Early development of congeneric sea urchins (Heliocidaris) with contrasting life history modes in a warming and high CO2 ocean.

Marine Environmental Research xxx (2014) 1e10

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Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev

Early development of congeneric sea urchins (Heliocidaris) with contrasting life history modes in a warming and high CO2 ocean Natasha A. Hardy a, *, Maria Byrne a, b a b

School of Medical Sciences, University of Sydney, NSW 2006, Australia School of Biological Sciences, University of Sydney, NSW 2006, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

The impacts of ocean change stressors e warming and acidification e on marine invertebrate development have emerged as a significant impact of global change. We investigated the response of early development to the larval stage in sympatric, congeneric sea urchins, Heliocidaris tuberculata and Heliocidaris erythrogramma with contrasting modes of development to ocean warming and acidification. Effects of these stressors were assessed by quantifying the percentage of normal development during the first 24 h post fertilization, in cross-factorial experiments that included three temperature treatments (control: 20 C; þ4: 24 C; þ6: 26 C) and four pHNIST levels (control: 8.2; 0.4: 7.8; 0.6: 7.6; 0.8: 0.4). The experimental treatments were designed in context with present day and near-future (~2100) conditions for the southeast Australia global warming hotspot. Temperature was the most important factor affecting development of both species causing faster progression through developmental stages as well as a decrease in the percentage of normal development. H. erythrogramma embryos were less tolerant of increased temperature than those of H. tuberculata. Acidification impaired development to the larval stage in H. tuberculata, but this was not the case for H. erythrogramma. Thus, outcomes for the planktonic life phase of the two Heliocidaris species in response to ocean warming and acidification will differ. As shown for these species, single-stressor temperature or acidification studies can be misleading with respect to determining species' vulnerability and responses to global change. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Climate change Ocean warming Ocean acidification Life history Stress tolerance Embryo Larva Echinoid

1. Introduction Anthropogenic carbon dioxide (CO2) emissions are driving ocean acidification and increasing ocean temperatures (Caldeira and Wickett, 2003, 2005; IPCC, 2013). These changes are expected to impact the planktonic developmental stages of marine animals over coming decades (Byrne, 2011). Temperature is highly significant to marine invertebrate development in controlling developmental timing, the length of the dispersive stage, larval energetics and survival (Sewell and Young, 1999; O'Connor et al., 2007; Byrne et al., 2011a; Hardy et al., 2014). Ocean acidification and associated reduction in carbonate mineral saturation will have wide-ranging impacts on survival, calcification and physiological €rtner, 2008, 2010; Kroeker et al., 2013). As processes in general (Po ocean temperature and acidification are changing simultaneously, with potential for interactive effects, it is critical to investigate the

* Corresponding author. Room 121, Edgeworth David Building A11, University of Sydney, NSW 2006, Australia. Tel.: þ61 2 9351 2482; fax: þ61 2 9351 2813. E-mail address: [email protected] (N.A. Hardy).

effects of simultaneous exposure to both stressors on marine species (Przeslawski et al., 2008; Widdicombe and Spicer, 2008; Byrne, 2011; Nguyen et al., 2012; Byrne and Przeslawski, 2013). The sensitivity of the developmental stages of marine invertebrates to increased temperature and acidification varies among species with some being more tolerant than others (Byrne and Przeslawski, 2013). This differential sensitivity will influence species persistence, invasive potential, faunal shifts and community function in a changing ocean (Byrne, 2011). Differences in species survival through past climate-driven change in planktonic communities are likely to have been influenced by life history characteristics according to studies investigating mass extinction events where changes in global temperature were a factor (Valentine and Jablonski, 1986; Uthicke et al., 2009). For echinoderms, there appears to have been differential survival of species with non-feeding larvae through past extinction events (Uthicke et al., 2009). Due to dependence on an exogenous food source and a long planktonic phase when predation is high, species with feeding planktotrophic larvae are suggested to have a high-riskehigh-gain life history (Uthicke et al., 2009). In contrast, species with non-feeding lecithotrophic larvae with enhanced maternal nutrients are suggested

http://dx.doi.org/10.1016/j.marenvres.2014.07.007 0141-1136/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Hardy, N.A., Byrne, M., Early development of congeneric sea urchins (Heliocidaris) with contrasting life history modes in a warming and high CO2 ocean, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.007

