Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

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Journal of Experimental Marine Biology and Ecology

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Ocean warming will mitigate the effects of acidification on calcifying seaurchin larvae (Heliocidaris tuberculata) from the Australian globalwarming hot spot

Maria Byrne a,⁎, Shawna Foo b, Natalie A. Soars b, Kennedy D.L. Wolfe b, Hong D. Nguyen b,Natasha Hardy b, Symon A. Dworjanyn c

a Schools of Medical and Biological Sciences, University of Sydney, New South Wales 2006, Australiab School of Medical Sciences, University of Sydney, New South Wales 2006, Australiac National Marine Science Centre, Southern Cross University, PO Box 4321, Coffs Harbour, New South Wales 2450, Australia

⁎ Corresponding author.E-mail address: mbyrne@anatomy.usyd.edu.au (M. By

0022-0981/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.jembe.2013.07.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 May 2013Received in revised form 24 July 2013Accepted 26 July 2013Available online xxxx

Keywords:Calcifying larvaeEchinopluteusGlobal changeOcean acidificationOcean warming

The negative effect of ocean change stressors –warming and acidification – on calcifying invertebrate larvae hasemerged as a significant impact of global change. We assessed the arm growth response of the echinopluteuslarva of Heliocidaris tuberculata to simultaneous exposure to these stressors in cross-factorial experiments in-volving ambient and near future temperatures (control −20 °C; +4 °C: 24 °C) and pHNIST levels (control:pH 8.1; −3–7 pH units: pH 7.6–7.8). The more extreme pH treatment, pH 7.4, not a near-future condition,was used to assess tolerance levels. Experiments were designed with respect to present day conditions deter-mined for the habitat ofH. tuberculata and future (2100+) conditions for the southeast Australia globalwarminghot spot. Across near futurewarming-acidification treatments (24 °C/pH 7.6–7.8) therewas a 5–25% decrease innormal development and, at pH 7.4, this increased to 11–33%. Increased temperature facilitated larval growthacross all pH treatments with a 20–50% increase in arm length at +4 °C across all pH levels. Larval growthwas strongly reduced by acidification with a 15–25% decrease in arm length at pH 7.4–7.6 at control tempera-ture.Warmingmitigated the effect of pH on growth. Both stressors increased larval abnormality and asymmetry.The stunting effect of decreased pH on larval growth is typical of echinoplutei, indicating that similar mecha-nisms operate across species. The large proportion of normal and larger larvae in the +4 °C/pH 7.8 treatmentsindicate thatH. tuberculatamay tolerate near-future ocean change and this may be facilitated by acclimatizationor adaption.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Climate change is driving ocean change, increasing sea surfacetemperature and decreasing pH and carbonate mineral saturation(Caldeira and Wickett, 2005; Doney et al., 2012; Orr et al., 2005).These co-occurring stressors have strong impacts on fundamentalphysiological processes in organisms as seen in the bleaching ofcorals, dissolution of shells of live pteropods, reduced coral reef com-munity calcification and invasion of species as they follow changingmarine isotherms (Bednarsek et al., 2012; Frieler et al., 2012; Linget al., 2010; Silverman et al., 2012; Thatje et al., 2005). The extentand pace of warming and acidification differs locally and regionally,an important consideration in teasing out the potential interactiveeffects of these stressors and in predicting potential outcomes for spe-cies (Byrne and Przeslawski, 2013; McElhany and Busch, 2013). Inter-tidal and shallow water species that routinely experience fluctuations

rne).

ights reserved.

in temperature and pH, and species that naturally experience low pHwater due to upwelling or infaunal species inhabiting low pH sedi-ments, may have some resilience to warming and/or acidification(Matson et al., 2012; Talmage and Gobler, 2009, 2011; Wolfe et al.,2013). In contrast, species from relatively invariable habitats may bemore vulnerable (Melzner et al., 2009).

The planktonic embryos and larvae of benthic marine invertebratesare likely to be sensitive to changing ocean conditions (Pechenik, 1987),but we have a limited understanding about potential synergistic effectsof the two main global change factors impacting on development —

ocean warming and acidification (Byrne, 2011, 2012). There is particularconcern for calcifying larvae and their ability to make their skeletons ascarbonate mineral saturation declines (Byrne et al., 2013a; Dupont et al.,2010; Hofmann et al., 2010; Kroeker et al., 2013). Larval skeletons playkey functions in swimming, feeding and defence and so compromisedskeletogenesis in a lower pH ocean will have a negative effect on larvalperformance (Allen, 2008; Chan et al., 2011; Hart and Strathmann,1994). Increased temperature and pCO2 (organism hypercapnia) alsohave strong effects on development.Warming promotes faster progress

251M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

through ontogenetic stages controlling planktonic larval duration,survival and population connectivity, and results in larger larvae upto thermotolerance limits when larvae become smaller and develop-mental failure occurs (Chen and Chen, 1992; Sheppard Brennandet al., 2010; Staver and Strathmann, 2002). Hypercapnia alters me-tabolism andmay slowdevelopment or cause increased energy expendi-ture resulting in smaller larvae in ocean acidification conditions (Pörtner,2010, 2012; Stumpp et al., 2012). Predicting outcomes for marine inver-tebrate development in a changing ocean requires multistressor experi-ments (Byrne and Przeslawski, 2013).

