ORIGINAL PAPER

Effects of elevated pCO2 and the effect of parent acclimationon development in the tropical Pacific sea urchin Echinometramathaei

S. Uthicke • N. Soars • S. Foo • M. Byrne

Received: 10 December 2011 / Accepted: 17 July 2012 / Published online: 17 August 2012

� Springer-Verlag 2012

Abstract Effects of acclimation to projected near-future

ocean acidification (OA) conditions on physiology, repro-

duction and development were investigated in the tropical

sea urchin Echinometra mathaei. Following 6 weeks in

control or one of the three elevated pCO2 (pHNIST 7.5–8.1;

pCO2 *485–1,770 latm) conditions, adult urchins exhib-

ited a slight decline of growth in low pH treatments and

moderately reduced respiration at intermediate levels. At

7 weeks, gametes from adults were used to produce larvae

that were reared in their respective parental treatments. To

assess whether larvae from acclimated parents are more

resilient to elevated pCO2 than those not acclimated, larvae

from control animals were also reared in the elevated pCO2

treatments. There was no difference in female ‘spawna-

bility’ and oocyte size between treatments, but male

spawning ability was reduced in increased pCO2 condi-

tions. In elevated pCO2 treatments, the percentage of nor-

mal larvae and larval size decreased in the progeny of

control- and elevated pCO2-acclimated parents, and arm

asymmetry increased. Thus, acclimation of the parents did

not make the progeny more resilient or sensitive to OA

effects. Negative effects of increased pCO2 on reproduc-

tion and development may impact on recruitment and

population maintenance of this species.

Introduction

Present-day atmospheric CO2 concentrations are over 30 %

higher than the maximum observed in the previous 2 Mil

years (Honisch et al. 2009). A large percentage of CO2 is

absorbed into the world’s oceans leading to reduced car-

bonate ion concentrations in parallel with a reduction in

seawater pH (Ocean Acidification, OA). Many marine

invertebrates rely on calcification to build internal or

external skeletons, and calcification is expected to be

compromised as carbonate saturation state is reduced, as

shown for corals (De’ath et al. 2009). Seawater pH has

decreased by 0.1 units since pre-industrial times and is

likely to be further reduced by 0.3–0.5 units by the end of

this century (Caldeira and Wickett 2005). Coral reef eco-

systems are particularly vulnerable to OA and climate

change-induced ocean warming (Hoegh-Guldberg et al.

2007) with a range of effects on the ecosystem and asso-

ciated biota evident (e.g. Anthony et al. 2008; Munday

et al. 2009; Diaz-Pulido et al. 2011; Fabricius et al. 2011).

The change in ocean pH/pCO2 and decreased carbonate

saturation expected over coming decades will be deleteri-

ous for a large suite of marine calcifiers. Echinodermata is

an exclusively marine phylum whose high magnesium

calcite skeleton may render them particularly vulnerable to

the effects of OA (Kroeker et al. 2010; McClintock et al.

2011). Amongst Echinodermata, OA studies have largely

focussed on the echinoids (sea urchins) where adults and

larvae rely on carbonate structures in their skeleton. Recent

studies along natural pCO2 gradients on carbon dioxide

Communicated by S. Dupont.

S. Uthicke (&)

Australian Institute of Marine Science, PMB No 3, Townsville,

QLD 4810, Australia

e-mail: S.Uthicke@aims.gov.au

N. Soars � M. Byrne

Schools of Biomedical and Biological Sciences, University

of Sydney, Sydney, NSW, Australia

S. Foo

School of Medical Sciences, F13, University of Sydney, Sydney,

NSW 2006, Australia

123

Mar Biol (2013) 160:1913–1926

DOI 10.1007/s00227-012-2023-5

vents demonstrate a significant decrease in the density of

two echinoid species with increasing pCO2 (Hall-Spencer

et al. 2008). Survival and growth of adult echinoids can be

significantly reduced at higher pCO2 (Shirayama and

Thornton 2005; Miles et al. 2007; Ries et al. 2009, Byrne

2012). Fertilization in most studies of sea urchins investi-

gated, including with the species investigated here, Echi-

nometra mathaei, is robust to near-future pH/pCO2 changes

(Kurihara and Shirayama 2004; Carr et al. 2006; Byrne

et al. 2009; Byrne et al. 2010; Ericson et al. 2010; Martin

et al. 2011), but see Havenhand et al. (2008) and Gonzalez-

Bernat et al. (2012). Early development in sea urchins (pre

hatching) also appears to be relatively unaffected by

increased pCO2 (Byrne et al. 2009; Ericson et al. 2010).

Increased acidification and hypercapnia has negative

effects on echinoid larval development reported within

near-future projections (Byrne 2011, 2012, Clark et al.

2009; Doo et al. 2011, Dupont et al. 2012, Sheppard

Brennand et al. 2010; Martin et al. 2011; Gonzalez-Bernat

et al. 2012).

We investigated the physiological and reproductive

responses of the Pacific sea urchin E. mathaei in animals

acclimated for 6–7 weeks to near-future OA conditions.

This period was chosen because it coincided with active

gonad development leading up to the spawning season

(Byrne, pers obs). Echinometra species are common eco-

logically important sea urchins in shallow water coral reef

habitats where they play important roles as algal grazers

and bioeroders (McClanahan and Muthiga 2007; Norstrom

et al. 2008). Because these sea urchins are broadcast

spawners with planktotrophic larvae, they are vulnerable to

population size fluctuations (Uthicke et al. 2009). This

vulnerability is illustrated by population outbreaks of this

species in Indian Ocean reefs following predator overf-

ishing (McClanahan and Muthiga 1988; McClanahan and

Shafir 1990; McClanahan et al. 1994). As the oceans are

increasing in pCO2, there is a focus on the prospects of

keystone species like E. mathaei because of the potential

vulnerability of larval and adult skeletons to changing

ocean conditions. Growth of small adult E. mathaei was

significantly reduced with only a small increase of pCO2

(220 latm, pH 7.9) (Shirayama and Thornton 2005).

Illustrations of the larvae of this species suggest that they

are smaller when reared at higher pCO2 (Kurihara 2008).

Ontogeny is an integrative process with different

developmental stages exhibiting different sensitivities to

stressors (Pechenik 1987; Allen and Pechenik 2010). Early

exposure to stress can result in deleterious downstream

effects, the ‘developmental domino effect’ where the

physiological performance and cellular responses of later

ontogeny depend on the success of preceding stages (Byrne

2011). Thus, all embryo and larval cultures of E. mathaei

were reared in experimental conditions from the onset of

development (fertilization). Given that even small reduc-

tions in single steps from adult spawning to larval settle-

ment (i.e. ‘transition-probabilities’) are multiplicative and

can reduce the ability of population size maintenance

(Eckman 1996), an overarching aim of this study was to

assess the potential effect of OA on population size of this

ecologically important echinoid in the long term.

