units
below the current upwelling minimum as a pH minimum (7.2)
for our experiments. Thus, to assess the effects of current up-
welling conditions, as well as scenarios reflecting future acidifi-
cation and deoxygenation, we evaluated fertilization success
across 40 levels of pH ranging from 7.95 to 7.2 pH crossed
with two DO concentrations representing normoxic and hyp-
oxic conditions (5.7 and 1.97 mg/l DO, respectively) at 13C.
Effects of acidification and ocean warming on
fertilization rates
To assess the effects of pH and temperature typical for present-
day upwelling (Figure 2b, pH 7.5, 9C) and future ocean acidifi-
cation and warming scenarios (pH 7.2–7.5, 18C), we evaluated
fertilization success across a range of pH, 7.95 to 7.2 pH,
across two temperatures (n ¼45 for 9 and 18C). It is important
to note that experimental syringes were stored at 8.5, and 18.5C
overnight prior to inoculation (see ‘Preparation of seawater treat-
ments’ section) in order to achieve targeted temperatures (9, and
Figure 2. pH, dissolved oxygen, and temperature (1 m above the
bottom, 25 April, 2013 – 14 June, 2013) from a near shore upwelling
zone (6 m depth; 36.624 N; 121.905 W). A. pH (total scale)
versus temperature (SeaFET pH sensor, Satlantic, Halifax, Canada). B.
Dissolved oxygen versus temperature (SBE 16 CTDO, Sea-Bird
Electronics, Inc., Washington, USA). The extreme pH and dissolved
oxygen and associated temperature during upwelling at this site are
noted in both panels.
4C. A. Boch et al.
18C, respectively) while accounting for the temperature of the
seawater used in the process of adding gametes (13C).
Subsequently, experimental syringes were held at 9 and 18C
(Figure 1b), as appropriate, during the period of gamete exposure
(600 s).
Statistical analysis
To evaluate proportional fertilization success as a function of
measured pH in combination with DO or temperature groups,
response data were logit transformed and evaluated for any sig-
nificant response to pH, DO, and temperature using Generalized
Linear Models (GLMs) in R (Warton and Hui, 2011).
Homogeneity of the data was assessed via visual inspection of re-
siduals versus GLM predicted logit transformed data
(Supplementary Figure S3). These GLM models were then incor-
porated into Segmented Model analysis to determine the exist-
ence of a threshold or a breaking point, and any changes in the
response slopes as a function of pH and any additional factors
(Muggeo, 2003,2008). Additional comparison of GLM and
Segmented Model fits were assessed using corrected Akaike
Information Criterion (AICc) with a reduction of AICc >10 by
the Segmented Model used as a conservative estimate for report-
ing a significant improvement relative to the GLM and further
evidence for non-linearity and a possible threshold.
Seawater treatment measurements
Overall, the mean difference between predicted pH and measured
pH was 0.03 60.03 pH units for the ocean acidification and hyp-
oxia experiment and 0.08 60.06 pH units for the ocean acidifica-
tion and ocean warming experiment. For the former experiment,
the mean DO for the control group was 5.70 60.35 mg/l DO,
5.72 60.35 mg/l for the High DO group, and 1.97 60.33 mg/l
for the hypoxic Low DO group. For the latter experiment, the
temperature in the incubation bins during the fertilization time
remained stable at 13.6, 9.3, and 17.9C for the Control, Low,
and High Temperature groups (Table 1). Measured pH showed a
high correlation with predicted pH values from CO
SYS for both
experiments with r
¼0.99 and r
¼0.91 (Figure 3a and b). The
lower r
value in the second regression experiment is attributed
to 3 pH outliers that were likely caused by inconsistent volume
mixture during preparation.
Fertilization response to ocean acidification and hypoxia
Overall, fertilization success decreased from 59 to 4% as pH
decreased from 7.9 to 7.18 for the low DO group (1.97 mg/l
DO). For the normoxic group (5.72 mg/l DO), fertilization
dropped from 58 to 3% as pH decreased from 7.84 to 7.15. In the
control group (7.81–7.89 pH, 5.70 mg/l DO), fertilization suc-
cess ranged from 59 to 38%. These results are shown in Figure 4a.
Results of the GLM model (Table 2, Model A) showed that the
pH DO interaction was not significant, indicating that DO did
not interact with pH to affect variation in fertilization success.
Further evaluation with Segmented Model analysis revealed a sig-
nificant threshold at a pH of 7.56 (60.03 SE) and a significant
change in the intercept and slope below this threshold estimate
[Table 3(A)]. These results indicate a greater drop in fertilization
success per unit of pH change below this point. AICc comparison
Figure 3. Measured vs. predicted pH. A. Correlation between SentrOn-Line 8100-100 ISFET probe (ion-sensitive field-effect transistor;
Sentron, Netherlands) measured pH output (total scale) versus CO2Sys predicted pH (total scale) from source water mixing at 13 C. B.
Correlation between SentrOn-Line 8100-100 ISFET probe (ion-sensitive field-effect transistor; Sentron, Netherlands) measured pH output (y-
axis) versus CO2Sys predicted pH from source water mixing for 9, 13, and 18 C. For both panels, open circles represents each sample
treatment from the experiment (n=92 and n=108 respectively). Dashed line is the linear regression line fit and the solid black line represents
1 to 1 unity. SentrON-Line 8100-100 probe was connected via RS232 cable to a PC and the data logged via DataLogger Suite software.
Table 1. Dissolved oxygen and temperature measurements from
experimental treatments.
Experiment Group n mean SD SE
A. Ocean acidification
and hypoxia
Control 12 5.70 0.35 0.10
Low DO 40 1.97 0.33 0.05
High DO 40 5.72 0.35 0.06
B. Ocean acidification
and warming
Control 5 13.56 0.06 0.11
Low Temp. 5 9.30 0.25 0.02
High Temp. 4 17.90 0 0
n¼number of samples. Dissolved oxygen (mg/l) during experiment A was
measured using NeoFox Hyoxy dissolved oxygen sensor (OceanOptics, FL,
USA). Temperature (C) during experiment B was measured using Taylor
Digital Probe Thermometer 9842 (Taylor Precision Products, New Mexico,
USA). Temperature samples were taken every 20 minutes over 80 minutes
(the full duration of experiment B).
Effects of current and future coastal upwelling conditions 5
of GLM with Segmented Model fits showed that the Segmented
Model was a significant improvement over the GLM (SM, Table
3, AICc >10) indicating a non-linear response of fertilization
success to decreasing pH.
