The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius

MPB-07930; No of Pages 12 Marine Pollution Bulletin xxx (2016) xxx–xxx

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The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius Yaoyao Zhan a, Wanbin Hu a, Weijie Zhang a, Minbo Liu a, Lizhu Duan a, Xianya Huang a, Yaqing Chang a,⁎, Cong Li b,⁎ a b

Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian, Liaoning 116023, China College of Basic Medical Science, Dalian Medical University, Dalian, Liaoning 116044, China

a r t i c l e

i n f o

Article history: Received 26 February 2016 Received in revised form 21 July 2016 Accepted 2 August 2016 Available online xxxx Keywords: Ocean acidification Strongylocentrotus intermedius Early development Calcification

a b s t r a c t The impact of CO2-driven ocean acidification(OA) on early development and calcification in the sea urchin Strongylocentrotus intermedius cultured in northern Yellow Sea was investigated by comparing fertilization success, early cleavage rate, hatching rate of blastulae, larvae survival rate at 70 h post-fertilization, larval morphology and calcification under present natural seawater condition (pH = 8.00 ± 0.03) and three laboratorycontrolled acidified conditions (OA1, △pH = − 0.3 units; OA2, △pH = −0.4 units; OA3, △pH = − 0.5 units) projected by IPCC for 2100. Results showed that pH decline had no effect on the overall fertilization, however, with decreased pH, delayed early embryonic cleavage, reduced hatching rate of blastulae and four-armed larvae survival rate at 70 h post-fertilization, impaired larval symmetry, shortened larval spicules, and corrosion spicule structure were observed in all OA-treated groups as compared to control, which indicated that CO2-driven OA affected early development and calcification in S. intermedius negatively. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Ocean acidification (OA) after the greenhouse effect is another global environmental problem caused by increasing emissions of anthropogenic carbon dioxide (CO2). As a main sink for atmospheric CO2, the oceans have absorbed one third of the man-made CO2 since the industrial revolution era (see Sabine et al., 2004). The Intergovernmental Panel on Climate Change (IPCC) projected in 2013 that the average surface seawater pH value will be reduced about 0.3–0.5 units by 2100 (see IPCC, 2013). Even worse, recent studies implied that future atmospheric CO2 level might exceed current model-predictions and the degree of OA may be even worse than presently forecast, especially in polar, subpolar and cold-water region (e.g. Cooley and Donry, 2009; Doney, 2009; Comeau et al., 2015). The nearshore zone is the juncture belt of atmosphere, lithosphere and biosphere characterized by complex environment and abundant fishery resources. It is also an important place for artificial breeding of commercial marine species. To date, more and more studies on nearshore marine life, such as corals (e.g. Jokiel, 2015), mollusks (e.g. Fitzer et al., 2014; Fitzer et al., 2015), echinoderms (e.g. Yuan et al., 2015; Dupont et al., 2010), fish (e.g. Miller et al., 2015; Perry et al., 2015; Stapp et al., 2015), crustaceans (e.g. Taylor et al., 2015; Zheng, 2015), phytoplankton (e.g. Flynn et al., 2015; Collins et al., 2014)

⁎ Corresponding authors. E-mail address: [email protected] (Y. Chang).

and zooplankton (e.g. Cripps et al., 2014; Dam, 2013), suggested that ongoing OA can affect a number of bio-processes, including shell and skeleton calcification (see Falini et al., 2015), reproduction (e.g. Vehmaa

Fig. 1. The impact of CO -driven OA on fertilization success in S. intermedius. Percent fertilized eggs at 5 min,2 30 min, 60 min, and 90 min after insemination in control (Control, pH = 8.00 ± 0.03) and three CO treated-groups (OA , △pH = −0.3 units; 1 OA2, △pH = −0.4 units; OA3, △pH = −0.52units). Error bars = s.d.

http://dx.doi.org/10.1016/j.marpolbul.2016.08.003 0025-326X/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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Y. Zhan et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

Table 1 One-way ANOVA results for the impact of OA on fertilization success in S. intermedius.