Despite the pervasive effect of temperature on development, oceanwarming is rarely considered in studies of global change impacts onmarine larvae. Thus far, there are only two studies on the interactiveeffect of temperature and pH on calcification in larval sea urchins, onefor the tropical species Tripneustes gratilla, and one for the polar speciesSterechinus neumayeri (Byrne et al., 2013b; Sheppard Brennand et al.,2010). For T. gratilla warming (+3 °C) mitigated the negative effect ofpH on growth with a significant antagonistic interactive effect betweenthe two stressors (Sheppard Brennand et al., 2010). For S. neumayeriwarming (+2 °C) boosts larval growth across all pH treatments, butthere was no interaction between stressors (Byrne et al., 2013b). Theeffect of temperature on increased growth in these species is likelydue to the stimulatory effect of warming on physiological processes,particularly calcification. Here we extend this research to a temperatesea urchin, H. tuberculata.

H. tuberculata is a conspicuous component of the temperate rockyreef fauna of New South Wales (Keesing, 2013), in a region where theocean is warming much faster than the global average (Hobday andLough, 2011). We characterised the temperature and pH conditionsthat this species experiences in its shallow subtidal habitat. Our ex-periments were placed in a habitat and regionally relevant settingof projected future change for this species in southeast Australia in the“business as usual” scenario (Hobday and Lough, 2011; InternationalPanel on Climate Change, IPCC, 2007). H. tuberculata has a comparativelynarrowdistribution along theAustralian coast being limited toNewSouthWales from ~Byron Bay to Eden and is also found on off shore islands(Keesing, 2013; Museum Records — http://www.ozcam.org.au). InSydney where the animals were collected for this study, H. tuberculatais near the middle of its latitudinal range. This range ~1200 km(~8° latitude) is small for a species that has planktonic larval phaseof 3–4 weeks. It is not known if strong warming in easternAustralia N 4 °C by 2100 (Hobday and Lough, 2011), will impactthe survival of developmental stages and future recruitment successin H. tuberculata and, if the effects of warming will be exacerbated byacidification. These considerations are addressed here in fully facto-rial experiments where the embryos of H. tuberculata were rearedfrom the outset of development (fertilization) in experimental treat-ments encompassing the range of near-future temperature–pH levelsfor open waters (2100: +4 °C/pH 7.6–7.8) where development occurs.More extreme conditions (+4 °C/pH 7.4) were used to assess stress tol-erance levels. A previous study of fertilization inH. tuberculata, using thegametes from multiple parents (population approach), indicated thatfertilization, at optimal sperm levels, is resilient to near-future oceanwarming and acidification (Byrne et al., 2010).

We predicted that increased temperature would result in fastergrowth and increased larval size, but that a 4 °C warming might alsoapproach the thermotolerance limits for embryonic development inH. tuberculata, as shown for other echinoids including the sympatriccongener Heliocidaris erythrogramma (Byrne et al., 2009, 2011a). Devel-opment in low pH conditions commensurate with near-future oceanacidification conditions results in smaller echinoplutei (Byrne et al.,2013a). We expected a similar outcome for H. tuberculata. Increasedtemperature, commensurate with projections for southeast Australia(Hobday and Lough, 2011), was expected to reduce the negative effectof decreased pH on larval growth through stimulation of physiologicalprocesses.

2. Methods

2.1. Specimen collection and water chemistry

H. tuberculatawere collected from 1 to 5 mdepth at low tide at LittleBay, Sydney (33°58′S, 151°14′E), New South Wales, Australia inNovember 2011 during their spring spawning period. Animalswere transported in ambient seawater in a cool box and placed inaquaria at ambient sea surface temperature (SST). They were usedfor experiments within days of collection. The seawater for experi-ments was collected at high tide near the collection site. Just priorto and during the collection period SST ranged between 19 and 20 °Cas determined from the Physical Oceanography DAAC Ocean ESIP Tool(POET) website (http://poet.jpl.nasa.gov/) and a local reference station(www.mhl.nsw.gov.au). This provided an indication of the conditionsthat the embryos and larvae might experience in local waters while inthe plankton.