Parental environmental history influences the outcomes

of development in echinoderms (O’Connor and Mulley

1977; Johnson and Babcock 1994; Byrne et al. 2010; Byrne

et al. 2011), and we investigated the potential that accli-

mation (phenotypic plasticity) of parents on OA conditions

would convey greater tolerance to progeny reared in

parental acclimation conditions. A recent study of

Strongylocentrotus purpuratus suggested that parental

acclimation to elevated pCO2 on OA conditions conveyed

resilience to progeny, a response dominated by maternal

effects that are largely due to phenotypic (plasticity) dif-

ferences in egg quality with potential for a genetic influ-

ence (Sunday et al. 2011). A study on a different species

from the same genus (S. droebachiensis) detected carry-

over effects from adults to larvae after exposure to high

pCO2 but also stated that short-term (4 months) acclima-

tion is not sufficient for full expression of plasticity

(Dupont et al. 2012). In a recent study where oysters were

acclimated in OA conditions for 5 weeks, progeny of

selectively bred oysters were more resilient than those of

wild-type oysters, a potential genetic (adaptive) effect, but

maternal (phenotypic) effects were not discounted (Parker

et al. 2012). Our study of E. mathaei considers trans-gen-

erational acclimation (phenotypic) effects. To assess the

effect of parental acclimation on development, gametes

from urchins conditioned for 7 weeks in control and three

elevated pCO2 conditions were used to generate larval

cultures reared in the respective parent acclimation treat-

ment, and larvae from control animals were also reared in

elevated pCO2 treatments. This approach using gametes

from urchins held in present-day ambient to generate larval

cultures in OA conditions parallels that used in recent

studies (Byrne 2011; Byrne 2012) and provided a basis to

place our results in context with previous research. Use of

progeny from parents acclimated in OA conditions pro-

vided the basis to test our hypothesis that parental accli-

mation would ameliorate the effects of OA on development

of progeny. Coral reef habitats show some fluctuation of

carbon chemistry parameters driven by respiration, pro-

duction and calcification (Anthony et al. 2011). The same

authors suggested pCO2 can rise above 600 latm during

the nighttime if water residence time is high. Thus, it is

possible that E. mathaei is adapted to increase pCO2 to

some extent.

1914 Mar Biol (2013) 160:1913–1926

123

In the present study, adult E. mathaei were held in near-

future OA conditions for 7 weeks to assess the effect of

acclimation in near and far future OA conditions on

physiology, reproduction and development. After 6 weeks

of exposure, we measured growth and respiration as

physiological parameters and hypothesized that acclima-

tion to elevated pCO2 would cause reduced growth of adult

E. mathaei through reduced calcification rates and reduce

respiration through metabolic depression (Portner 2008).

Materials and methods

Specimen collection and experimental setup

Echinometra mathaei (test diameter 34–53 mm) were col-

lected in October 2010 at Rib Reef, a midshelf reef in the

central section of the Great Barrier Reef (146�52.490E,

18�28.860S) and held for 2 weeks in flow through outdoor

aquaria in ambient conditions before use in experiments. The

size range used represents ‘intermediate sized’ animals at

Rib Reef, omitting smallest and largest specimens. To reduce

bias due to size of the animals, the size range was evenly

distributed across treatments. During the acclimation period

and experiment, the urchins were fed ad libitum on brown

macroalgae (Sargassum spp., Dictyota sp.). In addition, three

pieces (6–10 cm) of coral rubble encrusted with crustose

coralline algae were offered as additional food and attach-

ment source in each treatment aquarium. The pCO2 manip-

ulation experiment was set up in a temperature controlled

(25 ± 1 �C) aquarium room. Prior to entering the system

described below, water was filtered through three bag filters

(WATERCO, Australia) in series (25, 10, 5 lm).

Different pCO2 levels were simulated using a computer-

controlled CO2 dosing system (AquaMedic, Germany). One

pH probe (Aqua Medic, accuracy 0.01 pH units) was placed

into each of the four header tanks and connected to the

control system. Values for the three treatments with

increased pCO2 concentrations were set to pHNIST 7.90,

7.70 and 7.50 ± 0.05. These values were chosen to repre-

sent pCO2 concentrations in the next 2 centuries under

various representative concentration pathways (Moss et al.

2010). The control aquaria received unmanipulated water

from the flow through aquarium system. Solenoid valves

were connected to a standard CO2 cylinder (GE 082, BOC,

Australia) equipped with a flow regulator. CO2 gas was

introduced into the header tank though a gas diffuser

(AquaMedic). The diffuser and a submerged stirrer pump in

each header tank ensured rapid mixing of the water and gas.

Water from each of the four header tanks was pumped

into a separate water line. From each of these lines, three

16-L glass treatment aquaria (randomly allocated along the

bench space) were supplied via a flow controller set to

450–500 mL min-1. Water flow within each treatment

aquarium was enhanced with a small submersible aquarium

pump. Water in the header tanks was aerated, and dissolved

oxygen (DO) levels were checked approximately weekly

with an optode (Hach). Experimental treatment water never

fell below 95 % DO.

The temperature in the header tanks was left at ambient

temperature (26–28 �C), but was capped at 28 �C using a

commercial chiller unit (Carrier, USA). Light was supplied

through 50:50 actinic 420 nm: 10 K trichromatic daylight

(Catalina Compact, 12-h dark:12-h light cycle, similar to

field conditions) at 180–200 lmol photons m-2s-1. To

allow specimens to shelter from the light, each treatment

aquarium was half covered with 70 % shade cloth.

The pH probes in the header tanks (including the con-

trol) measured (pHNIST) every 30 s. The pHNIST of each

treatment aquarium was also monitored manually every

24 h using a temperature-corrected pH metre (OAKTON,

USA; pH probe: EUTECH, USA). Manual and control pH

probes were calibrated frequently using NIST buffers 7.0

and 10.0 (Ajax) at treatment temperatures. However, for

quality control, we also measured Dickson’s pH standard

(Batch 5) daily and recorded converted pH readings and

raw potential (mV). Water samples for total alkalinity (AT)

and dissolved inorganic carbon (CI) determination were

collected in 250-mL Schott bottles following Dickson et al.