Fertilization response to ocean acidification and
Similar to the ocean acidification and hypoxia experiment at
13C, fertilization rates decreased with decreasing pH when gam-
etes were exposed to the upwelling-like temperature of 9C
(Figure 4b). At 9C, fertilization decreased from 65 to 6% as
pH decreased from 7.85 to 7.14, with the Segmented Model ana-
lysis indicating a threshold at a pH of 7.52 (60.02 SE), below
which with a much steeper reduction in fertilization occurred. In
contrast to 9 and 13C treatment and an equivalent range of pH
exposure, fertilization success decreased from 74 to 21% as pH
decreased from 7.95 to 7.1 at 18C(Figure 4c). The results from
the GLM evaluation showed a significant increase in the intercept
and a decrease in the slope when the 18C exposure interacts with
the range of pH examined (Table 2B,p<0.001). Further evalu-
ation with the Segmented Model analysis showed a lack of break-
ing point or a significant change in the intercept and slope at the
warmer temperature [Table 3(C)] thus indicating the linear pre-
diction evaluated by GLM model is an appropriate predictor of
fertilization rates at this temperature. AICc comparison of GLM
with Segmented Model fits showed that the Segmented Model
was not a significant improvement over the GLM predictions
supporting the results of the Segmented Model analysis
(Supplementary Table S3, AICc <10).
This is the first evidence of direct and interactive effects of vari-
ation in pH, DO and temperature on fertilization success in red
abalone. Specifically, the results indicate that: (i) fertilization gen-
erally decreases with declining pH, with the presence of a thresh-
old and the difference in the rate of change in fertilization success
dependent on the temperature and pH interaction; (ii) warming
above ambient temperature interacts with pH, and ameliorates
the negative impact of low pH; (iii) DO has no discernable effect
on fertilization success, at least within the range of oxygen vari-
ation investigated in this study.
Both negative and resistance to low pH on marine inverte-
brate fertilization success have been reported in the literature
but these diverse outcomes are likely due to the range of pH
examined and to the limited understanding of the fertilization
mechanism that is being affected. Similar to our study, reduc-
tions in fertilization success with decreasing pH have been re-
ported for several species of molluscs and echinoderms
(Kurihara and Shirayama, 2004;Moulin et al., 2011;Barros
et al., 2013;Frieder, 2014;Scanes et al., 2014). In these studies,
low fertilization success was evident as gametes were exposed to
pH levels below 7.6. In contrast, fertilization success remained
high with decreasing pH for echinoderms (Heliocidaris tubercu-
lata, Heliocidaris erythrogramma, Tripneuestes gratilla,
Centrostephanus rodgersii, Patirriella regularis) and the abalone
Haliotis coccoradiata (Byrne et al., 2010). Although fertilization
success in these organisms was found to be resistant when
exposed to pH levels ranging from 8.2 to 7.6, the authors sug-
gested that this resistance might change under more severe levels
of pH. In a mechanistic context, these previous studies suggested
Figure 4. Abalone fertilization response to multiple stressors. A.
Open circles represent proportional fertilization at pH (total scale)
ranging from 7.9 to 7.2 and 6.0 mg/l dissolved oxygen. Black
solid dots represent responses to pH ranging from 7.9 to 7.2 and
1.5 mg/l dissolved oxygen. For all the treatments in this experiment,
temperature was maintained at 13 C. Solid black line represents
segmented model fit with dashed lines representing lower and upper
95% confidence limits. B. Proportional fertilization success (solid
blue circles) to pH ranging from 7.9 to 7.2 and 9 C. Solid black
line represents segmented model fit with dashed lines representing
lower and upper 95% confidence limits. C. Proportional fertilization
success (squares) to pH ranging from 7.9 to 7.2 and 18 C. Solid
black line represents GLM fit with dashed lines representing lower
and upper 95% confidence limits. For panels B-C, dissolved oxygen
was maintained at normoxic levels. For all panels,y-axis represent
proportional fertilization success and x-axis represent the measured
pH in each experimental sample (n¼92 for C and n¼108 for C). For
panels A and B, black solid circles represent break-point estimates
with error bars.
6C. A. Boch et al.
that lower pH alters sperm swimming behavior and or sperm
kinetics, ultimately negatively affecting fertilization outcomes.
For example, a reduction of sperm swimming speed and percent
motility was found to be significantly correlated with reduced
fertilization success for H. erythrogramma at a pH level of 7.7
(Havenhand et al., 2008). However, these results become con-
founding when compared with the negligible effects of pH re-
ported by Byrne et al. (2010) for the same species and therefore,
indicate that fertilization processes may be more complex. For
example, changes in pH may also affect sperm attractant chem-
icals released by eggs (Riffell et al., 2002), the activation process
during egg fertilization via interference of lysine dissolution of
the egg membrane (Kresge et al., 2001), or the increase in H þ
may ionically interfere with the explosive wave of calcium neces-
sary for signalling egg activation and initiation of mitotic div-
ision (reviewed by Whitaker, 2006). Thus, while there is
evidence for negative effects of low pH on fertilization success,
the underlying physical or biological mechanisms require further
evaluation—e.g. via biochemical tracing experiments—to fully
understand when pH induces negative versus resistant
The ameliorating effect of warming over the negative effects of
pH below a threshold point has not been previously reported in
invertebrate fertilization studies but this may be due to differ-
ences in our experimental design relative to prior studies. For ex-
ample, in order to evaluate the effects of changing temperature
and pH, Byrne et al. (2010) examined fertilization success with a
4-degree warming from ambient (20C) coupled with 0.6 U de-
crease in pH from ambient (8.2 pH) for the tropical abalone H.
coccoradiata and a 6-degree warming from ambient (20C)
coupled with 0.6 U decrease in pH from ambient (8.2 pH) for
several species of echinoids. Under these conditions, fertilization
success was found to be resistant to both drivers and without ap-
parent interaction—i.e. fertilization rates remained high under all
conditions. Haliotis coccoradiata and the echinoids examined by
Byrne et al. (2010) are distributed at latitudes where near-shore
temperatures average 20C and where optimal fertilization suc-
cess in these organisms have demonstrated to be 20C(Wong
et al., 2010) and as such, an experimental temperature exposure
of 18–26C may not adequately capture the interactive effects of
warming temperature and lower pH. That is, an ameliorating ef-
fect may be only observed if the experimental treatments include
both warming and cooling comparisons of the same magnitude
from the optimal temperature in combination with a fuller range
of pH. Thus, experiments that examine co-variates moving in
both directions from the optimal may reveal different patterns of
multiple driver effects.