Temperature loggers were placed in the subtidal habitat (~1 mbelow MLW) of H. tuberculata in 2011 with measurements takenevery 20 min. In situ pHNIST measurements were also taken on 8 springtide series in 2011 at low tide ca. 30 min before sunrise and 1 h beforesunset with temperature corrected pH meter and electrode (WTW —

Wissenschaftilich-TechnischeWerkstätten GmbG P4). The probe (WTWSenTix® 41 pH electrode; precision ± 0.01 pH units) was calibrated fre-quently using high precision buffers (pHNIST 4.0, 7.0 at 20 °C, ProScitec)(Table 1). These times were chosen to characterise pH extremes due tothe diel pattern of photosynthesis, which tracks sunlight (McElroy et al.,2012; Wootton et al., 2008). The pHNIST in the open water of the baywas also recorded each time (n = 16) to provide an indication of surfacesea conditions that the developmental stages experience. In parallel,water samples were collected and preserved with mercuric chloridefor chemical analysis. Total alkalinity (TA) was measured using anopen-cell potentiometric titration and total dissolved inorganic car-bon (DIC) was measured by coulometry using certified referencestandards (Dickson et al., 2007), which indicated measurement ac-curacy and precision of ±2 μmol/kg for both parameters. Salinity wasmeasured using the WTW multimeter. The data from TA, DIC tempera-ture and salinity were used to determine pCO2 and pHT conditions inthe habitat of H. tuberculata at low tide using CO2SYS (Pierrot et al.,2006) with the constants of Mehrbach et al. (1973) as refitted byDickson and Millero (1987) (Table 1). As expected (Zeebe and Wolf-Gladrow, 2001), the values determined for pHT are slightly below(0.08–0.11 pH units) that determined in the field for pHNIST with thesame water samples (Table 1).

2.2. Experimental treatments

In multifactorial stressor experiments, the effects of 2 temperature(control: 20 °C, +4 °C: 24 °C) and 4 pHNIST (control: 8.1, −3–7 pHunits: 7.8, 7.6, 7.4) levels on development of H. tuberculatawere investi-gated in all combinations (Table 2). The control temperature 20 °Creflected the recent thermal history of the adults, which inH. tuberculata,determines the thermal tolerance of the progeny (O'Connor andMulley,1977). Experimental treatments were designed within the context ofthe “business as usual” emission scenario where SSTs in southeastAustralia are projected to increase 2–3 °C by 2070 and up to 4 °C by2100 (Hobday and Lough, 2011). For the habitat of H. tuberculata, thisis likely to be more extreme due to strong aerial warming (5 °C by2070) (Hobday and Lough, 2011). While we do not know the detailsof future local pH/pCO2 conditions in the habitat of H. tuberculata, weused our in situ data (see Results) and modelled estimates for the “busi-ness as usual” emission scenario (2100 and 2300 pHdecrease−0.40 andup to −0.70 units, respectively) (Caldeira and Wickett, 2005; Hobdayand Lough, 2011; International Panel on Climate Change, IPCC, 2007) toguide our experimental design (Table 2). The more extreme pH treat-ment, pH 7.4, not a near-future condition, was used to assess tolerance

Table 1Seawater conditions of the shallow subtidal habitat of H. tuberculata at Little Bay. Salinity, temperature and pHNIST were measured in situ 30 min before sunrise (AM), 1 h before sunset(PM) and at high tide. pHT and pCO2were calculated fromDIC and TAusing CO2SYS (seeMethods). Values aremeanswith standard error given inparentheses (n = 8 for low tide, n = 10for high tide).

Time Salinity Temp TA DIC pHNIST pHT pCO2

(ppt) (°C) (μmol/kg) (μmol/kg) (μatm)

Low tide (AM) 34.8 (0.25) 17.3 (0.79) 2301.2 (6.91) 2105.9 (4.13) 8.03 (0.02) 7.95 (0.02) 509.6 (29.98)Low tide (PM) 34.9 (0.41) 19.8 (0.83) 2273.4 (19.08) 1940.4 (22.82) 8.27 (0.01) 8.18 (0.02) 276.7 (17.25)High tide 35.2 (0.22) 20.0 (0.66) 2301.7 (4.77) 2022.6 (10.15) 8.20 (0.02) 8.08 (0.02) 364.9 (16.14)

252 M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

levels. Water baths were used to maintain constant temperature andthese were monitored using temperature loggers. Experimental pHNIST

and dissolved oxygen (DO) N 90% were achieved by bubbling CO2 gasand air into the FSW until the desired pH was reached as monitored bythe pH meter. This method allows for rapid equilibrium of CO2 to therequired levels and stability (Table 2).