(2007) four times throughout the experiment from treat-

ment aquaria and/or header tanks (see Table 1 for times

and number of replicates). In addition, we collected

aquarium water used for fertilization experiments and

during larval rearing for AT and CI determination. Alka-

linity and CI samples were fixed with 125 lL of saturated

(7 g in 100 mL) mercuric chloride until analysis. Samples

for AT and CI were analysed at 25 �C using a VINDTA 3C

titrator (Marianda, Germany). Alkalinity was analysed by

acid titration (Dickson et al. 2007) and CI by acidification

and coulometric detection (UIC 5105 Coulometer) of the

evolved CO2. Calibration was conducted using Certified

Reference seawaters (A. G. Dickson, Scripps Institute of

Oceanography, Dixon, Batch 106).

At the start of the experiment, five E. mathaei were

placed into each treatment flow through aquarium, and

thus, 15 (3 9 5) individuals per pCO2 level were available

for physiological measurements and experiments on fer-

tilization and development.

Physiological parameters

Growth and respiration rates were measured as indicators

of physiological fitness. Growth of individual E. mathaei

was based on a comparison of the initial wet-weight and on

day 45 (±0.1 g). Measurements were taken after a 24-h

period of no food to reduce the influence of gut contents on

Mar Biol (2013) 160:1913–1926 1915

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weight. Respiration rates of E. mathaei were measured

after 43–44 day of exposure in 10 urchins from each

treatment. Thus, respiration rates presented here represent

those of echinoids acclimated to the respective conditions.

Respiration was measured in a custom build respirometer

which used an OXY-4 (Presens, Germany) fibre-optic

oxygen metre to measure and record oxygen concentration

in enclosed Perspex chambers (volume: 638 mL). A

detailed description of a smaller version of the setup was

described in Uthicke et al. (2011). Each of the four

chambers was placed in a water bath (volume: 6.8 L)

placed on a stirring unit. The water bath had an inlet and an

outlet connected via Nalgene tubes to the pump of tem-

perature control (±0.1 �C) unit (Lauda, Germany) set to

27 �C. Water flow was 4.2 ± 0.1 L min-1 to ensure con-

sistent temperature in each chamber. Oxygen concentration

in each chamber was recorded every 15 s. The optodes

were calibrated at the beginning and the end of the mea-

surements using water-saturated air as 100 % O2 and water

treated with Na2SO4 as 0 % O2. During each run, indi-

vidual E. mathaei were placed into a jar containing the

respective treatment water. Water used was re-filtered

using a 0.5-lm cartridge filter. Samples were incubated for

a minimum of 25 min to allow for an initial period to

Table 1 Echinometra mathaei. Water chemistry during the experiment

Date/treatment (N) pHNIST Salinity Temperature (�C) AT (lmol/kgSW) CI (lmol/kgSW) pCO2 (latm) XAr

9-11-2010

8.1 (3) 8.11 (0.01) 34.5 27.2 (0.2) 2,295 (1) 2,027 (7) 501 (14) 3.11 (0.07)

7.9 (3) 7.93 (0.03) 34.5 27.3 (0.1) 2,295 (1) 2,120 (12) 833 (58) 2.18 (0.11)

7.7 (3) 7.79 (0.04) 34.5 27.3 (0.1) 2,298 (1) 2,177 (15) 1,176 (108) 1.68 (0.14)

7.5 (3) 7.70 (0.00) 34.5 27.5 (0.2) 2,298 (3) 2,212 (4) 1,492 (5) 1.39 (0.01)

19-11-2010

8.1 (2) 8.11 (0.02) 34.5 27.2 (0.2) 2,226 (1) 1,964 (9) 488 (28) 3.01 (0.10)

7.9 7.90 34.5 27.4 2,227 2,065 858 2.03

7.7 (2) 7.82 (0.01) 34.5 27.4 (0.2) 2,229 (0) 2,102 (5) 1,071 (31) 1.71 (0.05)

7.5 (2) 7.54 (0.01) 34.5 27.6 (0.0) 2,229 (1) 2,170 (0) 1,726 (1) 1.17 (0)

06-12-2010

8.1 (2) 8.14 (0.01) 33.9 26.4 (0.0) 2,240 (1) 1,972 (4) 456 (10) 3.08 (0.04)

7.9 (2) 7.92 (0.01) 33.9 26.3 (0) 2,253 (0) 2,092 (3) 832 (14) 2.03 (0.03)

7.7 (2) 7.72 (0.01) 33.9 26.5 (0.0) 2,247 (5) 2,162 (2) 1,378 (31) 1.36 (0.03)

7.5 (2) 7.57 (0.03) 33.9 26.4 (0) 2,260 (11) 2,226 (1) 2,012 (128) 0.99 (0.06)

13-12-2010

8.1 (F) 8.12 33.5 27.4 2,169 1,912 466 2.96

7.9 (F) 7.96 33.5 27.3 2,184 2,005 728 2.19

7.7 (F) 7.71 33.5 27.3 2,193 2,112 1,406 1.32

7.5 (F) 7.57 33.5 27.7 2,179 2,142 1,968 0.99

15-12-2010

8.1 (F) 8.11 33.5 26.4 2,235 1,985 493 2.90

7.9 (F) 7.97 33.5 26.8 2,223 2,040 719 2.24

7.7 (F) 7.77 33.5 26.2 2,221 2,121 1,200 1.47

7.5 (F) 7.61 33.5 27 2,232 2,186 1,843 1.07

Total averages pHNIST pHTOTAL pCO2

8.1 8.12 (0.01) 7.97 (0.01) 487 (18)

7.9 7.94 (0.03) 7.79 (0.03) 785 (71)

7.7 7.76 (0.05) 7.61 (0.05) 1,261 (126)

7.5 7.60 (0.06) 7.45 (0.06) 1,768 (247)

(F) extra filtered water from the aquaria line used for fertilization experiments and flow through larval rearing, HT: water from header tanks.

Replicate samples (N) are given in brackets in the first column if not one; values in brackets in other columns represent standard deviations.

pHNIST represents the pH value as calculated from measured alkalinity (AT) and dissolved inorganic carbon measurements (CI) values. pH values

on the total scale were in each case 0.15 units below those on the NIST scale. Total averages (treatment aquaria only) below the table are

averaged over sample days

1916 Mar Biol (2013) 160:1913–1926

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stabilize temperatures and for individuals to settle, as

indicated by a linear decrease of oxygen over the last

15 min. Blank chambers were run every 2–3 runs to control

for background respiration, but in each case, background

respiration was in detectable. Respiration rates were cal-

culated from slopes of oxygen concentration over time

during the last 15 min of the incubations. All regressions

thus obtained were highly significant (p [ 0.001) and had a

high coefficient of determination (R2 C 0.95 in 63 % of all

models, R2 C 0.90 in 95 %).