5 min post-fertilization

30 min post-fertilization

60 min post-fertilization*

90 min post-fertilization

SS

df

MS

F

P

Tukey/Tamhane's T2

Between groups Within groups Total Between groups Within groups Total Between groups

61.07 168.15 229.22 136.06 142.92 278.97 12.33

3 8 11 3 8 11 3

20.36 21.02 – 45.35 17.87 – 4.11

0.97 – – 2.54 – – 0.51

0.45 – – 0.13 – – 0.69

Within groups Total Between groups Within groups Total

64.34 76.67 9.95 52.61 62.56

8 11 3 8 11

8.04 – 3.32 6.58 –

– – 0.50 – –

– – 0.69 – –

Control = OA2 = OA3 = OA1 – – Control = OA1 = OA3 = OA2 – – Control vs OA1: p = 0.91; Control vs OA2: p = 0.94; Control vs OA3: p = 0.62 – – Control = OA1 = OA2 = OA3 – –

SS: sum of squares; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. *: the data were not equal variances, Tamhane's T2-test was employed for statistical analysis. n = 3.

et al., 2013), development (e.g. Xu et al., 2015; Hardy and Byrne, 2014), metabolism (e.g. Lannig et al., 2010; Torstensson et al., 2015), intracellular acid-base and ion regulation (e.g Gutowska et al., 2010), immunity (e.g. Hernroth et al., 2011) and apoptosis (e.g. Kvitt et al., 2015). Although there are ~2% marine biota has been assessed under different futureprojected CO2 levels (see Doney, 2009), our understanding of the profound consequences of OA on nearshore areas is still limited, therefore, more data are urgently needed to quantify the potential effects of OA on

marine biota, especially commercial species, and marine ecosystems of the nearshore zone. Sea urchins, one of representative marine animals of shallow coastal areas, play a very important role in ecology. Because the edible part (gonad) serves as a good and nutritious food for people, some sea urchins are major fishery resources in Asian, Mediterranean countries and Western Hemisphere countries (see Andrew et al., 2003). Over the past one hundred years, sea urchins have been widely used as classic

Fig. 2. The impact of OA on early embryonic cleavage in S. intermedius. Percentages of 1-, 2-, 4-, 8-,16-cell stage embryos in fertilized eggs cultured for 60 min(A), 90 min(B), 120 min(C), 150 min(D), 180 min(E), and 210 min(F) after fertilization in each experimental group (Control, pH = 8.00 ± 0.03; OA1, △pH = − 0.3 units; OA2, △pH = − 0.4 units; OA3, △pH = −0.5 units). Error bars = s.d.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

Y. Zhan et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

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Table 2 One-way ANOVA results for the impact of OA on early embryonic cleavage in S. intermedius.