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N.A. Hardy, M. Byrne / Marine Environmental Research xxx (2014) 1e10

to have a buffered life history and be less vulnerable to climate change stressors (Uthicke et al., 2009). The increase in the number of species with lecithotrophic development over evolutionary time may be due to higher speciation rates and the unidirectional switch to lecithotrophy in many taxa, where once a feeding larval form is lost, it is not regained (Duda and Palumbi, 1999; Jeffrey and Emlet, 2003). In studies that investigate the interactive effects of increased temperature and acidification on marine invertebrate development, early embryos appear to be most sensitive to warming (Byrne and Przeslawski, 2013). If thermal thresholds are breached, or if there are deleterious interactions between temperature and acidification, embryos may not reach the larval stage (Byrne, 2011), thus forming a bottleneck of mortality with negative flow-on effects for persistence of populations. Identifying these bottlenecks for species with different development modes using multi-stressor experimental designs is crucial to understanding the impacts of projected near-future climatic change on species persistence (i.e. range shifts, local extinction). We investigated the effects of increased temperature and acidification on early development in sympatric, congeneric sea urchins with contrasting life histories. Heliocidaris tuberculata has the ancestral-type mode of development for echinoderms in possession of a small egg and a planktotrophic larva (Raff and Byrne, 2006). In contrast, Heliocidaris erythrogramma has the derived life history with a large egg and a lecithotrophic larva. These species are common in shallow water rocky habitats in southeastern Australia (Keesing, 2013), an ocean warming hotspot (Hobday and Lough, 2011). These species provided an ideal model to compare the impacts of warming and acidification on early embryos and larvae with contrasting maternal provisioning because their close phylogenetic relationship (5e8 mya apart), allows for comparisons not € rtner, 2001; confounded by disparate phylogeny (Sokolova and Po Raff and Byrne, 2006). For H. erythrogramma, fully developed larvae are more sensitive to warming than acidification while for H. tuberculata, acidification exerts a greater influence (Byrne et al., 2011a,b, 2013a). Here, we focused on very early stages to determine the dynamics of cleavage to the development of the early larva (first 24 h) in temperature and acidification treatments commensurate with projections for the local ocean over coming decades (Hobday and Lough, 2011). Increased temperature was predicted to result in faster progression through developmental stages, as typical of marine invertebrate embryos (O'Connor et al., 2007; Hardy et al., 2014). On the other hand we expected that increased acidification might slow this progression due to alteration of embryo physiology. Due to enhanced nutritive provisioning, and potential possession of a greater titre of protective maternal factors (Hamdoun and Epel, 2007), H. erythrogramma embryos were predicted to be more tolerant to warming and acidification than those of H. tuberculata.

the larvae of these species are likely to experience in the plankton (Wolfe et al., 2013; Byrne et al., 2013a). The seawater for experiments was collected from the open ocean near Sydney (mean pHNIST 8.26 ± 0.02; salinity 34.6 psu ± 0.2, n ¼ 8). The water was filtered (1 mm Millipore) (filtered seawater, hereafter FSW) and total alkalinity (TA) was determined in samples (300 mL) fixed with mercuric chloride (mean TA: 2294.28 mmol kg1 seawater, SE ± 10.30; n ¼ 8) by potentiometric titration using the Gran method. pHTotal and pCO2 were calculated using CO2SYS (Pierrot et al., 2006) (Table 1). Experimental treatments were designed in context with present day and near future (2100) projections for the region (Hobday and Lough, 2011). Embryos were reared for 24 h in three temperature treatments (20 C, 24 ¼ þ4 C, 26 þ6 C) and four pHNIST levels (pHNIST: ambient pH 8.2, pH 7.8: 0.40 units, pH 7.6: 0.60 units, and pH 7.4: 0.80 pH units) in all combinations in a fully orthogonal experimental design. The pH 8.2 and 20 C treatments were the controls. Large volumes (20e30 L) of seawater were equilibrated to the desired temperature/pH level to ensure that all embryos were placed in the same conditions. The water was brought to temperature in water baths (±0.25 C) and bubbled with CO2 gas until the required pHNIST was reached as determined by pH meter (WTW Multiline 3400i; Table 1). The probe was calibrated daily with NIST (high precision) buffers pH 4, 7 and 10 (ProSciTech). Rearing containers (100 mL jars, see below) were filled with experimental water with minimal headspace to avoid out-gassing of CO2 until gametes were placed in treatments and throughout the experiments. At the end of the experiment pHNIST, measured at 24 h had remained stable (Table 1). Dissolved oxygen (DO) was >90% and salinity was checked and remained stable (34.4 ± 0.1 psu) over the 24 h experiments. Temperature loggers (Onset Thermodata) placed in the water baths with effective water flow confirmed that the temperature was stable throughout the experiments (change over 24 h < ±0.025 C). 2.2. Fertilization and rearing Spawning was induced by injection of 2e4 mL of 0.5 M KCl into the coelom and the released gametes were collected using a pipette. Gametes from three to four of the most fecund males and females were chosen based on production of copious amounts of high quality gametes, as determined by microscopic examination