Reproductive response

After a 7-week reproductive conditioning period, the

E. mathaei were spawned in mid-December 2010 coin-

ciding with the spawning period in the field (Byrne pers

obs). The urchins were induced to spawn by injecting

2–3 mL of 0.5 M KCl through the peristome. The spawning

response of each urchin from the control and experimental

conditions was scored as non-responsive and responsive,

with the latter category divided into three categories: (1)

Minute amount of spawn (not usable for development

experiments), (2) poor—small amount of spawn, but usable

for development experiments and (3) Good—copious vol-

ume of gametes released. This score was used to determine

a spawning index. The gametes of each individual were

checked microscopically for quality (e.g. spherical uniform

eggs and motile sperm). The gonads of the non-spawners

were dissected for microscopic examination to determine

gender. A sample of eggs from 5 females from each

pH/pCO2 treatment was set aside for photography using a

microscope (Zeiss Axioscope) mounted digital camera

(Axiocam) to measure the diameter of 30–60 eggs per

female using ImageJ (Abramoff et al. 2004).

Sperm was collected dry from the surface of urchins and

was placed in a Petri dish until use. The eggs spawned by

females from each treatment were placed in 250 mL of

filtered sea water (0.5 lm, FSW) at the same pH as the

acclimation treatment. For each pH treatment, gametes

from multiple males and females (at least three individuals

of each sex) were pooled to represent a population of

spawners as might occur in the field. This was also done to

avoid strong experimental variance caused by maternal

(environmental) and paternal (genetic) effects, and male x

female interactions seen in single dam-sire crosses (Byrne

2012; Evans and Marshall 2005).

For fertilization, 10,000 eggs (combined from 5

females) were transferred to 250-mL beakers of fresh FSW

with the parent acclimation pH/pCO2 level (‘acclimated’

treatment). In addition, we transferred eggs from the con-

trol (ambient) treatment to water from each of the four

increased pH/pCO2 treatments (‘non-acclimated’ treat-

ment). After 15 min, sperm was added at ca. 106 sperm

mL-1 based on haemocytometer counts. After a further

15 min, the eggs were rinsed in experimental FSW.

Examination of the cultures after 2 h indicated C80 %

fertilization. The number of embryos arrested at fertiliza-

tion (e.g. fertilization envelope only) was low (\1 %)

indicating that polyspermy was minimal.

Embryos from: (1) parents held at ambient conditions

and (2) from parents acclimated in OA conditions were

reared in experimental conditions from the outset of

development (fertilization). This experimental design

provided a comparison of the response of progeny of

acclimated and non-acclimated urchins. Equally important,

the design provides a data set with which to compare the

outcome of recent studies where gametes from urchins

from ambient (control) conditions are used to generate

embryo and larval cultures reared in OA conditions.

Nineteen millilitres of the embryos from each beaker

was added to six falcon tubes (ca. 15 embryo mL-1) with

mesh covered holes (63 lm mesh) and fixed to the bottom

of a tank with flow through FSW (aquarium water as used

in the experiments, but with additional filtration through a

0.5-lm cartridge filter). Embryos from each treatment were

held in water of the respective treatment condition. At eight

hr (hatched blastula stage), a random sample of embryos

was removed with a pipette and examined microscopically

to score the percentage of normal hatched embryos. Forty-

eight hours after fertilization (2-arm echinopluteus stage),

larvae (Fig. 1a–g) were removed from the tube and fixed in

1 % formalin. Under a microscope, the first 50 randomly

selected specimens were scored as normal (well-developed

arms and only slight arm asymmetry) (Fig. 1a–d) or

abnormal (clearly underdeveloped arms, arrested develop-

ment) (Fig. 1e–g) to determine the percentage of normal

larvae. The first 20–30 larvae scored as normal from each

of the six replicates per treatment were photographed using

a Zeiss microscope (Axioscope). This was done within 4 h

after fixation to avoid post-fixation changes in morphology.

The length of both post-oral arm rods was measured, and

the sum of these measurements was used as a proxy for the

amount of calcite skeleton produced. The difference in

length of the post-oral arms rods (longer arm length—

shorter arm length) in relation to the average arm length,

expressed as %, was used as a measure of arm asymmetry,

a common response of echinoplutei to perturbation (e.g.

Sheppard Brennand et al. 2010) (Fig. 1e, f).

Statistical analyses

Changes in respiration rates and growth of E. mathaei were

tested using linear models with average pH levels for each

individual aquarium as explanatory variable. Because

responses to increased pH/decreased pCO2 can be strongly

nonlinear, we tested linear-, polynomial (quadratic) and

Mar Biol (2013) 160:1913–1926 1917

123

power functions following Ries et al. (2009). The best

model was chosen by model comparisons (ANOVA) and

highest R2 values. To accommodate differences depending

on the initial weight of the specimens, growth was

expressed as percentage growth. For respiration, weight of

the urchin was included as a co-variable in the model;

inclusion of an interaction term between pH and weight did

not improve the model (ANOVA). Power functions were

fitted by fitting linear functions to log–log transformed

data. Average pH values from 30 readings per aquarium

were obtained by averaging H? ion concentrations and

back-transforming those to pH. Total averages per treat-

ment (30 times by 3 replicates = 90 readings) were

obtained in a similar fashion. For those averages, we also

present asymmetric confidence intervals based on back-

transformed H? Ion concentrations.

Differences in sex ratios were tested by Chi-square test.

Differences in mean egg diameter (average diameter per

replicate), % hatched blastulae (8 h), % normal two armed

echinoplutei (48 h) and total arm length and arm asym-

metry were analysed using linear models with pH levels as

explanatory variable. For parameters where the overall

linear model was significant, we tested the following linear

contrasts using the DoBy package in R. The average of

each of the non-acclimated treatment levels (7.9, 7.7, 7.5)

was tested against the control (8.1). A second set of con-

trasts tested whether averages of the parameters mea-

sured in progeny from urchins held in control conditions

(non-acclimated) differed from progeny from parents

acclimated in OA conditions in each of the treatments with

increased pCO2.

Prior to all analyses, assumptions of normality and

homogeneity of variances were checked using box plots

and residual plots. No deviations from these assumptions

were detected, but a small number (2) of outliers (defined as

[2 SD from average of remaining replicates) were

removed. Several parameters based on percentages (%

hatched blastulae, normal larvae, arm asymmetry) were

arcsine transformed for statistical analysis. All statistical

analyses were performed in (R Development Core Team

2010).