Three sources of freshly collected filtered seawater (1 μm)(FSW) from the open coast were used. They exhibited similar pa-rameters (Mean, SE: pHNIST: 8.11 ± 0.01, salinity: 34.83 ± 0.07;TA = 2316.49 ± 6.04). Seawater pHNIST and salinity were mea-sured using the WTW meter. Total alkalinity (TA) was measuredby titration (as above). Experimental pCO2 and aragonite (Ωar)and calcite (Ωca) saturation values for each temperature–pH combi-nation (Table 2) were determined from TA, pHNIST and salinity datausing CO2SYS (Pierrot et al., 2006). Both aragonite (Ωar) and calcite(Ωca) were used to assess carbonate mineral saturation because thesaturation states for the high magnesium–calcite echinoderm skele-ton have not been determined. High (N12%) magnesium–calcite ismore soluble than aragonite and calcite (Bischoff et al., 1987).

Gametes frommultiplemales and females (minimum three of each)were used to establish independent populations of developing embryos.This approach was taken so that the outcomes might reflect the re-sponse of progeny generated from multiple spawners as in nature.This multiple parent approach avoids the strong effects of individualmales and females and variable gamete compatibility (Evans andMarshall, 2005; Foo et al., 2012; Palumbi, 1999; Schlegel et al., 2012).The urchins were induced to spawn by injection of 1–3 ml of 0.5 M KCland the gametes were collected from the surface using pipettes. Theeggs were pooled in beakers (500 ml) of fresh, filtered seawater (FSW;1 μm) at 20 °C. The eggs were checked microscopically for shape andintegrity and sperm were checked for motility before use. Egg densitywas determined in counts from the egg suspension of 100 μl aliquots.Sperm were collected dry and sperm concentration was measuredusing haemocytometer counts. Pooled sperm from multiple maleswere placed in a small dish and stored dry at 4 °C until use.

2.3. Fertilization and development

Prior to fertilization, the eggs (ca. 3 ml−1) were placed in 500 ml ofexperimental FSW for 15 min. Just prior to fertilization, 1 μl of thesemen sample was added to 1 ml of experimental FSW in a 10 mltube. The sperm were briefly (1–2 s) activated in experimental waterand then added to the eggs. The amount of the sperm solution to addto each rearing container to achieve the optimal sperm to egg ratio

Table 2Parameters for experimentswithHeliocidaris tuberculata to the 72 h pluteus larva in eight tempaverage pHNIST (8.11, SE = 0.01, n = 3), total alkalinity (2316.49, SE = 6.04, n = 3), salinity

pH 8.1 pH 7.8

20 °C 24 °C 20 °C 24 °C

pCO2 338.1 (1.19) 334.5 (1.21) 757.2 (2.51) 761.8 (2.56)Ωca 4.92 (0.01) 5.62 (0.01) 2.77 (0.01) 3.22 (0.01)Ωar 3.19 (0.01) 3.69 (0.01) 1.80 (0.00) 2.11 (0.00)

(25:1, 102 sperm/ml) (Byrne et al., 2010) was determined from ahaemocytometer count of the semen sample. After 15 min, theeggs were rinsed 2–3 times in experimental FSW using reverse filtra-tion to remove excess sperm. The eggs were checkedmicroscopicallyin counts of 50 embryos to ensure acceptable levels of fertilization(ca. N80%).

At 2 h, approximately 100 embryos (as determined from originalegg counts) were placed into 140 ml jars (b1 embryo/ml) filled withexperimental FSW at 20 °C and 24 °C. The jars were sealed leaving mini-mal headspace. Eight independent populations of embryoswere reared inexperimental conditions to the 24 h gastrula stage (2 temp × 4 pH × 8populations = 64 jars) and 8 populations were reared to the 72 h larvalstage (n = 64 jars). We used 3 day unfed 4-armed larvae to determinethe influence of experimental treatments on arm growth, as a proxy oflarval calcification. The larvae were not fed, because after food is intro-duced to cultures of H. tuberculata, and other echinoid larvae, arm exten-sion is influenced by phenotypic plasticity, a feedbackmechanism linkingphytoplankton density (which may itself be effected by treatments) andarm growth (Soars et al., 2009).

The pH of the gastrula stage jars checked prior to harvest of embryosindicated that this parameter was stable (Table 3). The experimentalwater in the jars for larval development was changed at 24 h and 48 h.Seawater pHNIST was measured from a random subsample (n = 6) ofjars per treatment at 24 h (prior to renewal of water) (Table 3).