Table 1 Parameters for water used in experiments with H. erythrogramma and H. tuberculata in 12 temperatureepH treatments. Mean values (±SE; n ¼ 8) for pHNIST and change in pH within experimental jars after 24 h are included, as well as mean values for pCO2 (matm) determined from average pHNIST, total alkalinity (2294.3 ± 10.3, n ¼ 8), salinity (34.5 ± 0.2, n ¼ 8) and temperature using CO2SYS (Pierrot et al., 2006).

2. Methods

pH treatment

Temp. treatment

pHNIST

pH (change after 24 h)

2.1. Specimen collection and experimental treatments

pH 8.2

20 C 24 C 26 C

8.16 ± 0.02 8.18 ± 0.01 8.18 ± 0.01

±0.05 ±0.07 ±0.08

291.2 ± 20.7 266.9 ± 10.2 260.1 ± 11.0

pH 7.8

20 C 24 C 26 C

7.79 ± 0.01 7.80 ± 0.00 7.80 ± 0.01

±0.03 ±0.03 ±0.02

767.6 ± 11.2 751.6 ± 9.6 757.7 ± 12.2

pH 7.6

20 C 24 C 26 C

7.59 ± 0.00 7.60 ± 0.00 7.59 ± 0.00

±0.03 ±0.03 ±0.04

1274.4 ± 12.4 1260.7 ± 15.0 1274.1 ± 17.5

pH 7.4

20 C 24 C 26 C

7.39 ± 0.00 7.39 ± 0.00 7.40 ± 0.00

±0.08 ±0.08 ±0.08

2088.5 ± 16.3 2094.1 ± 21.6 2107.5 ± 21.8

H. erythrogramma (5e10 cm test diameter) and H. tuberculata (10e20 cm test diameter) were collected in MarcheApril 2012 from Little Bay, Sydney (33 580 S, 151140 E), an open ocean site where these species co-occur in shallow-water boulder habitat. They were transported and placed in aquaria at ambient SST (20e21 C) and used for experiments within 48 h of collection. At the time of collection average sea surface temperature (SST) of 20e21 C was determined from the Physical Oceanography DAAC Ocean ESIP Tool (POET) web site (http://poet.jpl.nasa.gov) and a local reference station (http://mhl.nsw.gov.au). This is also the temperature that