Results

Seawater chemistry

Aquaria with E. mathaei were monitored daily for pH and

water chemistry to ensure that pH/pCO2 were maintained

at desired levels throughout the experiment (Table 1).

Control (ambient) pH was on average 8.10 (CI 8.05–8.16).

The averages for the OA treatments were pH 7.9 (average

pH 7.89, CI 7.85–7.94), pH 7.7 (average pH 7.75, CI

7.70–7.80) and pH 7.5 (average pH 7.55, CI 7.52–7.60),

close to the target values. These values were also in close

correspondence to values determined from AT and CI data

Fig. 1 Echinometra mathaei 2-day larvae in control a pH 7.90 b, pH 7.70, c and pH 7.50 d. Abnormalities included asymmetrical arm

development (e, f) and arrested development at the prism stage (g) or earlier

1918 Mar Biol (2013) 160:1913–1926

123

from the same water (Table 1). Average pCO2 values were

487 latm in the control treatment, and 785, 1,261 and

1,768 latm in the pH 7.9, pH 7.7 and pH 7.5 treatments,

respectively (Table 1). During the experiment, the urchins

were frequently observed feeding on the food sources

offered with abundant faeces produced and removed from

the aquaria every 2–3 days.

Physiology

Growth

Between initial and final measurement after 6 weeks in

experimental conditions, individual E. mathaei increased in

weight (Mean 0.94 g wet-weight, SD = 0.67, N = 60).

Although growth of individual E. mathaei was variable,

linear models of the growth data (normalized to % growth

of initial weight) showed a marginally significant reduction

of growth at lower pH levels (Fig. 2a), and a linear func-

tion provided the best fit (F1,58 = 3.29, p = 0.075).

Respiration

Respiration rates of 10 urchins per treatment were mea-

sured after 6 weeks in experimental conditions. Pooled

across all treatments, whole individual respiration rates

strongly depended on individual size. Using a power

function, wet-weight of the individuals explained 67 % of

the variation in respiration rates (Fig. 2b, F1,38 = 78.5,

p \ 0.001).

Wet-weight-specific respiration rates were negatively

related to wet-weight (power function, weight-specific

respiration = 235.22 9 weight0.520; R2 = 0.70, F1,38 = 91.7,

p \ 0.001). To accommodate this, wet-weight was used as

a co-variable in a linear model testing the dependency of

respiration on pCO2 levels. A polynomial function exhib-

ited the best fit to the data. This function suggests slightly

reduced respiration rates at intermediate pH ranges

(Fig. 2b). In the overall model (R2 = 0.64), pH explains

a significant amount of the variation in respiration

(MS = 46.15, F2,36 = 4.16, p = 0.024). Although the

amount of variation is relatively small when compared to

the covariable (MS = 719.30, F1,36 = 64.91, p \ 0.001),

this provides some evidence that respiration at pH 7.7 and

7.9 was reduced when compared to pH 8.1 and 7.5.

Reproduction

Spawning response

Most (53/60) specimens of E. mathaei released gametes in

response to KCl injection and the gender of most specimens

(57/60) could be determined during spawning or by post-

spawning dissections (Table 2). Although there were more

males (32) than females (25), this did not differ from a 1:1 sex

ratio (Chi-square test, v2 = 0.86, p = 0.3538). Nearly all

females spawned in each of the pCO2 treatments (Table 2)

and they all released copious amounts of eggs (i.e spawning

category 3) to generate larval cultures. There was also no

apparent difference in the quality of the eggs amongst

treatments on microscopic examination, and there was no

difference in egg size between the five females held in each

of the different pCO2 conditions (linear model, F3,16 = 0.15,

p = 0.9266). Egg diameter was relatively invariable with a

mean of 71.8 lm (SD = 1.7 lm, n = 20 mean values from

30 eggs per female) amongst individuals and treatments.

8

(A) Growth

Gro

wth

(%

of I

nitia

l wt.) p = 0.094

8.120

2530

3540

4550

55

(B) Respiration

Res

pira

tion

(mg

O2

h-1 g

-1)

p = 0.026

pH Units7.87.98.0 7.7 7.6

42

06

Fig. 2 Echinometra mathaei.

Linear model fits (grey-shaded

areas: 95 % confidence

intervals) for growth (a) and

respiration (b) of E. mathaei.

The best fit for growth was

given by a linear function,

respiration was best described

by a second-order polynomial;

animal wet-weight is included

as a co-variable in the model.

p values shown reflect the

overall p values of the models

Mar Biol (2013) 160:1913–1926 1919

123

In contrast to females, the spawning response of males

differed amongst pCO2 treatments (Table 2). Males held in

the control (pH 8.1) treatment released copious amounts

(*[50 lL) of sperm. We scored male spawning ability in

three categories and associated a weighting factor (little or

no sperm: 1, poor: 2, good: 3). These scores were averaged

for each treatment to determine a male spawning index

(Table 2). This index was 2.6 (just below good on average)

for pH 8.1, and ca. 2 (poor) for all treatments with

increased pCO2; indicating a reduction in male gamete

output at all treatments with increased pCO2.

Development

At 8 h, a high percentage (total average: 97.1 %,

SD = 2.6, n = 42) of embryos were hatched blastulae

(Fig. 3a), with no significant difference between treat-

ments (linear model, F6,33 = 0.77, p = 0.5989). Thus,

there was no difference in the percentage of hatching in

blastulae between controls and OA treatments, and those

derived from ambient non-acclimated parents and those

derived from acclimated parents for each of the OA

treatments.

In contrast, the percentage of normal larvae (48 h)

varied between treatments (linear model, F6,34 = 3.45,

p = 0.0091) and exhibited a decline with increasing pCO2

(Fig. 3b). Planned contrasts comparing each OA treatment

to the ambient control revealed that larvae derived from

non-acclimated parents had significantly reduced percent-

age of normal development in all three increased pH/pCO2

treatments (Table 3).

A second set of planned contrasts compared the per-

centage of hatched blastulae derived from acclimated and

non-acclimated parents for each of the OA treatments

separately. In none of the OA treatments was the per-

centage of normal development significantly different

between the progeny of urchins acclimated in OA condi-

tions and the progeny of parents from control conditions

(Table 3). However, it is noteworthy to point out that the

percentage of normal larvae was higher in all three OA

treatments for larvae derived from acclimated parents, and

that this effect was marginally significant for pH 7.9.