The percentage of normal gastrulae (24 h) and prefeeding larvae(72 h) were scored in the first 50 specimens collected with a pipettefrom each jar. These samples were placed in plastic tubes and fixedwith 2% formalin. Abnormal gastrulae included arrested embryos andembryoswith an irregular profile. At 72 h, the percentage of normal lar-vae were scored in the first 50 specimens collected from each replicatepopulation (jar). Abnormal larvae included arrested embryos, armlesslarvae and larvae with marked arm asymmetry (arms of differentlengths) (Fig. 1). Fixed larvaewere promptly photographed. A drop of lar-vae from each replicate was placed on slides with small modelling claysupports on the corners of cover slips to prevent the larvae being flat-tened. Digital photographs were taken of the first 30 randomly collectedlarvae (normal or abnormal) at 20×magnification using a compoundmi-croscope (Olympus DP70). Care was taken so that larvaewere orientatedflat to the plane of focus so that the full postoral (PO) arm length could bevisualized before photos were taken. Length of both PO arms was docu-mented using Image J (http://imagej.nih.gov/ij/). The mean length ofthe two PO armswas determined for each larva and themean arm lengthfor each population was used as the datum for analysis. The difference inthe length of the two arms was calculated as a measure of arm

erature–pH treatments.Mean values (±SE) for pCO2 (μatm),Ωca andΩar determined from(34.83, SE = 0.07, n = 3) and temperature using CO2SYS (Pierrot et al., 2006).

pH 7.6 pH 7.4

20 °C 24 °C 20 °C 24 °C

1257.1 (4.05) 1273.8 (4.16) 2053.9 (6.50) 2091.5 (6.68)1.83 (0.00) 2.14 (0.00) 1.19 (0.00) 1.40 (0.00)1.19 (0.00) 1.41 (0.00) 0.77 (0.00) 0.92 (0.00)

Table 3Average pH in experiments with Heliocidaris tuberculata measured at 24 h for gastrulae (n = 8, ±SE) and prior to water change for larval rearing (n = 6, ±SE) measured in a randomsubsample of jars.

pH 8.1 pH 7.80 pH 7.6 pH 7.4

20 °C 24 °C 20 °C 24 °C 20 °C 24 °C 20 °C 24 °C

pH gastrulae 8.13 (0.02) 8.1 (0.02) 7.8 (0.01) 7.78 (0.01) 7.63 (0.01) 7.61 (0.01) 7.48 (0.02) 7.48 (0.01)pH larvae 8.08 (0.02) 8.04 (0.02) 7.80 (0.01) 7.77 (0.02) 7.62 (0.02) 7.59 (0.01) 7.44 (0.02) 7.44 (0.02)

253M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

asymmetry. The mean asymmetry of each population was used as thedatum for analysis.

2.4. Statistical analysis

Each population of gastrulae or larvae was a separate replicate. Foreach replicate, the percentage of normal gastrulae or larvae,mean larvalarm length and arm asymmetry were determined. These data and thepercent fertilization data were analysed using two-way analysis of var-iance (ANOVA) with pH and temperature as fixed factors. Percentagedata were arcsine transformed prior to analysis. Normality and homo-geneity of the data were checked graphically (Quinn and Keough,2002). Tukey's post hoc testwas used to determine differences amongstexperimental treatments. SPSS software (SPSS 17.0, SPSS Inc., Chicago,IL) was used for all analyses.

3. Results

3.1. Environmental conditions in the habitat of H. tuberculata

Loggers placed in the subtidal habitat ofH. tuberculata recorded tem-peratures ranging from 14 to 25 °C (Fig. 2) with the average annualtemperature to be ca. 19 °C (n = 40,613, SE ± 0.01). During Oct–Nov2011 when the experiments were undertaken, the average SST at hightide was 20.9 °C (n = 5136, SE ± 0.02). During this time the loggersindicated a range of 15.5–18.5 °C during night time low tides (mean =16.8 °C, n = 10, SE ± 0.29) and 16–20 °C during daytime low tides(mean = 18.0 °C, n = 10, SE ± 0.45). The pHT of the seawater in thehabitat of H. tuberculata measured during 8 spring tides ranged from7.88 to 8.06 during predawn low tides (n = 8) to 8.06–8.25 duringpre-sunset low tides (n = 8), with an average pHT 8.08 (n = 10,SE ± 0.02) in the open water. The greatest difference in pHT between2 consecutive low tides was 0.31 units.

Fig. 1. Phenotypes of larvae in the 8 temperature–pH treatments. Larvae reared at 8.1 and 7.8 amixture of small larvae with a normal body profile and larvae with asymmetrical arms or othe

3.2. Development

There was a significant effect of temperature (F1,64 = 6.94, p =0.011) and pH (F3,64 = 6.63, p = 0.001) on the percentage of normalgastrulation (Table 4, Fig. 3a). Tukey's HSD indicated that the percent-age of normal gastrulae were reduced at 24 °C and at pH 7.4 and 7.6compared with controls (Table 4, Fig. 3a). There was no interactionbetween stressors. Across the near future warming-acidification treat-ments (24 °C/pH 7.6–7.8) there was a 5–19% decrease in normal gas-trulae and in the most extreme pH 7.4 treatments this increased to11–24% (Fig. 3a).