pCO2



(i.e. egg shape, sperm motility). The eggs were pooled in a 500 mL beaker of FSW and sperm from several males was collected by a pipette from the surface of the urchins and pooled in a Petri dish that was then covered and kept cool and dry until use to avoid activation of sperm by seawater. The multiple parent approach was taken to reflect the response of progeny generated from a population of spawners and to avoid the strong maternal and paternal effects characteristic of single dam-sire crosses (Palumbi, 1999; Foo et al., 2012; Evans and Sherman, 2013). Egg density was determined from an average of five counts of 100 mL aliquots for H. erythrogramma and 10 mL aliquots for H. tuberculata, using a Sedgwick Rafter Counting Chamber. The eggs (10 mL1) were distributed among 12 glass beakers (250 mL) at 3 temperature and 4 pH treatments for 15 min prior to fertilisation. The optimal sperm to egg ratio was used (H. erythrogramma: 200:1; H. tuberculata 25:1), as determined using a haemocytometer (see Byrne et al., 2010, 2011a). Sperm was activated briefly in treatment water just prior to addition to the beakers containing eggs. After 15 min, the eggs were checked to ensure suitable fertilisation levels (90%) and then rinsed 2e3 times in fresh experimental FSW. Following rinsing, 100e200 eggs from the fertilization beakers were placed in experimental FSW in 100 mL glass jars with 3 replicates per treatment, per time point, for each experiments. Experiments were repeated three times with n ¼ 3 for each treatment for each experiment, thus n ¼ 9 replicates over the three experiments. 2.3. Embryonic development The embryos were reared to 24 h. For each species 108 jars were used (3 time points 3 temperature 4 pH 3 replicates). At each time point, water was reverse aspirated and a 2 mL sample of embryos was fixed in 10% formalin for scoring of embryo stages with the exception of the hatching stages which were scored live to determine the percentage of normal motile embryos. The containers were discarded after sampling, thus embryos from each time point were raised in independent containers to avoid repeat sampling, and replicates were raised in separate jars to avoid pseudoreplication. Development of H. erythrogramma was scored at 6 h, 12 h and 24 h, and for H. tuberculata embryos were scored at 4 h, 16 h and 24 h. Because the two species have very different early development with H. erythrogramma having a wrinkled blastula stage that does not exist in H. tuberculata (Raff and Byrne, 2006) the very early embryos were scored at different times. In H. erythrogramma this was 6 h for the wrinkled blastula stage and 12 h for the hatched blastula stage. In H. tuberculata this was 4 h for the early multi-celled stage and then 16 h for the hatched blastula stage. By 24 h both species are swimming larvae and so at this time point are more comparable. For both species, samples were scored according to developmental stage achieved as well as for normal and abnormal or arrested development and mortality in random counts of the first 50 specimens encountered in each sample. Normal development included symmetrical cleavage embryos, considerations of shape in blastulae, gastrulae and larvae, which contrasted quite clearly with abnormal development (see Hardy et al., 2014). 2.4. Statistical analysis All percentage data were arscine transformed prior to analysis. Data on the percentage of the most advanced developmental stages achieved at each time point were analysed by 2-way analysis of variance (ANOVA) for each species, with temperature and pH as fixed orthogonal factors. Data on the percentage of normal development over time were analysed by 2-way ANOVA for each

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species and each time point with temperature and pH as fixed orthogonal factors. ANOVAs were followed by a posteriori StudenteNewmaneKeuls (SNK) tests on appropriate terms of the model found to be significant with P < 0.05, in order to identify significant differences between treatments. For the 24 h time point analysis of the percent normal development data, species was also included in a 3-way ANOVA. The assumption of normality was checked for each dataset by plotting residuals against normal distribution, but normality was not always met due to trends in the dataset being driven by extreme treatments. The ANOVAs were still performed as these tests are robust to deviations from normality with large datasets (Quinn and Keough, 2002). The assumption of homogeneity of variance was also checked using Cochran's C, and was met for all analyses except for the percentage developmental stage achieved data for H. erythrogramma, for these data the analyses were carried out because ANOVAs performed for large datasets (here 12 treatment levels and n ¼ 9) are robust to deviations from this assumption (Sokal and Rolfe, 1995; Underwood, 1997). All data were analysed in WinGMAV5 (Underwood, 1997). 3. Results 3.1. Impact of temperature and acidification on H. tuberculata embryos 3.1.1. Normal development The percentage of normal development of H. tuberculata was significantly affected by temperature and pH (Table 2), with no interactions between these stressors at any time point. Very early embryos (4 h) were robust to both stressors, with a slight decrease in normal development at pH 7.4 (SNK). By 16 h at the hatching stage, increased temperature was deleterious, particularly 26 C and there was also a decrease in normal development at pH 7.4 (Table 2, Fig. 1). However, percentage of normal development remained >80% in all but the extreme treatments (26 C, pH 7.4) (Table 2, Fig. 1). At 24 h, the percentage of normal development was lowest in the high temperature treatment (26 C) and in all the experimental pH treatments (Table 2, Fig. 1). Overall, however, percentage of normal development remained 80% across all temperature and pH treatments (Fig. 2). 3.1.2. Progression through developmental stages Both temperature and pH had a significant effect on progression through developmental stages in H. tuberculata with no interactive effect of stressors. At increased temperature there were more advanced stages at each time point with a similar effect of 24 C and 26 C. At each time point the lowest pH treatment (pH 7.4) retarded development (Table 3, Fig. 2 and Supplementary Tables S.1 and S.2). The effect of temperature on hatching and gastrulation was evident at 16 h, with 55.0% prism and early 2-armed larvae present in the 24 C and 26 C treatments compared with 24 C > 26 C 8.2 ¼ 7.8 ¼ 7.6 > 7.4 e