Total arm length of E. mathaei echinoplutei reared in

flow through conditions under four different pH regimes

differed significantly between treatments (linear model,

F6,34 = 10.86, p \ 0.0001). Control larvae derived from

parents maintained in ambient conditions and reared in

ambient conditions (pH 8.1) had the longest arm rods

(Fig. 1a). Larval size declined with increasing pCO2 con-

centrations (Figs. 1b–d, 3c). Compared to the control

treatment, the length of the arm rods in echinoplutei from

non-acclimated adults was significantly reduced in all

experimental OA treatments (linear contrasts, Table 3).

Larvae in increased acidification/hypercapnia conditions

were stunted, with a reduction in arm length of *7 % in

the pH 7.9 treatment and *22 % in the pH 7.5 treatment

(Table 3). The second set of planned contrasts showed that

acclimation of adults for 7 weeks in OA conditions did not

influence the size of the echinoplutei reared in the

respective parental pCO2 levels. In none of the OA treat-

ments did the size of larvae differ in the progeny of

acclimated and non-acclimated parents.

The pattern of increased larval arm asymmetry was sim-

ilar to that seen for larval size with increased asymmetry in

experimental OA conditions (linear model, F6,32 = 10.58,

p \ 0.0001). The larvae exhibited increased asymmetry in

higher pCO2 treatments (Fig. 2d). Although the effect size

was large (up to 87 %, Table 3), arm asymmetry in larvae

derived from non-acclimated urchins was only significantly

different from the ambient control for larvae reared at the

highest pCO2 treatment (linear contrasts, Table 3). Similar

to the other larval parameters, there was no significant dif-

ference in arm asymmetry between larvae from acclimated

and non-acclimated adults at each OA level (Table 3).

Thus, development in embryos and larvae fertilized and

held in ambient or experimentally low pH/high pCO2 condi-

tions differed significantly for all three parameters measured,

with impaired development of those reared in OA conditions.

Surprisingly, 7-week acclimation of the parents prior to

spawning did not improve the performance of the larvae.

Table 2 Echinometra mathaei. The number of females, males and indeterminate specimens (Indet.) of identified during spawning and post-

spawning dissections

Treatment Indet. Females Males

Spawner Non-spawn Little/no spawn (1) Poor (2) Good (3) Total no. Average category

7.5 2 5 1 2 2 3 7 2.1

7.7 1 7 0 1 5 1 7 2.0

7.9 0 6 0 5 3 1 9 1.6

8.1 0 5 1 1 2 6 9 2.6

Male individuals were grouped into three categories: little or no sperm detected, small amounts of sperm not useful for fertilization experiments

(poor) and large amounts *[50 lL (good). Categories were weighted with a value of 1, 2 or 3 respectively and then averaged for each treatment

1920 Mar Biol (2013) 160:1913–1926

123

Discussion

The present study illustrated that impacts of living in an

ocean acidification (OA) environment on somatic fit-

ness (e.g. growth and respiration) in E. mathaei were

smaller than effects on reproduction and development.

The high sensitivity of planktonic life history stages of

echinoids and other invertebrates to ocean acidification and

other stressors (e.g. increased temperature, pollution) is

well known (Pechenik 1987; Byrne 2011; Byrne 2012). As

one of the first studies, we determined the influence of

acclimation of adults to OA on somatic fitness, reproduc-

tion and the performance of progeny reared in the respec-

tive adult acclimation conditions. Our hypothesis that

parental acclimation would ameliorate the negative effects

of OA on development was not supported. Overall, larvae

produced by adults acclimated to OA conditions were

equally vulnerable to those from non-acclimated adults.

Physiology

As found for adult echinoid Arbacia punctulata (Ries et al.

2009) maintained in similar OA conditions (present day to

2,856 latm CO2, XA B 0.5) for 60d, we found that growth

in adult E. mathaei was relatively insensitive to increased

pCO2. Arbacia punctulata had a slight increase in growth

at intermediate pCO2 levels (600–900 latm) and a reduc-

tion in the highest pCO2 concentration (Ries et al. 2009).

Somatic and reproductive growth under increased pCO2

was also decreased in Strongylocentrotus droebachiensis

(Stumpp et al. 2012). In our study, specimens achieved

positive growth in low pH treatments (pH = 7.5 pCO2

1,770 latm, XA = 1.5–1.8). Thus, adult E. mathaei

maintained in ambient controls and three OA conditions for

6 weeks exhibited some resilience to living in an OA

environment. A similar result was obtained in a previous

study of subadult E. mathaei where in a 6-month experi-

ment in OA conditions (pH 7.90, pCO2: 560 latm), growth

of urchins in low pH and control treatments did not differ

for the first 12-14 weeks of the experiment, although this

was then followed by a period of reduced growth in the low

pH treatment (Shirayama and Thornton 2005). Thus, we

might have seen a difference in growth and respiration of

E. mathaei in a longer-term experiment. Indeed, linear

models suggested a decline in growth at higher pCO2

concentrations. Growth of young adult Hemicentrotus

pulcherrimus (wet-weight: 0.84–4.1 g) was also reduced

after 12 weeks in slightly elevated (560 latm) pCO2 levels

(Shirayama and Thornton 2005). The cidaroid urchin

Eucidaris tribuloides showed a threshold response with

reduced growth only evident at high (2,856 latm) pCO2

levels (Ries et al. 2009). Thus, the present and previous

studies show that effects of OA on echinoid growth are not

strong, but might still be relevant, especially for small

individuals and long exposure.

Many marine invertebrates and vertebrates exhibit

reduced oxygen consumption rates (‘metabolic depres-

sion’) under low pH/hypercapnic conditions (Langenbuch

and Portner 2002; Portner et al. 2004; Portner 2008), but

Hat

ched

(%

)

80

85

90

95

Nor

mal

dev

elop

men

t (%

)

80

85

90

95

100

Arm

leng

th (

mm

)

0.20

0.25

0.30

0.35

0.40

Arm

asy

mm

etry

(%

)

8.1 7.9N 7.9A 7.7N 7.7A 7.5N 7.5A

4

6

8

10

12

14

16

(A)

(B)

(C)

(D)

Fig. 3 Echinometra mathaei. a The percentage of hatched blastulae

(8 h), b percentage of normal larvae (48 h), c total arm length and

d arm asymmetry, of echinopluteus larvae in seven different treatment

groups. Control treatments (pH 8.1, larvae derived from parents held

at ambient pH/pCO2 and grown in those conditions) are represented

by dark grey boxes. Larvae reared under decreased pH/increase pCO2

conditions are either derived from adults previously kept in control

conditions (N, ‘non-acclimated’, white boxes) or from those kept

under the respective pCO2 condition (a, ‘acclimated’, light grey

boxes)

Mar Biol (2013) 160:1913–1926 1921

123

usually only at extreme pCO2 increases beyond the range

tested here. Comparison between studies is hampered

because of different methods used. Studies apply different

pH levels, and the effect of hypercapnia can also strongly

depend on temperature (Lannig et al. 2010; Wood et al.