The phenotypes of larvae from the 8 treatments are illustrated inFig. 1. Most larvae reared at pH 7.8–8.1 at both temperatures were nor-mal plutei. In the other treatments the larvae were a mixture of smalllarvae with a normal body profile and larvae with asymmetrical armsor other abnormalities (Fig. 1). There was a significant effect of temper-ature (F1,54 = 20.818, p b 0.05) and pH (F3,54 = 35.544, p b 0.05) onthe percentage of normal H. tuberculata larvae, but no interaction be-tween stressors (Table 3, Fig. 3b). The highest abnormality was seen inthe most extreme 24 °C/pH 7.4 treatments. Tukey's HSD indicated asignificant reduction in the percentage of normal larvae at 24 °Cand at pH 7.4–7.6 (Table 4, Fig. 3b). Across the near future warming-acidification (24 °C/pH 7.6–7.8) treatments therewas a 5–25%decreasein normal larvae and in the most extreme pH 7.4 treatments this in-creased to 13–33% (Fig. 3b).

There was a significant effect of temperature (F1,52 = 207.726,p b 0.05) and pH (F3,52 = 11.015, p b 0.05) on postoral arm lengthwith no significant interaction between stressors (Table 4; Fig. 3c).Larvae reared at 24 °C had significantly longer arms across all pHlevels (e.g. pH 8.1–25% longer; pH 7.4–52% longer) compared withtreatments at control temperature (Tukey HSD: 20 °C b 24 °C). In con-trast larvae reared at pH 7.4 and 7.6 had shorter arms than those rearedat pH 7.8 and 8.1 (Table 4; Fig. 3c). Larvae reared at pH 7.4 and 7.6 incontrol temperature (20 °C) had 25% and 14% shorter arms comparedwith pH 8.1 controls (Fig. 3c).

t both temperatures were largely normal plutei. In the other treatments the larvae were ar abnormalities. Scale = 200 μm.

7.80

7.90

8.00

8.10

8.20

8.30

10

15

20

25

30

01-Jan 01-Apr 30-Jun 28-Sep 27-Dec

pH

T

Tem

per

atu

re (

°C)

Fig. 2. Temperaturesmeasured by an in situ data logger deployed in the subtidal habitat ofHeliocidaris tuberculata at Little Bay in 2011. In situ pHTmeasurements (grey circles)weredetermined from water chemistry of water samples taken during 8 spring tides.

254 M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

Arm asymmetry was a conspicuous feature of larvae in the lower pHtreatments especially at 24 °C (Figs. 1, 3d). There was a significanteffect of temperature (F1,64 = 6485, p b 0.05) and pH (F3,64 = 5.461,p b 0.05) on arm asymmetry with no significant interaction betweenstressors (Table 4; Fig. 3d). Tukey's HSD indicated greater arm asymme-try at 24 °C in the pH 7.4 and 7.6 treatments (Table 4; Fig. 3d). Thelower arm asymmetry at pH 7.4 (Fig. 3d) is due to the fact that manyof these larvae only had arm nubbins thereby reducing the differencebetween the two arms.

4. Discussion

In this study of the effects of increased temperature and acidificationon development of H. tuberculata, we show that both stressors had anegative effect on gastrulae and larvae. There was a decrease in normaldevelopment in the near future (24 °C/pH 7.6–7.8) treatments. Larvalgrowth was positively correlated with increased temperature acrossall pH treatments. This supported our prediction, but with the caveatof increased abnormality and arm asymmetry with a 4 °C warming. Asexpected, larval growth was negatively correlated with decreased pH.

Table 4Two-way ANOVA of data on percentage normal gastrulae (24 h) and echinoplutei (72 h) ofHelments. df: degrees of freedom. n = 8.

Factor Mean square df

A. % normal gastrulaeTemperature 0.160 1pH 0.153 3Temperature × pH 0.010 3Residual 0.023 56Total 63

B. % normal larvaeTemperature 0.254 1pH 0.397 3Temperature × pH 0.025 3Residual 2.066 54Total 62

C. Arm lengthTemperature 1892287.318 1pH 10037.569 3Temperature × pH 1630.003 3Residual 911.234 52Total 60

D. Arm asymmetryTemperature 1779.036 1pH 150.425 3Temperature × pH 7.196 3Residual 56Total 64

The boost in growth caused by increased temperature resulted in larvaethat were larger than would be observed if acidification was examinedin the absence of warming. This shows the importance of consideringboth major ocean change stressors in assessing potential species' re-sponses, especially in a regionwherewarming is themost important con-temporary ocean change stressor (Hobday and Lough, 2011).