6.66 13.75 1.27

0.002 0 0.2772

20 C ¼ 24 C > 26 C 8.2 > 7.8 > 7.6 ¼ 7.4 e

176.65 0.39 1.92

0 0.757 0.0848

20 C ¼ 24 C > 26 C e e

138.12 4.94 2.55

0 0.0031 0.0247

20 C > 24 C > 26 C 8.2 ¼ 7.8 ¼ 7.6 ¼ 7.4 20 C pH 8.2 > 20 C pH 7.8, 7.6, 7.4 ¼ 24 C all pH's ¼ 26 C all pH's

72.93 0.73 0.99

0.0001 0.5363 0.4362

20 C > 24 C > 26 C e e

3.2.2. Progression through developmental stages Temperature and but not pH had a significant effect on the progression through developmental stages in H. erythrogramma with more advanced stages at 24 C and 26 C, up to 24 h with high mortality occurred in the 26 C treatment (Table 3, Fig. 2, Supplementary Tables S.3 and S.4). The percentage of advanced stages was always highest at 24 C and lowest in controls. The effect of temperature on hatching and gastrulation was evident at 12 h. At 20 C, most embryos were unhatched blastulae (>90.5%), while at 24 C and 26 C many were hatched blastulae (41e52%)

Fig. 1. Mean percentage of normal development in H. tuberculata embryos reared to 4 h, 16 h and 24 h in twelve treatments (3 temperature 4 pH); n ¼ 9 (±SE).



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Fig. 2. H. tuberculata percentage developmental stage achieved data at 24 h, percentage (±SE) of dead/arrested embryos, gastrula and prisms/early larvae; n ¼ 9.

(20 C < 26 C < 24 C) (Table 3, Supplementary Table S.4). By 24 h, the percentage of early larvae was similar in the 20 C and 26 C treatments (20 C: 34.1e42.9%; 26 C: 25.4e41.0%), and these treatments differed from the 24 C treatment, which had the highest percentage of early larvae (54.1e60.7%) (Table 3, Fig. 4). Although the percentage of larvae did not differ between the 20 C and 26 C treatments (30e40% early larvae in both), the remaining embryos reared at 20 C were normal albeit slower to development, whiles those reared at 26 C were dead (48.7e54.8%) (Fig. 4).

3.3. Comparative impact of temperature and acidification on H. tuberculata and H. erythrogramma at 24 h At the early larval stage, both species responded differently to temperature and pH stressors (Table 4, Fig. 5). The early plutei or larvae of H. tuberculata were more affected by decreasing pH than were the early larvae of H. erythrogramma with percentage of normal development dropping steeply as a response to this factor in H. tuberculata larvae (Fig. 5). The opposite was found for temperature, as H. erythrogramma larvae were more affected by

increasing temperature than were the early plutei of H. tuberculata which were barely affected at all whilst H. erythrogramma larvae experienced a steep drop in developmental success (Fig. 5).

4. Discussion This study of the effects of warming and acidification on developmental success and progression through ontogenetic stages in sympatric, congeneric species with contrasting modes of development revealed different sensitivities of H. erythrogramma and H. tuberculata, depending on the stressor tested. The hypothesis that development to the early larval stage in H. erythrogramma (large egg) would be more tolerant to stress than in H. tuberculata (small egg) was not supported with respect to projected temperature increase. As expected, time to reach the larval stage was shorter at increased temperature, with thermal tolerance limits evident for H. erythrogramma. A shorter planktonic duration at higher temperature is characteristic of marine invertebrate embryos (Pechenik, 1987; O'Connor et al., 2007; Byrne et al., 2013b; Davis et al., 2013; Pecorino et al., 2013; Hardy et al., 2014).