The negligible effect of low DO or hypoxia on fertilization
observed in this study was unexpected. Oxygen is a critical driver
of metabolic processes at multiple organismal scales. As oxygen is
the terminal electron acceptor during mitochondrial energy pro-
duction, loss of available oxygen would be expected to reduce the
metabolic energy needed for flagellum activity or sperm propul-
sion. Thus, a reduction in sperm motility is expected to have
negative consequences for sperm:egg interactions and ultimately
fertilization success. For example, low levels of DO have been re-
ported to reduce sperm swimming kinetics in marine species
(Shin et al., 2014;Graham et al., 2016). However, as the overall
role of oxygen in the metabolism involves creating a proton gra-
dient, a decrease in seawater pH, or related changes in seawater
carbonate parameters, may disrupt this proton gradient, or the ef-
fects of pH may dominate any effects of oxygen variation, at least
over the scales examined. Indeed, Graham et al. (2016) observed
an increase in the swimming speed of sea urchin sperm under
lower pH and hypoxic conditions. However, despite the antagon-
istic interactive effects of pH and hypoxia on sperm motility, they
also observed a synergistic decrease in fertilization under these
combined drivers. Those results differ from our findings and may
be indicative of differing species-specific responses. Furthermore,
seawater conditions to which our adult brooders were acclimated
to during reproductive development—which we did not
characterize—may also influence fertilization outcomes. Negative
impacts of seawater conditions during this phase are unlikely be-
cause the aquaculture tanks are highly aerated and the brooders
are cultured separately and under low densities to minimize oxi-
dative stress (Boch, pers. commun. with American Abalone
Farms). Thus, while it may be possible that the effects of seawater
pH may be greater than the effects of DO, future experiments
Table 2. Statistical results for proportion fertilized in each experiment.
Model Estimate SE z-value p-value
A. Model for pH and DO experiment: (Intercept) 24.69 1.16 21.22 ***
y¼pH þDO Group þpH * DO Group þepH 3.19 0.16 20.41 ***
Low DO 1.66 1.61 1.03 NS
pH x Low DO 0.23 0.22 1.07 NS
Null Deviance: 1316.42 (79 d.f.)
Residual Deviance: 464.05 (76 d.f.)
AIC: 879.25
B. Model for pH and temperature experiment: (Intercept) 20.83 0.89 23.37 ***
y¼pH þTemp. Group þpH * Temp. Group þepH 2.67 0.12 22.46 ***
High Temp. 16.75 1.13 14.88 ***
pH x High Temp 2.13 0.15 14.15 ***
Null Deviance: 2829.6 (89 d.f.)
Residual Deviance: 1514.4 (86 d.f.)
AIC: 2023.00
A. GLM model evaluation of pH and dissolved oxygen effects with 5.9 mg/l dissolved oxygen group as the reference (surface water) data and 1.9 mg/l group as
the comparative hypoxic group. Both data are constant at 13 C. B. GLM evaluation for pH and temperature dual stressor experiment with 9 C group (upwell-
ing) as the reference temperature and 18 C data as the comparative group (ocean warming). y= proportional fertilization response; e¼error term; DO ¼dis-
solved oxygen; Temp. ¼temperature; * ¼p<0.05; ** ¼p<0.01; *** ¼p<0.001.
Effects of current and future coastal upwelling conditions 7
should control for conditions during reproductive development
to limit pre-existing exposures and to clarify experimental
Prevailing theory based on aerobic scope suggests that expos-
ure to anomalously high temperature and a secondary driver such
as decreasing pH conditions would have a synergistic or an addi-
tive effect on fertilization success—i.e. dual stressors are predicted
to narrow the window of biological performance (Portner and
Farrell, 2008). However, our study shows that the effects of pH
can be dominant over a secondary stressor such as hypoxia and in
addition, the effects of ocean warming can ameliorate the effects
of decreasing pH for fertilization success. Furthermore, it is
unclear how warming temperatures in combination with hypoxic
exposure may affect fertilization success. Altogether, our experi-
mental results suggest that a synergistic or an additive response
may not adequately describe the full scope of biological perform-
ance under multiple drivers or stressors—at least in the context
of fertilization success. Based on our and other experimental
studies on multiple environmental drivers, we instead suggest
that the effects of multiple drivers can be complex and lead to re-
sistance, dominance or amelioration. Importantly, our results
show that non-linear responses and thresholds can also occur,
highlighting the need to examine biological responses across con-
tinuous stressor gradients.
Table 3. Break-Point estimation.
A. Ocean acidification and hypoxia (all data)
Segmented model y¼pH þUþpsi þe
Estimate SE z-value p-value
(Intercept) 32.78 1.68 19.55 ***
pH 4.28 0.23 18.85 ***
U3.10 0.47 6.67 NA
Null seviance: 1316.42 (79 d.f.)
Residual deviance: 413.25 (76 d.f.)
AIC: 828.46
Segmented model BP estimate 7.56 60.03 SE
Davies test BP estimate 7.56***
Estimate SE LCI (95%) UCI (95%)
Slope segment 1 4.29 0.23 3.83 4.74
Slope segment 2 1.18 0.41 0.37 1.99
B. Ocean acidification and warming (9 C dataset)
Segmented model y¼pH þUþpsi þe
Estimate SE z-value p-value
(Intercept) 35.62 2.20 16.19 ***
pH 4.69 0.30 15.69 ***
U4.13 0.49 8.36 NA
Null deviance: 1094.28 (44 d.f.)
Residual deviance: 495.05 (41 d.f.)
AIC: 748.04
Segmented model BP estimate 7.52 60.02 SE
Davies test BP estimate 7.52***
Estimate SE LCI (95%) UCI (95%)
Slope segment 1 4.69 0.30 4.08 5.29
Slope segment 2 0.56 0.39 0.24 1.35
C. Ocean acidification and warming (18 C dataset)
Segmented model y¼pH þUþpsi þe
Estimate SE z-value p-value
(Intercept) 2.44 1.01 2.41 *
pH 0.32 0.14 2.33 *
U 1.69 0.71 2.39 NA
Null deviance: 977.09 (44 d.f.)
Residual deviance: 933.94 (41 d.f.)