60 min post-fertilization

1 cell fert−1

2 cell fert−1*

90 min post-fertilization

1 cell fert−1

2 cell fert−1

4 cell fert−1*

−1

120 min post-fertilization

1 cell fert

120 min post-fertilization

2 cell fert−1

4 cell fert−1

8 cell fert−1*

150 min post-fertilization

−1

1 cell fert

2 cell fert−1

4 cell fert−1

150 min post-fertilization

180 min post-fertilization

8 cell fert−1*

−1

1 cell fert

2 cell fert−1

−1

4 cell fert

8 cell fert−1

210 min post-fertilization

1 cell fert−1

210 min post-fertilization

2 cell fert−1

4 cell fert−1

8 cell fert−1

16 cell fert−1*

SS

df

MS

F

P

Tukey / Tamhane's T2

Between groups Within groups Total Between groups

6.58 130.02 136.60 0.08

3 8 11 3

2.19 16.25 – 0.03

0.14 – – 3.94

0.94 – – 0.05

Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups

0.06 0.14 257.07 39.93 297.00 386.55 25.82 412.37 3.13

8 11 3 8 11 3 8 11 3

0.01 – 85.69 4.99 – 128.85 3.23 – 1.04

– – 17.17 – – 39.92 – – 852.49

– – 0.00 – – 3.69 × 10−5 – – 2.32 × 10−10

Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups

0.01 3.14 180.57 14.56 195.12 761.04 10.19 771.22 996.49 3.72 1000.21 4.47

8 11 3 8 11 3 8 11 3 8 11 3

0.00 – 60.19 1.82 – 253.68 1.27 – 332.17 0.46 – 1.49

– – 33.08 – – 199.24 – – 715.16 – – 89.81

– – 7.39 × 10−5 – – 7.45 × 10−8 – – 4.68 × 10−10 – – 1.68 × 10−6

Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups

0.13 4.61 56.38 12.74 69.11 4.95 30.68 35.63 4.37 49.85 54.22 23.06

8 11 3 8 11 3 8 11 3 8 11 3

0.02 – 18.79 1.59 – 1.65 3.84 – 1.46 6.23 – 7.69

– – 11.81 – – 0.43 – – 0.23 – – 245.68

– – 0.00 – – 0.74 – – 0.87 – – 3.26 × 10−8

Within groups Total Between groups Within groups Total Between groups

0.25 23.31 13.23 46.26 59.48 51.88

8 11 3 8 11 3

0.03 – 4.41 5.78 – 17.29

– – 0.76 – – 41.58

– – 0.55 – – 3.17 × 10−5

Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups

3.33 55.21 1992.44 9.86 2002.30 2697.01 32.89 2729.90 71.17 17.59 88.76 27.88 3.10 30.98 10.73 3.91 14.64 1726.19 47.99 1774.18 1046.37

8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3

0.42 – 664.15 1.23 – 899.00 4.11 – 23.72 2.20 – 9.29 0.39 – 3.58 0.49 – 575.40 6.00 – 348.79

– – 539.06 – – 218.65 – – 10.79 – – 23.95 – – 7.32 – – 95.93 – – 103.29

– – 1.44 × 10−9 – – 5.16 × 10−8 – – 0.00 – – 2.38 × 10−4 – – 0.01 – – 1.30 × 10−6 – – 9.77 × 10−7

Control = OA1 = OA2 = OA3 – – Control vs OA1: p = 0.71; Control vs OA2: p = 0.71; Control vs OA3: p = 0.71 – – Control = OA1 b OA2 = OA3 – – Control = OA1 N OA2 = OA3 – – Control vs OA1: p = 0.01; Control vs OA2: p = 0.01; Control vs OA3: p = 0.01 – – OA3 N OA2 N Control = OA1 – – OA1 N OA2 N OA3 N Control – – Control N OA1 N OA2 = OA3 – – Control vs OA1: p = 0.06; Control vs OA2: p = 0.06; Control vs OA3: p = 0.06 – – OA3 = OA2 = OA1; OA1 = Control; OA3 = OA2 N Control – – OA1 = Control = OA2 = OA3 – – Control = OA2 = OA1 = OA3 – – Control vs OA1: p = 0.02; Control vs OA2: p = 0.02; Control vs OA3: p = 0.02 – – Control = OA2 = OA3 = OA1 – – OA2 = OA1; OA1 = Control; OA3 N OA2 = OA1; OA3 N OA1 = Control;OA2 N Control – – OA3 N OA1 = OA2 N Control – – Control N OA1 = OA2 N OA3 – – OA1 = Control = OA2; OA2 = OA3; OA1 = Control N OA3 – – OA1 N Control = OA2 = OA3 – – OA2 = OA3 = OA1; OA3 = OA1 = Control; OA2 N Control – – OA3 N OA2 N OA1 = Control – – Control vs OA1: p = 0.07; Control vs OA2: p = 0.03; Control vs OA3: p = 0.00 (continued on next page)

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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Table 2 (continued)

Within groups Total

SS

df

MS

F

P

Tukey / Tamhane's T2

27.01 1073.39

8 11

3.38 –

– –

– –

– –

SS: sum of squares; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. *: the data were not equal variances, Tamhane's T2-test was employed for statistical analysis. n = 3.

model organisms in many fields of developmental biology and ecological research. Due to their world-wide distribution, the planktonic life stage which is considered the most vulnerable to OA, and the calcified endoskeleton in larvae and exoskeleton in adults, sea urchins are considered ideal models to study the potential effects of OA on marine life (see Dupont and Thorndyke, 2013). Recent research efforts on OA impacts on sea urchins have mainly focused on fertilization kinetics (Hardy and Byrne, 2014; Dupont and Thorndyke, 2013), larval development (e.g. Dupont and Thorndyke, 2013), skeleton biomineralization