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Table 3 Two-way ANOVAs of data on percentage of developmental stage achieved of embryos of H. tuberculata and H. erythrogramma for three time points and across twelve temperature and pH treatments. SS: sum of square; df: degrees of freedom; F: F-value; P: P-value; SNK: a posteriori StudenteNewmaneKeuls tests; n ¼ 9. Species

Developmental stage

Source

SS

df

F

P

SNK

H. tuberculata

4 h %multiecelled

Temperature pH Temperature pH Residual Total Temperature pH Temperature pH Residual Total Temperature pH Temperature pH Residual Total

74,183.89 2008.575 408.8563 4167.274 80,768.6 12,243.52 1782.458 1036.612 13,074.62 28,137.2 48,380.1 1266.018 138.235 10,325.09 60,109.44

2 3 6 96 107 2 3 6 96 107 2 3 6 96 107

854.47 15.42 1.57

0 0 0.1643

20 C < 24 C ¼ 26 C 8.2 ¼ 7.8 ¼ 7.6 > 7.4 e

44.95 4.36 1.27

0 0.0063 0.2791

20 C < 24 C ¼ 26 C 8.2 ¼ 7.8 ¼ 7.6 > 7.4 e

224.91 3.92 0.21

0 0.0109 0.9715

20 C < 24 C ¼ 26 C 8.2 ¼ 7.8 ¼ 7.6 > 7.4 e

Temperature pH Temperature pH Residual Total Temperature pH Temperature pH Residual Total Temperature pH Temperature pH Residual Total

57,828.41 264.6686 188.9068 6954.91 65,236.89 25,129.72 312.9441 408.9154 22,583.95 48,435.53 4668.558 325.9586 595.0911 12,619.99 18,209.6

2 3 6 96 107 2 3 6 96 107 2 3 6 96 107

399.11 1.22 0.43

0 0.3075 0.8541

20 C < 26 C < 24 C e e

53.41 0.44 0.29

0 0.7225 0.9405

20 C < 26 C < 24 C e e

17.76 0.83 0.75

0 0.4824 0.6074

26 C ¼ 20 C < 24 C e e

16 h %gastrulae

24 h % prism & early larvae

H. erythrogramma

6 h %blastulae

12 h %hatched blastulae

24 h % late gastrula & early larvae

Temperature was the more important factor for both Heliocidaris species with faster progression through developmental stages from the first time point scored and increased mortality. By 24 h 50% of the H. erythrogramma embryos reared at þ6 C (¼26 C) were dead or had arrested development (which is fatal). In contrast, at this temperature most H. tuberculata embryos had progressed to the early larval stage and appeared normal. With regard to pH, H. tuberculata, but not H. erythrogramma was negatively affected by acidification at the most extreme level tested (pH 7.4). This low pH

level is not expected to occur in the ocean in the near future and may be toxic to H. tuberculata. Developmental delay at pH 7.4 suggests a narcotic effect of pCO2 on embryo physiology. Early cleavage in both species was fairly robust to increased temperature and acidification initially, particularly for the þ4 C treatments across all pH levels. Tolerance of echinoderm embryos to these stressors is reported in several studies (Byrne et al., 2011a,b, 2013a; Nguyen et al., 2012) and may be due to the presence of maternal protective factors deposited in the egg (Hamdoun

Fig. 3. Mean percentage of normal development in H. erythrogramma embryos reared to 6 h, 12 h and 24 h in twelve treatments (3 temperature 4 pH); n ¼ 9 (±SE).



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Fig. 4. H. erythrogramma percentage developmental stage achieved at 24 h data, percentage (±SE) of dead/arrested embryos, early gastrulae and late gastrulae/early larvae; n ¼ 9.

and Epel, 2007). However, from the 12e16 h time point, and certainly by 24 h, deleterious effects were evident and this differed between the two species. Mortality of H. erythrogramma was high in the þ4e6 C treatments across all pH levels. This sensitivity to increased temperature in this species is also reported in Byrne et al. (2011b). For H. tuberculata, development over the first 24 h tolerated þ4e6 C treatments across pH levels (pH 7.6e7.8) projected for the local ocean (Hobday and Lough, 2011). Early embryos of a range of marine invertebrate species exhibit some tolerance to these stressors, but by 24 h, deleterious effects are often evident (Nguyen et al., 2012; Byrne et al., 2013b; Byrne and Przeslawski, 2013). Assessment of propagule loss and developmental success of survivors in ocean change studies requires consideration of early embryos (12e24 h) while dead and dying embryos are still present to be scored (e.g. sea stars: Byrne et al., 2013b; this study). Disintegration and loss of embryos can confound survivorship assessment. Outcomes of ocean change experiments with marine invertebrate development also depend on the stage at which experimental animals are placed in treatments (Byrne, 2012). Many studies start at the larval stage (Byrne and Przeslawski, 2013) and