AIC: 1197.5
Segmented model BP estimate 7.72 60.06 SE
Davies test BP estimate 7.73*
Estimate SE LCI (95%) UCI (95%)
Slope segment 1 0.32 0.14 0.04 0.6
Slope segment 2 2.01 0.70 0.61 3.42
For each experiment, data are evaluated according to Table 2 GLM results with Segmented Model regression and Davies Test Breaking-Point estimation
(Muggeo, 2008). A. Results for ocean acidification + hypoxia experiment data evaluated as a single dataset. B. Results for ocean acidification + 9 C experiment
data evaluated as an independent dataset. C. Results for ocean acidification + 18 C experiment data evaluated as an independent dataset. U ¼difference in
slopes of the two segments; psi = breaking point estimate at each step with standard error; e¼error term; BP ¼Breaking Point; S.E. ¼6standard error; NA ¼
Not Applicable. LCI ¼Lower Confidence Interval; UPI ¼Upper Confidence Interval; * ¼p<0.05; ** ¼p<0.01; *** ¼p<0.001.
8C. A. Boch et al.
Understanding the influence of multiple co-varying environ-
mental factors on the success of red abalone populations or simi-
lar marine organisms with complex life cycles requires that we
disentangle the individual and combined effects of variation in
key environmental drivers. In addition, it requires an understand-
ing of the net outcomes that integrate the vulnerability of each
life history stage. For example, although we found that fertiliza-
tion rates increased with higher temperatures and decreased with
upwelling-like conditions, H. rufescens have been found to have
higher gonadal development under cooler phases of the
California current (Vilchis et al., 2005). Furthermore, male aba-
lone exhibited a significant reduction of sperm production after 6
months of exposure to 18C(Rogers-Bennett et al., 2010). Thus,
if abalone populations, which have been observed to be gravid
from a few months to year-round (Boolootian et al., 1962) re-
spond to selection from the positive, negative, and other effects of
environmental variation on gamete production and fertilization,
the net demographic outcome may either be lessened or amplified
by the ensemble of environmental drivers acting differentially on
each life stage.
Our integrated approach to examine the effects of multiple en-
vironmental drivers provide new insights concerning the expected
consequences of future changes in ocean conditions for abalone
populations that were unlikely to be detected in single factor ex-
periments. For the CCLME, our results suggest that red abalone
fertilization success is at a possible tipping point under current
upwelling events—i.e. 7.5 pH and 9–13C. Our results also sug-
gest that the effects could be further detrimental to fertilization
success if ocean acidification causes a further reduction in sea-
water pH below 7.5. As gaps in our understanding remain, ex-
panding integrated approaches will be critical to disentangle the
effects of climate change and natural variability in multiple envir-
onmental drivers as we endeavor to predict and manage changes
in marine populations.
Supplementary data
Supplementary material is available at the ICESJMS online ver-
sion of the manuscript.
We are grateful to Kurt Buck, Patrick Whaling, Dale Graves,
Joshua Lord, Jody Beers, Peter Hain, Tom Ebert, and numerous
volunteers who helped with the experiments.
This study was supported by the US NSF-OA Programme (award
no. OCE-1416934) and through the US NSF-CNH Programme
(award no. DEB-1212124), and support from the David and
Lucile Packard Foundation.
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Handling editor: David M. Fields
10 C. A. Boch et al.
... For example, low DO and low pH act synergistically to reduce the survival and growth of juvenile red abalone (Haliotis rufescens) 23 , while increased temperature counteracts the negative impacts of low pH on fertilization rates in the same species 23 . Such interactions suggest that understanding how temperature, DO, and pH co-vary and how these conditions affect the local biota is critical for predicting biological responses to climate change 3,9,24,25 . Here, we show how variability in co-varying environmental drivers (T, DO, pH) may change under future conditions (increasing levels of CO 2 ) and how these scenarios affect exposure of nearshore organisms to potentially stressful conditions. ...
... Fertilization response. We estimated fertilization success using results of Boch et al. 25 where the fertilization response of red abalone (Haliotis rufescens) was quantified in response to multiple stressor climate conditions (high temperature, low DO, and low pH). Fertilization in abalone occurs over relatively short periods, therefore E int would not provide an appropriate estimate in such cases. ...
... While the process of fertilization occurs over short periods, adult red abalone exhibit an extended spawning season, over which environmental conditions may vary greatly based on our modeling results. Thus, we used the equations from Boch et al. 25 to examine how fertilization success over a one-month period might be affected by environmental variability, specifically the interactive effects of ph and temperature. Changes in DO did not show a strong effect on fertilization in their experiments ( Fig. 4 where β 0 and β pH are intercepts, β and β 2 are slope segments, β A is slope of the pH-temperature interaction (pH × Temperature Group), β B is accounts for high temperature effects, and BP is the curve breaking point. ...
Full-text available
Climate change is expected to warm, deoxygenate, and acidify ocean waters. Global climate models (GCMs) predict future conditions at large spatial scales, and these predictions are then often used to parameterize laboratory experiments designed to assess biological and ecological responses to future change. However, nearshore ecosystems are affected by a range of physical processes such as tides, local winds, and surface and internal waves, causing local variability in conditions that often exceeds global climate models. Predictions of future climatic conditions at local scales, the most relevant to ecological responses, are largely lacking. To fill this critical gap, we developed a 2D implementation of the Regional Ocean Modeling System (ROMS) to downscale global climate predictions across all Representative Concentration Pathway (RCP) scenarios to smaller spatial scales, in this case the scale of a temperate reef in the northeastern Pacific. To assess the potential biological impacts of local climate variability, we then used the results from different climate scenarios to estimate how climate change may affect the survival, growth, and fertilization of a representative marine benthic invertebrate, the red abalone Haliotis rufescens, to a highly varying multi-stressor environment. We found that high frequency variability in temperature, dissolved oxygen (DO), and pH increases as pCO2 increases in the atmosphere. Extreme temperature and pH conditions are generally not expected until RCP 4.5 or greater, while frequent exposure to low DO is already occurring. In the nearshore environment simulation, strong RCP scenarios can affect red abalone growth as well as reduce fertilization during extreme conditions when compared to global scale simulations.
... Therefore, the roles of multiple stressor combinations, and of temporal patterns of exposure to these stressors, need to be better studied experimentally. Researchers have begun to tackle these questions for different stressors in various organisms and ecosystems (e.g., Kim et al. 2013;Ferrari et al. 2015;Britton et al. 2016;Clark and Gobler 2016;Boch et al. 2017), but more studies addressing these effects over a wide range of marine ecosystems, ecological processes and taxonomic groups will be needed to inform predictions of global change impacts on ecosystems. ...