2010; Christensen et al. 2011; McElroy et al. 2012) or the

time (if any) of acclimation (Michaelidis et al. 2005).

Here, we measured oxygen consumption rates for

E. mathaei after an acclimation period of 6 weeks. We

noted a slight decrease (5–7 %) in the respiratory rate in

response to intermediate pCO2 increase. Although the

decrease is small, it is in the same range as that measured

for other invertebrates in similar pH conditions, such as

isolated tissues of Sipunculus nudus at pH 7.6 (Langenbuch

and Portner 2002). However, other invertebrate species

exhibit an increase in respiration at pH 7.6–7.7 (Lannig

et al. 2010).

Only a few studies have investigated potential metabolic

depression in echinoderms. After 40 day of exposure to

reduced pH (to pH 7.3), the ophiuroid Ophiura ophiura

displayed no change in oxygen consumption rates at a high

temperature/low pH treatment, and a slight increase at the

low temperature/low pH treatment (Wood et al. 2010).

Similarly, oxygen consumption rates increased with pH in

Amphiura filiformis acclimated for 6 weeks to a range (to

6.8) of conditions (Wood et al. 2008). In the subtidal

species Ophionereis schayeri, oxygen consumption in

acute exposure decreased at intermediate pH levels in non-

acclimated anima, but increased at pH 7.6 (Christensen

et al. 2011). A similar pattern of metabolic depression at

intermediate pH levels and marked increase in respiration

at lower pH was also observed for an intertidal asteroid in

non-acclimated animals on exposure to acute stress

(McElroy et al. 2012). Thus, data presented here for the

echinoid E. mathaei show some similarity to those found in

other echinoderms, which either showed a slight depression

and subsequent increase, or direct respiration increase in

lower pH conditions. Metabolic depression at intermediate

levels of pH/pCO2 has previously been interpreted as the

narcotic effect of hypercapnia, where subsequently

increased oxygen consumption in lower more extreme pH

conditions (e.g. pH = 7.6) is a stress response (Christensen

et al. 2011). Deviations from a model of metabolic

depression have also been observed in the bivalve Mytilus

edulis (Thomsen and Melzner 2010). Studies on other

echinoderm species are required to investigate whether this

deviation from patterns of metabolic depression in

response to low pH/hypercapnia as observed in other

invertebrates is a general feature of the phylum.

Reproduction and development

For female E. mathaei, there was no apparent effect of

living in a high pCO2 environment (1,770 latm) with

respect to spawning response or egg size. The eggs were

similar in size to that reported in other studies (Kominami

and Takata 2003), and not significantly different between

OA treatments and control. The latter authors also noted

that the size of the eggs of E. mathaei is small compared

with that of other echinoids with planktotrophic develop-

ment. In contrast, S. droebachiensis exhibited a marked

decline in female fecundity following 4-month incubation

at 1,200 latm, although this was not observed in urchins

held for 16 months in these conditions (Dupont et al., this

volume). The short-term response of S. droebachiensis may

reflect a stress response while the lack of a negative effect

after 16 months may be due to reproductive acclimation.

Clearly, more long-term OA experiments encompassing

Table 3 Echinometra mathaei. Results of planned contrasts following linear model analysis for three larval development parameters

% Normal Arm length Arm asymmetry

Effect size (%) t value p Effect size (%) t value p Effect size (%) t value p

Contrasts

Comparison to control

7.9 -14.29 -2.64 0.0126 -6.91 -2.06 0.0476 -1.96 -0.13 0.8966

7.7 -17.27 -3.18 0.0031 -16.17 -4.81 0.0000 18.56 1.31 0.2010

7.5 -20.33 -3.75 0.0007 -21.66 -6.44 0.0000 87.04 5.83 <0.0001

Acclimated versus not

7.9 11.65 1.84 0.0744 2.32 0.61 0.5452 18.81 1.24 0.2256

7.7 5.64 0.82 0.4178 -2.16 -0.54 0.5939 1.89 0.16 0.8774

7.5 10.35 1.52 0.1375 8.63 2.01 0.0523 -7.20 -0.86 0.3940

For each parameter, the first set of contrasts (‘Comparison to Control’) test whether larvae derived from non-acclimated parents at each of the

three OA treatments are significantly different from larvae reared in control conditions. The second set of contrasts (‘Acclimated vs. not’) tests

for each of the three OA conditions whether larvae derived from parents acclimated for 7 weeks to the OA conditions differ from those derived

from non-acclimated parents. Bold type indicates significance at a = 0.05

1922 Mar Biol (2013) 160:1913–1926

123

the entire sea urchin gametogenic cycle from gonial pro-

liferation to gametic maturation are required to determine

the impacts of hypercapnia on reproduction.

In contrast to the females, we noted a significant dif-

ference in male reproduction in E. mathaei between treat-

ments. Male spawning response was lowered in all

treatments with increased pCO2, including the pH 7.9

treatment (average pCO2: 785 pCO2) equivalent to values

expected towards the end of this century under most IPCC

scenarios (Caldeira and Wickett 2005). Ten-month expo-

sure to pH 7.8 caused delayed gonad development in

Hemicentrotus pulcherimus (unpublished data in Kurihara

2008). Gonad development of the green sea urchin

(S. droebachiensis) was reduced by 67 % in low pH/high

pCO2 conditions (pH \ 7.0) (Siikavuopio et al. 2007),

levels more extreme than used here. The authors of the

latter study suggested reduced food conversion efficiencies

or inability to convert organic material from the digestive

system to gonad tissue as likely causes. None of these

studies, however, indicate a differential susceptibility of

male and female gonads to OA. Thus, further studies are

required to determine the significance of this finding.

However, if OA has a negative effect on gonad develop-

ment in either (or both) of the sexes, this would result in

severe consequences for population replenishment.

The timing of early larval development of E. mathaei

observed here at *28 �C was similar to that reported

previously at a similar temperature, with well-developed

echinoplutei reached after 48 h (Kominami and Takata

2003). There was no difference in the percentage of

hatching in blastulae when OA treatments were compared

to ambient controls, or between embryos derived from

ambient non-acclimated parents and those derived from

acclimated parents for each of the OA treatments. Differ-

ences, however, were seen at the larval stage. The per-

centage of larvae with normal development was clearly

reduced in treatments with decreased pH/increased pCO2,

with only small differences between larvae derived from

acclimated or non-acclimated parents.