thus cannot discern sensitivities of early embryos, which as shown here for Heliocidaris, can have a significant influence on survival. This study investigated the influence of life history strategy of closely related, sympatric echinoids with contrasting modes of development to climate change stressors. Species like H. erythrogramma with a large egg and non-feeding larvae are suggested to have a buffered life history and be more robust to climatic change than species like H. tuberculata, with feeding larvae (Valentine and Jablonski, 1986; Uthicke et al., 2009). The embryos of H. erythrogramma, however, were less stress tolerant than the species with the small egg, H. tuberculata, with respect to increased temperature. Acidification however does not appear to affect the planktonic phase of H. erythrogramma, whereas this stressor does impair development of the larvae of H. tuberculata. Thus, outcomes for the planktonic life phase of the two Heliocidaris species in a changing ocean will differ. Different sensitivity to increased temperature is particularly important where these species reside in southeastern Australia, an ocean warming hotspot (Hobday and Lough, 2011). Predictions for this region are of þ5 C by 2070, which will push the early


8


Table 4 Three-way ANOVAs of data on percentage of normal development of the 24 h early larval stage for H. tuberculata and H. erythrogramma across twelve temperature and pH treatments. SS: sum of square; df: degrees of freedom; F: F-value; P: P-value; SNK: a posteriori StudenteNewmaneKeuls tests; n ¼ 9. Factor

SS

df

F

P

Species Temperature pH Species temperature Species pH Temperature pH Species temperature pH RES TOT

9245.7231 6617.5739 1451.4415 2330.1819 709.6494 647.9636 185.6313 10,534.611 31,722.7757

1 2 3 2 3 6 6 192 215

168.51 60.3 8.82 21.23 4.31 1.97 0.56

0 0 0 0 0.0057 0.072 0.7587

SNK for interaction terms (see Tables 1 and 3 for single factors) Sp Te Each species had a significantly different response to each temperature treatment, graphically: H. erythrogramma 20 C ¼ H. tuberculata 20 C, 24 C, 26 C > H. erythrogramma 24 C > H. erythrogramma 26 C Sp pH Each species had a significantly different response to each pH treatment, graphically: H. erythrogramma (all pH treatments) ¼ H. tuberculata pH 8.2, 7.8 and 7.6 > H.tuberculata pH 7.4

developmental stages of H. erythrogramma to their upper thermal thresholds. For later developmental stages only ~20% of larvae develop to the juvenile stage under near-future conditions although, those that do produce a juvenile, appear normal, indicating the presence of a subset of tolerant larvae (Byrne et al., 2011a). Further, juvenile H. erythrogramma settle into tide pools where temperature and pH can fluctuate diurnally during spring tides up to 10 C and 0.45 pH units and juvenile survival and growth were not found to be significantly affected by near-future ocean change conditions (Wolfe et al., 2013). It is suggested that animals adapted to these conditions may be tolerant to changing ocean conditions (Melzner et al., 2009; Byrne, 2011; Sanford and Kelly, 2011). Thus, it appears that the propagules of H. erythrogramma that survive to the juvenile stage in warm-low pH conditions may