... This experiment aimed to quantify temperature effects on abalone critical oxygen thresholds by exposing them to progressively decreasing DO levels, at different seawater temperatures. These DO levels and the different temperatures represent a range of conditions that have been recorded in both central and Baja California, though not always simultaneously (Boch et al. 2017(Boch et al. , 2018.We implemented an experimental schedule in which DO was lowered through 6 decreasing levels from 7 to 2 mg/L. At each level, we maintained the DO concentrations for a period of 30 min to allow for metabolic rate measurements to be conducted, before allowing DO concentrations to continue decreasing. ...
Marine organisms and ecosystems face multiple, temporally variable stressors in a rapidly changing world. Realistic experiments that incorporate these aspects of physiological stress are important for advancing our ability to understand, predict, and manage their ecological impacts. However, the experimental systems needed to conduct such experiments can be costly. Here, we describe a low‐cost, modular control system that can be used with seawater sensors and actuators to dynamically manipulate multiple seawater variables. It enables researchers to run a variety of realistic multiple‐stressor, variable exposure experiments with a range of marine organisms. This tank controller system is based on the open‐source Arduino prototyping platform and features a custom‐made circuit board with a 16‐bit analog‐to‐digital converter, a real‐time clock, a MicroSD memory card reader, a high‐voltage transistor array, and solderless screw terminal connectors for easy connection of sensors, actuators, and power supplies. The assembly and use of this controller system does not require extensive electronics engineering or programming experience, and each module can be assembled for under 80 USD in parts. To demonstrate the system's capabilities, we present seawater manipulations from experiments involving (1) simultaneous manipulations of dissolved oxygen and pH; (2) fluctuating dissolved oxygen levels; and (3) a controlled stepwise decrease in dissolved oxygen at different temperatures. The low cost and high customizability of this Arduino‐based control system can contribute to expanding capacities for running global change experiments for researchers and students worldwide.
... The majority of studies, which predominantly evaluate echinoderms (largely urchins) and bivalve molluscs, conclude that acidification negatively affects fertilization rate when gametes are directly exposed (Smith and Clowes, 1924;Havenhand et al., 2008;Parker et al., 2009;Ericson, 2010;Parker et al., 2010;Kimura et al., 2011;Moulin et al., 2011;Schlegel et al., 2012;Van Colen et al., 2012;Barros et al., 2013;Gonzales-Bernat et al., 2013;Uthicke et al., 2013;Foo et al., 2014;Frieder, 2014;Scanes et al., 2014;Suckling et al., 2014;Sung et al., 2014;Riba et al., 2016;Boch et al., 2017;Shi et al., 2017a;Shi et al., 2017b;Szalaj et al., 2017;Zhan et al., 2017;Basallote et al., 2018;Garcıá et al., 2018;Sẃiezȧk et al., 2018;Zhan et al., 2018;Smith et al., 2019;Sui et al., 2019;Wang et al., 2020), or parents are exposed prior to fertilization (Graham et al., 2015). The pH level at which fertilization rate becomes compromised varies considerably across taxa, and also depends on factors such as sperm and egg concentration (Ericson, 2010;Albright, 2011a;Ericson et al., 2012;Albright and Mason, 2013;Gonzales-Bernat et al., 2013;Ho et al., 2013;Frieder, 2014), gamete incubation time (Bechmann et al., 2011;Gianguzza et al., 2014;Garcıá et al., 2018;Kong et al., 2019), inter-individual and mating pair variability (Schlegel et al., 2012;Foo et al., 2014;White et al., 2014;Smith et al., 2019), collection location and time of year (Martin et al., 2011;Cohen-Rengifo et al., 2013;Pecorino et al., 2014;Riba et al., 2016;Basallote et al., 2018;Garcıá et al., 2018;da Silva Souza et al., 2019;Pereira et al., 2020;Caetano et al., 2021), and species hybridization (Striewski, 2012). ...
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Sexual reproduction is a fundamental process essential for species persistence, evolution, and diversity. However, unprecedented oceanographic shifts due to climate change can impact physiological processes, with important implications for sexual reproduction. Identifying bottlenecks and vulnerable stages in reproductive cycles will enable better prediction of the organism, population, community, and global-level consequences of ocean change. This article reviews how ocean acidification impacts sexual reproductive processes in marine invertebrates and highlights current research gaps. We focus on five economically and ecologically important taxonomic groups: cnidarians, crustaceans, echinoderms, molluscs and ascidians. We discuss the spatial and temporal variability of experimental designs, identify trends of performance in acidified conditions in the context of early reproductive traits (gametogenesis, fertilization, and reproductive resource allocation), and provide a quantitative meta-analysis of the published literature to assess the effects of low pH on fertilization rates across taxa. A total of 129 published studies investigated the effects of ocean acidification on 122 species in selected taxa. The impact of ocean acidification is dependent on taxa, the specific reproductive process examined, and study location. Our meta-analysis reveals that fertilization rate decreases as pH decreases, but effects are taxa-specific. Echinoderm fertilization appears more sensitive than molluscs to pH changes, and while data are limited, fertilization in cnidarians may be the most sensitive. Studies with echinoderms and bivalve molluscs are prevalent, while crustaceans and cephalopods are among the least studied species even though they constitute some of the largest fisheries worldwide. This lack of information has important implications for commercial aquaculture, wild fisheries, and conservation and restoration of wild populations. We recommend that studies expose organisms to different ocean acidification levels during the entire gametogenic cycle, and not only during the final stages before gametes or larvae are released. We argue for increased focus on fundamental reproductive processes and associated molecular mechanisms that may be vulnerable to shifts in ocean chemistry. Our recommendations for future research will allow for a better understanding of how reproduction in invertebrates will be affected in the context of a rapidly changing environment.
... Some organisms appear to be highly susceptible to changes in carbonate chemistry (e.g., molluscs, corals, echinoderms, calcified seaweeds) (Kroeker et al., 2013). In particular, exposure of early life stages to low pH water may have disproportionate effects on calcifying species, such as abalone, due to the sensitivities of larval development and juvenile performance to changing environmental conditions (Byrne et al., 2011;Kim et al., 2013;Boch et al., 2017). Impairment of early development can have long lasting carryover effects on fitness that may affect growth, physiological performance, disease resistance, and other traits important to maintaining a high product quality for the aquaculture industry. ...