(e.g. Byrne et al., 2013a, 2013b; Sheppard Brennand et al., 2010), gene expression pattern alteration (e.g. Martin et al., 2011; Todgham and Hofmann, 2009; Hammond and Hofmann, 2012), and the response of acid-base regulatory system (e.g. Calosi et al., 2013; Stumpp et al., 2012). To date, ~2.7% of sea urchin species have been studied to increase our understanding of the impact of OA on different life-history stages, such as gametes, embryos, larvae, juveniles or adults (see Dupont and Thorndyke, 2013) in echinoderms. It is noteworthy that recent laboratory and field studies indicated species-specific variability in response to CO2-induced OA, i.e. some tended to be more tolerant than others (e.g. Byrne and Przeslawski, 2013). Thus, we need to broader our survey and investigate more sea urchin species to clarify the consequences of near future CO2-induced OA. Strongylocentrotus intermedius, which was transplanted from Japan in 1989 by Dalian Ocean University, is widely cultivated along the coastal areas of Liaoning and Shandong Provinces and is the dominant important cultured sea urchin species in China (see Chang et al., 2004). Far exceeding its ecological value, the annual output of the sea urchin S. intermedius is more than 200 tons because of the large demand for its gonad as a highly valuable local and export product (see Chang et al., 2012). In order to evaluate the extent of the impact of CO2-driven OA on S. intermedius and provide information about (1) whether the near future CO2-driven OA affects S. intermedius ecological function and the industry of S. intermedius, (2) whether there is any species-specific differences in early life stages between S. intermedius and other sea urchins when exposure to elevated CO2, four lab-based experimental groups were setup including one present natural oceanic condition group (as control) and three near-future OA-scenarios groups with △pH values were − 0.3, − 0.4, − 0.5 units, respectively, in order to cover the range of IPCC projected for 2100 in the current study. Fertilization success, early embryonic cleavage rate, percent hatched blastulae and four-armed larvae survival rate at 70 h post-fertilization were calculated. Basic spicule elements of four-armed larvae were measured. Four-armed larvae morphology and spicule calcification ultrastructure were observed by microscopy. By comparison with the control, we attempted to find out to what extent that near future OA-scenarios affected the early life stages of the sea urchin S. intermedius.

2. Results Fig. 3. The impact of CO2-driven OA on percent hatched blastulae and larvae survival rate at 70 h post-fertilization in S. intermedius. A. Comparison of percent hatched blastulae between control and OA-treatments (Control, pH = 8.00 ± 0.03; OA1, △pH = − 0.3 units; OA2, △pH = − 0.4 units; OA3, △pH = − 0.5 units) 18 h after fertilization. B. Comparison of larvae survival rate between control and OA-treatments (Control, pH = 8.00 ± 0.03; OA1, △pH = −0.3 units; OA2, △pH = − 0.4 units; OA3, △pH = −0.5 units) 70 h after insemination. Error bars = s.d.

2.1. The impact of OA on fertilization in S. intermedius Mean percent fertilization success across all experimental groups ranged from 84.08 ± 5.75% (OA3) to 86.90 ± 4.44% (Control) 5 min post-fertilization (Fig. 1). 30 min after insemination, the percent

Table 3 One-way ANOVA results for the impact of OA on percent hatched blastulae and larvae survival rate at 70 h post-fertilization in S. intermedius.

% Hatched blastulae

% survival larvae at 70 h post-fertilization

Between groups Within groups Total Between groups Within groups Total

SS

df

MS

F

P

Tukey

1511.98 116.66 1628.64 3515.21 101.25 3616.47

3 8 11 3 8 11

503.99 14.58 – 1171.74 12.66 –

34.56 – – 92.58 – –

6.29 × 10−5 – – 1.50 × 10−6 – –

Control N OA1 = OA2 N OA3 – – Control N OA1 N OA2 N OA3 – –

SS: sum of squares; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. n = 3.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

Y. Zhan et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

fertilization success continued to increase in all experimental groups, high percent fertilization success (N 90%) were observed in both control and OA1-groups. After 60 min post-fertilization, the percent fertilization success was N90% in all experimental groups. No significant difference

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observed between control and OA-treatments during the course of fertilization (see Table 1), which indicated that OA delayed the process of fertilization at the very beginning 30 min after insemination but had no effect on overall process of fertilization.