The 4 °Cwarming resulted in faster growth inH. tuberculata larvae, aresponse to increased temperature expected for ectotherms (Gilloolyet al., 2001), and reported for many echinoids (Fujisawa, 1989; Sewelland Young, 1999; Sheppard Brennand et al., 2010). Despite this facilita-tion of growth, the increased abnormality and arm asymmetry withwarming indicates that+4 °Cmight approximate a threshold betweenfacilitation at certain levels of warming and failure at upper thermallimits for H. tuberculata. Later on in the year during summer, the popu-lation of H. tuberculata used for this study, occasionally experiences24 °C, and so adults may be adapted to tolerate this temperature.As baseline conditions in nature will increase by 3–4 °C by 2100 ata more gradual pace than in our experiments, the high percentageof normal larvae in the 24 °C treatments indicate that developmentin H. tuberculata may have some resilience to near future warming,and this would be facilitated if thermal acclimatization and/or adap-tation ensues. Similarly, as the shift to pH of 7.8 by ca. 2100 will alsobe more gradual in nature, the high percentage of normal larvae in thepH 7.8 °C treatments indicate that H. tuberculata larvae may also be re-silient to this level of acidification. Long-term (~12 months+) acclima-tion of urchins and oysters to moderately elevated pCO2 can result intrans-life-cycle enhancement of larval and juvenile resilience to reducedpH (Dupont et al., 2013; Parker et al., 2012). In addition, quantitative ge-netics studies with echinoids indicate the presence of genotypic traits tofacilitate resilience and adaptation to ocean acidification, (Sunday et al.,2011; Schlegel et al., 2012) ocean warming (Runcie et al., 2012) andboth stressors (Foo et al., 2012).

The interactive effects of warming and acidification on developmentremain a challenge for global change biology and, thus far, have beeninvestigated for 23 species (3 corals, 9 molluscs, 6 echinoderms, 5crustaceans), including 3 sea urchin species with echinopluteus larvae(Byrne and Przeslawski, 2013, this study). For abalone and bivalvelarvae both stressors exert an additive negative effect resulting in

iocidaris tuberculata and postoral arm length and asymmetry across temperature–pH treat-

F-value p-Value Tukey's HSD

6.940 0.011 20 N 246.631 0.001 8.1 N 7.6 = 7.4; 8.1 = 7.80.436 0.728

20.818 0.000 20 N 2435.544 0.000 8.1 = 7.8 N 7.6 = 7.42.066 0.115

207.726 0.000 24 N 2011.015 0.000 8.1 = 7.8 N 7.6 = 7.41.789 0.161

64.585 0.000 24 N 205.461 0.002 8.1 = 7.8, 7.8 = 7.6 = 7.40.261 0.853

0

20

40

60

80

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20 24

% N

orm

al g

astr

ula

e

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8.1 7.8 7.6 7.4

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asy

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Fig. 3. Response of Heliocidaris tuberculata embryos and larvae to rearing in 8 temperature–pH treatments. a. Mean percentage normal gastrulae. b. Mean percentage normal larvae c.Postoral larval arm length. d. Larval arm asymmetry (arm length 1–arm length 2). n = 8. Error bars are standard error.

255M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

unshelled or smaller-shelled larvae (Byrne et al., 2011b; Parker et al.,2010; Talmage and Gobler, 2011). For some crustacean and sea starlarvae, near future warming is the more important stressor to develop-ment (Arnberg et al., 2013; Nguyen et al., 2012; Walther et al., 2011).ForH. tuberculata, warmingmitigated the stunting effect of acidificationon larval growth. For the tropical echinoid T. gratilla a +3 °C warminginteracts antagonistically with acidification reducing the negative ef-fect of low pH on larval growth, but this was not evident at +6 °C(Sheppard Brennand et al., 2010). It would be interesting to determineif we would see a statistically significant antagonistic interaction be-tween warming and acidification in growth of H. tuberculata larvae ata lower level of increased temperature ca. 2–3 °C (2070 projection).

Decreased biomineralization in response to pCO2-driven acidifica-tion is reported for larval, juvenile and adult echinoids (Albright et al.,2012; Byrne et al., 2013a; Shirayama and Thornton, 2005; Wolfe et al.,2013). H. tuberculata larvae reared in pH 7.4–7.6 had significantlyshorter postoral arms than those reared in pH 7.8–8.1. Stunting of pluteireared in low pH conditions is a general feature of ocean acidificationexperiments, reported for ca. 15 species from the tropics to the poles(Byrne et al., 2013a). Calcification in sea urchin larvae occurs intracellu-larly, in a tightly controlled environmentwhere pH is regulated, but at aconsiderable energetic cost, which increases under ocean acidificationconditions (Dubois and Chen, 1989; Stumpp et al., 2012). Low pH, espe-cially pH 7.4, was deleterious to development. The abnormalities andpoor growth and calcification seen in larvae reared in pH 7.4 (not anear-future scenario) indicate that this level of acidification is toxic todevelopment. The larvae were able to calcify under these conditions,

but were also the smallest larvae. This pCO2/mineral saturation levelmay approach the limits for successful calcification and armdevelopment.