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Integrated multi-trophic aquaculture (IMTA) has the potential to enhance growth, reduce nutrient loads, and mitigate environmental conditions compared to traditional single-species culture techniques. The goal of this project was to develop a land-based system for the integrated culture of seaweeds and shellfish, to test the efficacy of integrated versus non-integrated designs, and to assess the potential for IMTA to mitigate the effects of climate change from ocean acidification on shellfish growth and physiology. We utilized the red abalone (Haliotis rufescens) and the red seaweed dulse (Devaleraea mollis) as our study species and designed integrated tanks at three different recirculation rates (0%, 30%, and 65% recirculation per hour) to test how an integrated design would affect growth rates of the abalone and seaweeds, modify nutrient levels, and change water chemistry. We specifically hypothesized that IMTA designs would raise seawater pH to benefit calcifying species. Our results indicated that juvenile abalone grew significantly faster in weight (22% increase) and shell area (11% increase) in 6 months in tanks with the highest recirculation rates (65%). The 65% recirculation treatment also exhibited a significant increase in mean seawater pH (0.2 pH units higher) due to the biological activity of the seaweed in the connected tanks. We found a significant positive relationship between the mean pH of seawater in the tanks and juvenile abalone growth rates across all treatments. There were no significant differences in the growth of dulse among treatments, but dulse growth did vary seasonally. Seawater phosphate and nitrate concentrations were depleted in the highest recirculation rate treatment, but ammonium concentrations were elevated, likely due to the abalone effluent. Overall, our results indicate that there are benefits to IMTA culture of seaweeds and abalone in terms of improving growth in land-based systems, which will reduce the time to market and buffer commercial abalone operations against the effects of ocean acidification during vulnerable early life stages.
... For example, pH is also known to correlate closely with temperature and dissolved oxygen during upwelling events 25,55 , and also to influence hypoxia tolerance 56 . Simultaneous exposures to oxygen, pH and temperature variation and extremes are likely to produce additive, synergistic or antagonistic effects on organisms 57,58 . ...
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Declining oxygen is one of the most drastic changes in the ocean, and this trend is expected to worsen under future climate change scenarios. Spatial variability in dissolved oxygen dynamics and hypoxia exposures can drive differences in vulnerabilities of coastal ecosystems and resources, but documentation of variability at regional scales is rare in open-coast systems. Using a regional collaborative network of dissolved oxygen and temperature sensors maintained by scientists and fishing cooperatives from California, USA, and Baja California, Mexico, we characterize spatial and temporal variability in dissolved oxygen and seawater temperature dynamics in kelp forest ecosystems across 13° of latitude in the productive California Current upwelling system. We find distinct latitudinal patterns of hypoxia exposure and evidence for upwelling and respiration as regional drivers of oxygen dynamics, as well as more localized effects. This regional and small-scale spatial variability in dissolved oxygen dynamics supports the use of adaptive management at local scales, and highlights the value of collaborative, large-scale coastal monitoring networks for informing effective adaptation strategies for coastal communities and fisheries in a changing climate.
... Once abundant populations of this species supported both commercial and recreational fisheries, but with recent widespread population collapses (22,23), commercial aquaculture now serves as the only source for abalone in the United States. OA poses a broad threat to the sustainable commercial and restoration aquaculture of this and other abalone species worldwide (5,(24)(25)(26)(27)(28)(29). During early life, the nonfeeding (lecithotrophic) larvae of abalone depend on maternally provisioned energy reserves, much of which occurs in the form of yolk lipids (30), which support numerous physiological processes (31). ...
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Significance The pH of the global ocean is decreasing due to the absorption of anthropogenically emitted CO 2 , causing ocean acidification (OA). OA negatively impacts marine shellfish and threatens the continuing economic viability of molluscan shellfish aquaculture, a global industry valued at more than 19 billion USD. We identify traits linked to growth and lipid regulation that contribute tolerance to OA in abalone aquaculture, with broader implications for adaptation efforts in other shellfish species. We also identify evolved heritable variation for physiological resilience to OA that may be exploited in commercial and restoration aquaculture breeding programs to offset the negative consequences of continuing climate change.
... Amongst the shelled molluscs, abalones (class Gastropoda, genus Haliotis) are important farmed and fished commercial species worldwide, considered a delicacy in many regions (Cook, 2014). Experiments suggest that Haliotids will be susceptible to future OA levels, including at fertilization (Boch et al., 2017but see Byrne et al., 2011, the larval (Byrne et al., 2011), and juvenile (Kim et al., 2013) stages. The New Zealand black footed abalone (Haliotis iris) or p aua is a key coastal species with cultural, economic, and ecological importance, with a large market both for abalone flesh and decorative shells. ...
Larval settlement is a key process in the lifecycle of benthic marine organisms; however, little is known on how it could change in reduced seawater pH and carbonate saturation states under future ocean acidification (OA). This is important, as settlement ensures species occur in optimal environments and, for commercially important species such as abalone, reduced settlement could decrease future population success. We investigated how OA could affect settlement success in the New Zealand abalone Haliotis iris by examining: (1) direct effects of seawater at ambient (pHT 8.05) and reduced pHT (7.65) at the time of settlement, (2) indirect effects of settlement substrates (crustose coralline algae, CCA) preconditioned at ambient and reduced pHT for 171 days, and (3) carry-over effects, by examining settlement in larvae reared to competency at ambient and reduced pHT (7.80). We found no effects of seawater pH or CCA incubation on larval settlement success. OA-induced carry-over effects were evident, with lower settlement in larvae reared at reduced pH. Understanding the mechanisms behind these responses is key to fully comprehend the extent to which OA will affect marine organisms and the industries that rely on them.
We present an approach for estimating drag coefficients for depth-averaged tidal flows that uses the ratio of observed RMS velocities to the RMS velocities that would be observed without bottom friction. We find that this ratio, R, depends on a single non-dimensional number, P=CDCη0/ωH2, where CD is the drag coefficient, and C is the phase speed of a tidal wave with amplitude η0 and frequency ω, in water of depth h. The function R(P) can be inverted to solve for CD using measured values of R. Taking advantage of a unique multi-year record of tidal flows on Isla Nativdad, Baja California, Mexico, during which time the kelp forest there varied between non-existent and dense, we use this method to quantify the effect of kelp biomass on drag. This analysis shows that a maximum value of CD ≈ 0.04 is reached for relatively low values of kelp biomass, which may be an effect of sheltering (reductions in the velocity creating drag due to the close proximity of bundles of kelp stipes). However, values as large as 0.015 were observed when the water column experienced strong secondary flows in the presence of strong density stratification. Given that the long-term measurements were made near a coastal headland, we argue that this may reflect variations in secondary flow strength due to stratification. Lastly, our measurements show little evidence of enhancement of drag by surface waves.