Fig. 4. The impact of CO2-driven OA on larval morphology and symmetry in S. intermedius. A. S. intermedius larval morphology 70 h after fertilization in each experimental group; (I, I′): Control group (pH = 8.00 ± 0.03); (II, II′): OA1 group (△pH = −0.3 units); (III, III′): OA2 group (△pH = −0.4 units); (IV, IV′): OA3 group (△pH = −0.5 units). Scale bar: 50 μm. B. Comparison of percent larvae with different asymmetry degree (Ad) between control and OA-treatments.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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2.2. The impact of OA on embryonic and early larval development in S. intermedius

in the OA3 group was greater than those of larvae in both OA1 group and OA2 group.

The impact of CO2-driven OA on early embryonic development in S. intermedius was determined by calculating fertilized egg cleavage rate at different post-fertilization time points in each experimental group. More than 90% of the fertilized eggs (fert) across all experimental groups were at the 1-cell stage 60 min after insemination (Fig. 2-A). While, deleterious effect was evident after 90 min post-fertilization, a dose-dependent cleavage rate delay was observed at 90-, 120-, 150-, 180-, 210-min post-fertilization (Fig. 2-B, C, D, E, F), respectively. It seemed that OA-treatment might prolong cell division process about 30 min in S. intermedius (see Table 2). The impact of OA on later embryonic and early larval development in S. intermedius was determined by calculating the hatching rate of blastulae and four-armed larvae survival rate at 70 h post-fertilization. The hatching rate of blastulae decreased linearly in OA-treated groups (Fig. 3-A) and differed significantly from that of the control (92.67 ± 4.05%) (see Table 3). After 70 h in culture, four-armed larvae survival rate showed a linear decline trend with decreased seawater pH (Fig. 3-B). Statistical differences observed in OA-treated groups as compared to control (see Table 3).

3. Discussion

2.3. The impact of OA on larval morphology and spicule structure in S. intermedius Four-armed larva of S. intermedius had well developed endoskeleton, such as the body rod (BR) and post oral (PO) arm, and kept a good symmetrical shape under natural oceanic condition 70 h after fertilization (Fig. 4-A(I, I′)). When compared with larvae reared in control, abnormal larvae were observed with an asymmetrically developed PO arm in all OA-treatments (Fig. 4-A (II, II′, III, III′, IV, and IV′)). By calculating the proportion of larvae with different asymmetry degree (Ad) in each experimental group (Fig. 4-B), we found that OA-treatment can increase the proportion of asymmetric larvae linearly as compared to that of control (see Table 4). Measurement data of 4 basic endoskeleton elements that included overall length (OL) (Fig. 5-A), BR length (LBR) (Fig. 5-B), PO length (LPO) (Fig. 5-C) and distance between skeletal rods (SD) (Fig. 5-D) showed a dose-dependent shortened OL and LPO with decreased seawater pH (see Table 5). SEM images revealed acidified sea water corroded not only the surface structure of larval spicule but also the cross section of larval spicule. Both the surface and the cross section of larval spicules cultured in the control condition were smooth, compact and unblemished (Fig. 6-B). As for four-armed larvae reared in the OA1 and OA2 groups, the spicule structure displayed a similar appearance to those in the control group, although there were some slight corrosion signs observed in the surface and cross section. The degree of dissolution in spicule structure of larvae