The stunting effect of OA conditions (pH 7.7–7.9) on

larval growth in E. mathaei is similar to that observed in

other studies of echinoplutei where larvae in OA treat-

ments have shorter larval arm rods (Kurihara and Shi-

rayama 2004; O’Donnell et al. 2010; Sheppard Brennand

et al. 2010; Moulin et al. 2011). For E. mathaei studied

here, progeny generated from parents maintained in con-

trol treatments and reared in the full range of OA condi-

tions and progeny from parents acclimated to OA

conditions and reared in the parental environment had a

similar response to OA conditions. The former approach

parallels similar experiments in previous studies (reviewed

in Byrne 2011, 2012) while the latter approach provided

the first test of the effect of acclimation in OA conditions

on the performance of echinoderm embryos. Regardless of

parent history, the total length of the larval arms (as a

proxy for the amount of calcite produced) in E. mathaei at

pH 7.9, 7.7 and 7.5 was ca. 7, 16 and 22 % smaller,

respectively, compared with controls. Arm asymmetry

showed a similar pattern, elevated by 87 % in the pH 7.5

treatment. Projected near-future (2,100) and beyond (ca.

2,300) ocean acidification has a general miniaturizing

effect on calcifying larvae, producing smaller larvae with

less skeleton and smaller shells, often combined with

abnormal development in more that 26 species of echi-

noderms and molluscs (Byrne 2012).

With regard to the mechanisms underlying altered

growth of echinoplutei in response to low pH/high pCO2

(pH B 7.4/pCO2 B 1,700), several may be involved: 1.

Reduced XC or XA saturation at low pH may have had

direct impacts on skeleton production, 2. Direct teratogenic

effects of low pH, 3. Hypercapnic suppression by increased

pCO2 may have depressed the physiological processes

involved in calcification and 4. A reduced scope for growth

resulting from higher larval metabolism (Portner 2008;

Martin et al. 2011; Stumpp et al. 2011a, b). A combination

of these mechanisms is likely to have influenced the out-

come for larval growth and skeletogenesis in E. mathaei.

Given that the low pH treatments resulted in both stunted

and abnormal larvae, it is doubtful that the effects of OA on

E. mathaei larvae were solely due to hypercapnic delay of

development or altered scope for growth. In contrast to

observations of isometric echinoplutei with no change in

larval profile at low pH (Martin et al. 2011; Stumpp et al.

2011a, b), the prevalence of arm asymmetry in the echin-

oplutei of E. mathaei shows that these larvae were not

isometric, similar to that reported for the larvae of another

tropical echinoid Tripneustes gratilla (Sheppard Brennand

et al. 2010). Regardless of the mechanism(s) involved in

OA-induced change in larval form, impaired ability of the

echinoplutei of E. mathaei and other echinoids to produce a

larval skeleton in a timely manner may be a weak link for

persistence of a broad suite of benthic invertebrates in a

changing ocean.

Reduced larval size in a high pCO2 ocean will have a

negative impact on feeding and swimming ability and

make larvae more vulnerable to predation (Allen 2008;

Przeslawski et al. 2008; Soars et al. 2009; Chan et al.

2011). The smaller arms of echinoplutei reared in ocean

acidification conditions will result in lower filter feeding

efficiency and reduced feeding performance, as also indi-

cated by the presence of smaller stomachs in the larvae of

Dendraster excentricus (Chan et al. 2011). If the phe-

nomenon of smaller larvae is associated with develop-

mental delay and increased time to metamorphosis, then

the risk of mortality due to predation in the plankton will

also increase (Lamare and Barker 1999).

Mar Biol (2013) 160:1913–1926 1923

123

Experimental methods used to investigate the effects of

climate change on development vary considerably between

studies (Byrne 2011; Byrne 2012). The approach used in all

studies published to date is to use gametes from adults

collected in present-day conditions to generate embryo

cultures (Byrne 2012, but see Parker et al. 2012; Dupont,

this volume). The outcomes of echinoderm development

are strongly influenced by the environmental history of the

parents, particularly that of the maternal parent (O’Connor

and Mulley 1977; Johnson and Babcock 1994; Bingham

et al. 1997). Here we used gametes derived from adults

acclimatized to three reduced pCO2 conditions for 7 weeks

prior to spawning and compared the performance of the

progeny of echinoids with different environmental (OA)

histories. The percentage of normal development, arm

length and arm asymmetry was significantly affected by

increased pCO2, in most cases under near-future conditions

of 785 latm. However, acclimation of parents for 7 weeks

did not convey a higher resilience in offspring to elevated

pCO2, with the possible (but only marginally significant)

exception of a higher percentage of normal developing

larvae when larvae were derived from acclimated parents.

We are not aware of other studies comparing the response

of acclimated and non-acclimated adult echinoids and their

progeny with respect to OA. Fertilization rates of Para-

centrotus lividus were more resilient to reduced pCO2

when spawners came from a tide pool with high daily pH

fluctuations compared to a pool with less fluctuation

(Moulin et al. 2011). As found here, further development of

P. lividus from both environments (larval size and nor-

mality) was similarly impacted by increased pCO2, as

found here for E. mathaei. In contrast, offspring generated

from Strongylocentrotus droebachensis held at high pCO2

(1,200 latm) for 4 months had decreased success at

metamorphosis (Dupont et al. 2012).

Gonad development and gamete production, fertilization

success, development and successful metamorphosis and

recruitment determine the population dynamics of a species

and future population size maintenance (Eckman 1996;

Uthicke et al. 2009). Reductions at only one or a few of

these stages could severely impact the maintenance of

future populations. Fertilization in E. mathaei is resilient to

pH \ 7.4 (Kurihara and Shirayama 2004). Thus, amongst

the factors investigated here and previously, both the

reduced spawning ability (and potentially gonad develop-

ment) in males and impaired development would be

expected to severely reduce future recruitment of this spe-

cies. A 7-week acclimation of the adults did not protect

offspring from the deleterious effects on larval growth in

our study. Future studies involving longer acclimation times

from the outset of gonad development through development

of progeny to metamorphosis and settlement are needed to

understand the trans-generational effects of OA.

Acknowledgments We are grateful to Murray Logan for help with

the statistical analysis, and Florita Flores, Paolo Momigliano and

Nikolas Vogel for assistance with the aquarium system. The work was

supported by a Discovery Grant from the Australian Research Council

and conducted with the support of funding from the Australian

Government’s National Environmental Research Program.

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