In recent decades, the marine environment has been seriously affected by various anthropogenic activities (e.g., deforestation, fossil fuel combustion, and disordered discharges of pollutants). As a consequence, a range of changes in seawater environmental factors have taken place in oceans around the world, including increased temperature, reduced pH and dissolved oxygen, salinity fluctuation, and many other anomalous alterations in environmental factors, and these changes have aroused concerns from scientists. It has been widely reported that these changes in environmental factors would impact marine organisms severely. Meanwhile, it is worth noting that the environmental stressors mentioned above are rarely occurring independently in nature. Thus marine organisms are usually threatened by many different environmental stressors, and there would be complex and unpredicted interactions among the stressors. Generally, the interactive effects varied among additive (total effect equal to the sum of individual effects), synergistic (total effect greater than the sum of individual effects), or antagonistic (total effect less than the sum of individual effects), depending on the species and life stages of the studied organism, and the nature of the stressors themselves. It is necessary to figure out the interactive effects among various environmental stressors on specific marine organisms to accurately predict their physiological states and population dynamics under future climate scenarios. Therefore in this chapter, we summarize the related experiments in the last 20 years to discuss the interactive effects of ocean acidification (OA) combined with four other typical environmental stressors, namely ocean warming, hypoxia, salinity fluctuation, and heavy metal pollution, on marine organisms according to previously published studies. The authors hope that the contents of this chapter provide some basic information about the interactive effects of OA and the other four environmental factors for readers who are interested in this subject area.
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Understanding spatial and temporal patterns in the recruitment of marine invertebrates with complex life histories remains a critical knowledge gap in marine ecology and fisheries. As marine invertebrates are facing multiple stressors from overfishing and climatic stress, it is important to evaluate the conditions that facilitate recruitment in low-density populations. The red abalone Haliotis rufescens historically supported an economically important fishery in California, but the fishery was sequentially closed as stocks declined, and the last fished area was closed in 2018 following the collapse of the kelp forests in Northern California. Here, red abalone recruitment was evaluated annually from 2012 to 2016 and monthly from 2016 to 2017 in Central California where red abalone naturally occur in highly aggregated but low-density populations because of sea otter predation. Trends in wind-driven upwelling, temperature, wave forces, and food resources (kelp) were evaluated over the same time period as factors that could affect recruitment patterns. Recruitment was annually consistent except in 2015, when recruitment declined by 76%, likely because of reproductive failure during the second year of the North Pacific marine heat wave. The monthly recruitment assessment was the first field assessment of red abalone recruitment over a full year, and it showed that red abalone can recruit year-round. There were no clear recruitment patterns associated with seasonal wind-driven upwelling or relaxation patterns, and kelp density was constant over the study period; however, conditions at the study sites included three key features that may provide optimal conditions for consistent recruitment: (1) spatial abalone aggregation, (2) a sheltered embayment that may retain larvae, and (3) persistent algal food resources. These results can inform statewide and global abalone recovery and management programs.
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Exposure of nearshore animals to hypoxic, low-pH waters upwelled from below the continental shelf and advected near the coast may be stressful to marine organisms and lead to impaired physiological performance. We mimicked upwelling conditions in the laboratory and tested the effect of fluctuating exposure to water with low-pH and/or low-oxygen levels on the mortality and growth of juvenile red abalone (Haliotis rufescens, shell length 5–10 mm). Mortality rates of juvenile abalone exposed to low-pH (7.5, total scale) and low-O2 (40% saturation, mg L−1) conditions for periods of 3 to 6 h every 3–5 days over 2 weeks did not differ from those exposed to control conditions (O2: 100% saturation, 12 mg L−1; pH 8.0). However, when exposure was extended to 24 h, twice over a 15-day period, juveniles experienced 5–20% higher mortality in the low-oxygen treatments compared to control conditions. Growth rates were reduced significantly when juveniles were exposed to low-oxygen and low-pH treatments. Furthermore, individual variation of growth rate increased when juveniles were exposed simultaneously to low-pH and low-O2 conditions. These results indicate that prolonged exposure to low-oxygen levels is detrimental for the survival of red abalone, whereas pH is a crucial factor for their growth. However, the high individual variation in growth rate under low levels of both pH and oxygen suggests that cryptic phenotypic plasticity may promote resistance to prolonged upwelling conditions by a portion of the population.
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Abalone have been exploited commercially at La Natividad Island, Baja California since about 1956. The fishery for Haliotis sorenseni collapsed after about 7 years, and those for Haliotis corrugata and Haliotis fulgens in 1984. Subsequently, the fishery recovered somewhat before the recent decline in 1994 to 1997. Egg-per-recruit (EPR) analyses for the two major species were carried out with information on growth rate, fishing mortality rate, and size at sexual maturity and other data obtained mostly during the 1990s. Egg production conserved before the 1984 collapse was probably somewhat low for H. corrugata at ~30-40% of the maximum possible in unfished conditions, and certainly low for H. fulgens at ~20%. After the collapse with better control of the fishery, the egg production improved slightly for H. corrugata to ~30-50%, and for H. fulgens to ~25-40%, but from 1995 has declined again as fishing mortality increased. The periodic El Ninos cause elevated sea temperatures and loss of Macrocystis in the region. The total abalone catch from 1965 to 1996 was correlated with mean sea surface temperature anomalies with a lag of 8 years, which is the average period from larval settlement until recruitment into the fishery. This implies that sea temperature anomalies have a positive effect on recruitment. On the other hand, there is also slight evidence of recruitment failure during severe El Ninos. Although environmental variables and recruitment overfishing can each cause reductions in catch, the presence of both makes a decline practically inevitable. Quota managed fisheries must take into account environmental effects on recruitment if they wish to avert declines.