tolerate near future conditions, with the caveats that conditions in the intertidal may approach stress tolerance limits in pulses at low tide (Byrne et al., 2011a; Wolfe et al., 2013). The most important planktonic life phase bottleneck for success to the adult stage in H. erythrogramma appears to be embryos and larvae due to the limited tolerance to ocean warming (Byrne et al., 2011a,b). There are no data on the response of adult H. erythrogramma to increased temperature and acidification. The response of H. erythrogramma embryos and larvae to pH is consistent with the responses documented for similar development in species with large eggs, non-feeding and non-calcifying larvae (e.g. sea stars, corals) (Anlauf et al., 2011; Byrne et al., 2011a,b; Nguyen et al., 2012; Chua et al., 2013). This stressor even facilitated development in the sea star Crossaster papposus with faster growth observed (Dupont et al., 2010). For species with lecithotrophic larvae, the life phase bottleneck for successful development in response to acidification may be the early juveniles with deleterious impacts on calcification processes such as spine growth (Anlauf et al., 2011; Wolfe et al., 2013). However, for species with non-feeding larvae that also produce a shell (e.g. mollusc veligers), both stressors have a negative impact on development (Byrne et al., 2011a; Crim et al., 2011). For H. tuberculata, embryonic thermal and pH tolerance is not a barrier to achieve the prism/early echinopluteus stage in nearfuture conditions and beyond (þ4e6 C/pH 7.4e7.6), but by the later echinopluteus stage growth is negatively affected by ocean acidification (pH 7.6e7.8), as typical of calcifying larvae (Byrne et al., 2013a; Ross et al., 2011; Scanes et al., 2014). However, increased temperature (þ4 C) can mitigate the negative effect of acidification on growth in the echinoplutei of H. tuberculata and other echinoids (Sheppard Brennand et al., 2010; Byrne et al., 2013a,b). The large proportion of normal and larger larvae reared at þ4 C/pH 7.8 indicated that H. tuberculata might tolerate nearfuture ocean change (Byrne et al., 2013a). Based on available data, the most important planktonic life phase bottleneck for success to the adult stage in H. tuberculata is likely to be the echinopluteus due

Fig. 5. Mean percentage of normal development of the 24 h early larval stage in H. erythrogramma and H. tuberculata in twelve treatments (3 temperature 4 pH); n ¼ 9 (±SE).



to sensitivity of calcification systems to pH/pCO2 (Byrne et al., 2013c). We have no data however on the responses of juvenile and adult H. tuberculata to increased temperature and acidification. Newly settled juveniles may also likely to be a bottleneck of mortality (Pechenik, 1987; Gosselin and Qian, 1997). The results of this study illustrate how single-stressor studies can be misleading in their conclusions on a species' vulnerability and responses to global change (Byrne, 2011; Byrne and Przeslawski, 2013). Marine invertebrate embryos and larvae live in a multi-stressor world (Doney et al., 2012) and our results for the two Heliocidaris echinoids and other species (Byrne and Przeslawski, 2013) show that cross-factorial experimental designs are essential to discern impacts of near-future ocean change on marine invertebrate development, especially with respect to the most important concurrent stressors e warming and acidification. This study covered just a small proportion of development of the two Heliocidaris species, but does compliment what we know about stress tolerance of later larval stages (Byrne et al., 2011a,b, 2013a). While we have an understanding of the responses of the pelagic phases of the Heliocidaris species to increased temperature and acidification, impacts on the requirements for metamorphic and settlement stages as well as for the species over longer time series are not known (Albright and Langdon, 2011; Nakamura et al., 2011; Bartolini et al., 2012) and we also do not know what sublethal effects these stressors have on development (e.g. change in metabolism, gene expression, disease susceptibility) (see Lenihan et al., 1999; Todgham and Hofmann, 2009; Stump et al., 2011; Walther et al., 2011). Ocean acidification may also alter the cues larvae require for settlement and metamorphosis (Albright and Langdon, 2011; Doropoulos and Diaz-Pulido, 2013; Uthicke et al., 2013).

5. Conclusions Finally, in the absence of phenotypic plasticity to acclimate to environmental change in the short term to allow time for genetic adaptation in the longer term, shifting baselines of environmental conditions in southeastern Australia may push extremes to suboptimal or lethal levels for invertebrate development, as seen in the ecosystem-wide species change following recent heat waves (Wernberg et al., 2011). Overall, warming of the Australian continent and coastal waters appears the most significant global change stressor for outcomes for Heliocidaris as a genus and other marine species in southeastern Australia (Hobday and Lough, 2011). Early development in H. erythrogramma can be expected to be more sensitive to near-future ocean conditions considered as whole, due to its sensitivity to temperature, than for its congener H. tuberculata, with differing impacts for these species.

Acknowledgements We gratefully acknowledge the assistance of Kennedy Wolfe at the University of Sydney. Sea urchins were collected under permit from the New South Wales Department of Primary Industries. Sydney Institute of Marine Science Contribution #135. This work was funded by an Australian Research Council.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.marenvres.2014.07.007.

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