The reduction in size of echinoplutei reared in ocean acidification con-ditions is suggested to be due to: 1) hypercapnic metabolic suppressioncausing delayed development (i.e. the larvae are normal and willcatch up given time); 2) decreased availability of carbonate mineralsfor skeletogenesis (physiological stress on calcification systems),3) the diversion of energy to acid/base regulation and away fromgrowth, inducing developmental delay and 4) teratogenic effects(cytotoxic, genotoxic) on larval development (Byrne et al., 2013b;Martin et al., 2011; O'Donnell et al., 2010; Sheppard Brennand et al.,2010; Stumpp et al., 2011; Uthicke et al., 2013). A recent review indi-cates thatmetabolic stress, due to elevated CO2, is themajormechanismunderlying decreased growth in echinoplutei in ocean acidificationtreatments (Byrne et al., 2013a). The significant increase in abnormalembryos, gastrulae and larvae in near-future low pH treatments indi-cates that acidification has teratogenic effects on cell dynamics andgene expression patterns that control larval growth.

Insights into the relative influence of pH/pCO2 and reduced mineralsaturation on echinoderm larvae are evident from studies of specieswith non-calcifying larvae (Byrne et al., 2011b, 2013c; Nguyen et al.,2012). Asteroid larvae are highly sensitive to warming especially inearly development.With long term rearing however, the negative effectof acidification in reducing larval growth is evident in bipinnaria larvae(Byrne et al., 2013c; Gonzalez-Bernat et al., 2013a). This result in larvaefor which carbonate mineral saturation is not likely to be a major directissue, shows the strong influence of pH/pCO2 on larval growth.

256 M. Byrne et al. / Journal of Experimental Marine Biology and Ecology 448 (2013) 250–257

Misshapen asymmetrical echinoplutei, as seen here for H. tuberculata,and in other ocean warming/acidification studies, would havecompromised performance in nature due to alteration to swimmingand feeding abilities (Chan et al., 2011; Chen and Chen, 1992; Clayand Grünbaum, 2011; Hart and Strathmann, 1994). Arm asymmetryin larvae reared in acidification conditions is also reported for theechinoplutei of two tropical species T. gratilla and Echinometra mathaei(Sheppard Brennand et al., 2010; Uthicke et al., 2013) and may be de-tected in other echinoid species with detailed assessment of larvalgrowth dynamics. Several studies also show altered morphometry inechinoplutei reared in ocean acidification experiments with changesin growth of body components relative to each other (O'Donnell et al.,2010; Gonzalez-Bernat et al., 2013b, but see Stumpp et al., 2011).Appropriate body allometry and functional arm rods are essential forfeeding, swimming and protection from predation and feeding successin echinoplutei is related to arm length (Allen, 2008; Chan et al., 2011;Clay and Grünbaum, 2011; Hart and Strathmann, 1994; Soars et al.,2009). Smaller larvae with a longer planktonic duration would also bemore vulnerable to predation in a changing ocean, decreasing chancesof survival and recruitment (Lamare and Barker, 1999). Production ofsmaller larvae with a longer larval period, larvae that have changedbody symmetry or abnormal larvae in a future ocean would compro-mise success of the pelagic life stage of H. tuberculata. The followingbenthic stage may also metamorphose as undersized juveniles withconsequences for postlarval success (Chen and Chen, 1992; Talmageand Gobler, 2009). Small changes in larval and early juvenile success ina changing ocean will have flow on impacts for the integrity of benthicpopulations (Eckman, 1996; Przeslawski et al., 2008;Uthicke et al., 2009).

As shown herewarming and acidification both had negative impactson development in H. tuberculata in a climate and regionally relevantsetting. For regions with significant warming such as eastern Australia,temperature is the most immediate and contemporary climate changestressor. This is also the case for mollusc larvae on the east coast ofnorth America (Talmage and Gobler, 2011), but not for those on thewest coast where acidification is the most important contemporarystressor (Service, 2012).

As the ocean will change gradually over coming decades our dataindicated that H. tuberculata may be able to acclimatize to warming inits habitat and thereby produce more resilient offspring (O'Connorand Mulley, 1977). This species may experience a potential contractionin the warmer parts of its range and poleward range extension in thesouthern part of its range. The latter is seen in the migration of thesympatric echinoid Centrostephanus rodgersii from New South Walesto Tasmania through larval dispersal facilitated by strong climate-driven warming and changes in ocean currents (Ling et al., 2010).Why H. tuberculata, whose larvae are in the plankton at the same timeas the larvae of C. rodgersii, has not also exhibited a similar range ex-tension is not known. There remains a possibility that migration ofH. tuberculata to higher latitudes is actually limited by the cool toler-ance of the larvae and adults of this species.

Acknowledgements

Researchwas funded by grants from theAustralian Research Council(MB, SD), the Sydney Institute ofMarine Science (SF) and theUniversityof Sydney (SF, NS, HDN) scholarships. Sydney Institute of Marine ScienceContribution #102. [SS]

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