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The distribution and function of many marine species is largely determined by the effect of abiotic drivers on their reproduction and early development, including those drivers associated with elevated CO2 and global climate change. A number of studies have therefore investigated the effects of elevated pCO2 on a range of reproductive parameters, including sperm motility and fertilisation success. To date, most of these studies have not examined the possible synergistic effects of other abiotic drivers, such as the increased frequency of hypoxic events that are also associated with climate change. The present study is therefore novel in assessing the impact that an hypoxic event could have on reproduction in a future high CO2 ocean. Specifically, this study assesses sperm motility and fertilisation success in the sea urchin Paracentrotus lividus exposed to elevated pCO2 for 6 months. Gametes extracted from these pre-acclimated individuals were subjected to hypoxic conditions simulating an hypoxic event in a future high CO2 ocean. Sperm swimming speed increased under elevated pCO2 and decreased under hypoxic conditions resulting in the elevated pCO2 and hypoxic treatment being approximately equivalent to the control. There was also a combined negative effect of increased pCO2 and hypoxia on the percentage of motile sperm. There was a significant negative effect of elevated pCO2 on fertilisation success, and when combined with a simulated hypoxic event there was an even greater effect. This could affect cohort recruitment and in turn reduce the density of this ecologically and economically important ecosystem engineer therefore potentially effecting biodiversity and ecosystem services.
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Ecosystem productivity in coastal ocean upwelling systems is threatened by climate change. Increases in spring and summer upwelling intensity, and associated increases in the rate of offshore advection, are expected. While this could counter effects of habitat warming, it could also lead to more frequent hypoxic events and lower densities of suitable-sized food particles for fish larvae. With upwelling intensification, ocean acidity will rise, affecting organisms with carbonate structures. Regardless of changes in upwelling, near-surface stratification, turbulent diffusion rates, source water origins, and perhaps thermocline depths associated with large-scale climate episodes (ENSO) maybe affected. Major impacts on pelagic fish resources appear unlikely unless couples with overfishing, although changes toward more subtropical community composition are likely. Marine mammals and seabirds that are tied to sparsely distributed nesting or resting grounds could experience difficulties in obtaining prey resources, or adaptively respond by moving to more favorable biogeographic provinces.
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Changes in ocean temperature can have direct and indirect effects on the population dynamics of marine invertebrates. We examined the impacts of warn water, starvation, and disease on reproduction in red abalone (Haliotis rufescens). We found that sperm production was highly sensitive to warm water and starvation, suggesting there may be a dramatic temperature threshold above which sperm production fails. Wild males from northern (72%) and southern (81%) California had sperm. In contrast, only 30% of the males exposed to warm water (18 degrees C) for 6 mo or starvation for 13 mo had sperm, with spermatogenesis dropping dramatically from 300,000 presperm cells/mm(3) (wild) to 46,000 presperm cells/mm(3) (warm water) and 84,000 presperm cells/mm(3) (starvation). In a longer warm-water experiment (12 mo), males had total reproductive failure in temperatures greater than 16 degrees C, irrespective of food treatment. Egg production was less sensitive to warm water, but was impacted more by starvation, especially food quantity relative to quality. Wild females from northern (97%) and southern (100%) California had mature oocytes averaging 3 million eggs and 21 million eggs, respectively. Females exposed to 18 degrees C water for 6 mo had diminished fecundity, averaging only 400,000 mature eggs whereas females in the starvation experiment did not produce any mature eggs. Normal sperm and egg production was found in abalone testing positive for Rickettsiales-like-prokaryote (12 LP), the agent of Withering Syndrome in cool water. However, abalone with RLP also exposed to warm water developed the disease withering syndrome and did not produce any mature gametes. The temperature-mediated lethal and sublethal effects on red abalone reproduction described here, combined with temperature's known impacts on abalone growth, kelp abundance, and disease status, clearly demonstrate population-level consequences. We suggest that temperature needs to be explicitly incorporated into red abalone recovery and management planning, because California's ocean has warmed and is predicted to warm in the future.
Chemical communication between sperm and egg is a key factor mediating sexual reproduction. Dissolved signal molecules that cause sperm to orient and accelerate towards an egg could play pivotal roles in fertilization success, but such compounds are largely undescribed. This investigation considered the behavioral responses of red abalone (Haliotis rafescens) sperm to soluble factors released into sea water by conspecific eggs. Sperm in proximity to individual live eggs swam significantly faster and oriented towards the egg surface. Bioassay-guided fractionation was employed to isolate the chemoattractant, yielding a single pure, fully active compound after reversed-phase and size-exclusion high-performance liquid chromatography. Chemical characterization by nuclear magnetic resonance spectroscopy indicated that the free amino acid L-tryptophan was the natural sperm attractant in H. rafescens. Eggs released L-tryptophan at concentrations that triggered both activation and chemotaxis in sperm, exhibiting significant activity at levels as low as 10(-8) mol l(-1). The D-isomer of tryptophan was inactive, showing that the sperm response was stereospecific. Serotonin, a potent neuromodulator and tryptophan metabolite, had no effect on sperm swim speeds or on orientation. In experimental treatments involving an elevated, uniform concentration of tryptophan (10(-7) mol l(-1)) or the addition of tryptophanase, an enzyme that selectively digests tryptophan, sperm failed to navigate towards live eggs. A natural gradient of L-tryptophan was therefore necessary and sufficient to promote recruitment of sperm to the surface of eggs in red abalone.
The northeastern Pacific Ocean is undergoing changes in temperature, carbonate chemistry, and dissolved oxygen concentration in concert with global change. Each of these stressors has wide-ranging effects on physiological systems, which may differ among species and life-history stages. Simultaneous exposure to multiple stressors may lead to even stronger impacts on organisms, but interacting effects remain poorly understood. Here, we examine how single- and multiple-stressor effects on physiology may drive changes in the behavior, biogeography, and ecosystem structure in coastal marine ecosystems, with emphasis on the California Current Large Marine Ecosystem. By analyzing the effects of stressors on physiological processes common to many marine taxa, we may be able to develop broadly applicable understandings of the effects of global change. This mechanistic foundation may contribute to the development of models and other decision-support tools to assist resource managers and policymakers in anticipating and addressing global change–driven alterations in marine populations and ecosystems.
The ocean moderates anthropogenic climate change at the cost of profound alterations of its physics, chemistry, ecology, and services. Here, we evaluate and compare the risks of impacts on marine and coastal ecosystems-and the goods and services they provide-for growing cumulative carbon emissions under two contrasting emissions scenarios. The current emissions trajectory would rapidly and significantly alter many ecosystems and the associated services on which humans heavily depend. A reduced emissions scenario-consistent with the Copenhagen Accord's goal of a global temperature increase of less than 2°C-is much more favorable to the ocean but still substantially alters important marine ecosystems and associated goods and services. The management options to address ocean impacts narrow as the ocean warms and acidifies. Consequently, any new climate regime that fails to minimize ocean impacts would be incomplete and inadequate. Copyright © 2015, American Association for the Advancement of Science.