Fertilization, embryonic development, and planktonic larval development stages are early developmental phases and play a significant role in determining population abundance, genetic diversity, distribution, and resilience to disturbances (see Grosberg and Levitan, 1992). The response of each early developmental stage to environmental stressors (such as pH, temperature and salinity) is various. Different from studies those experimental animals were placed in OAtreatments at larval stage (Dupont and Thorndyke, 2013), we focused on the response of S. intermedius to CO2-driven OA from fertilized eggs to four-armed larvae. Previous studies on the effect of OA on fertilization kinetics in sea urchins mainly focused on calculating fertilization rate at a single time point rather than the course of overall fertilization (Dupont and Thorndyke, 2013). While, we focused on the course of fertilization and found that fertilization success in S. intermedius seemed to decrease with increased seawater CO2 concentration in the first 30 min after insemination, while the overall process of fertilization was not affected by OA-treatment in S. intermedius. According to a recent report that CO2-induced OA affected sperm motility through reducing mitochondrial activity in the Australian sea urchin Centrostephanus rodgersii (Schlegel et al., 2015), we postulate that one possible explanation for OA-induced delay of fertilization in S. intermedius might due to the decreasing of sperm mitochondrial activity at the very beginning of fertilization and an adaptive restoration might be activated after 30 min post-fertilization, therefore, further research needs to be carried out to test if there is an adaptive restoration of sperm motility when exposure to CO2-driven OA in S. intermedius. On the other hand, the robustness of S. intermedius fertilization to decreased pH might be due to another reason, i.e. low pH is naturally associated with echinoderm reproduction because the internal pH of activated sperm is initially at pH 7.6 and acid is released by echinoderm eggs at fertilization (Morgan, 2011). Additionally, contrast with those previous reports that fertilization of some sea urchins were sensitive to CO2-induced OA (Dupont and Thorndyke, 2013), results observed in this study also support the concern that there is a species-specific difference in the effect of CO2-induced OA on fertilization success in sea urchins. Since a delayed cleavage might result in prolonged development duration, a dose-dependent delay of embryonic cleavage in S. intermedius was observed in the current study which consistent with many studies (Dupont et al., 2010; Hardy and Byrne, 2014), meanwhile, this result suggests that near future OA would make S. intermedius larvae more vulnerable to predation in a changing ocean. Furthermore, as shown in this study, a delayed cleavage decreased the percentage of hatched blastulae in S. intermedius subsequently since a small proportion of

Table 4 One-way ANOVA results for the impact of OA on percent larvae with different asymmetry degree (Ad) in S. intermedius.

0% b Ad ≤ 5%

5% b Ad ≤ 10%

10% b Ad ≤ 15%

15% b Ad

SS

df

MS

F

P

Tukey

Between groups

1010.38

3

336.79

14.69

0.00

Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total

183.45 1193.83 108.14 129.64 237.78 162.81 129.95 292.76 881.79 69.11 950.89

8 11 3 8 11 3 8 11 3 8 11

22.93 – 36.05 16.21 – 54.27 16.24 – 293.93 8.64 –

– – 2.22 – – 3.34 – – 34.03 – –

– – 0.16 – – 0.08 – – 6.66 × 10−5 – –

Control = OA1; OA1 = OA2; OA2 = OA3, Control N OA2 = OA3; OA1 N OA3 – – Control = OA1 = OA3 = OA2 – – Control = OA3 = OA2 = OA1 – – Control = OA1 = OA2 b OA3 – –

SS: sum of squares; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. n = 3.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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fertilized eggs were blocked to cleavage. As a subsequent consequence of decreased hatching rate of blastulae, outcomes in this study showed a significant dose-dependent decreased survival rate at 70 h postfertilization of S. intermedius planktonic larvae with increased CO2concentration in seawater. This is consistent with the results observed in Arachnoides placenta (e.g. Gonzalez-Bernat et al., 2013a, 2013b), Odontaster validus (e.g. Gonzalez-Bernat et al., 2013a, 2013b), and Patiriella regularis (e.g. Byrne et al., 2013a, 2013b). However, 9 of 13

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species showed a robust larval survival rate under OA conditions as reviewed by Dupont et al. (2010). In addition, it was reported that OA had no significant effect on larval survival in sea urchin A. lixula (see Gianguzza et al., 2014). Therefore, we conclude that there is OAinduced species-specific difference indeed on echinoderm larvae survival/mortality. Moreover, as results observed at later stages were outcomes of CO2-driven OA sustained effect, so we assumed here that the observations at later stages, such as decreased percent hatched

Fig. 5. The impact of CO2-driven OA on larval spicule elements in S. intermedius. A. Mean length of overall spicule. B. Mean length of body rod. C. Mean length of post oral arm, and D. Mean distance between skeletal rods. Error bars = s.d.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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Laboratory of Mariculture & Stock Enhancement in the North China' s Sea, Ministry of Agriculture at the Dalian Ocean University in September 2014. 5 days prior to experiments, they were maintained in laboratory circulating seawater tanks(~ 1000 L) at ambient temperature (20.06 ± 0.12 °C) and fed with kelp (Laminaria japonica).

blastulae and larval survival success, could be accumulative effect of CO2-driven OA from earlier stages. Furthermore, because of swimming upward is an important sign of the beginning of the free swimming blastula stage in embryonic development of sea urchins, percent hatched blastulae and larvae survival rate at 70 h post-fertilization reflect the efficiency of artificial seeding in traditional production process of S. intermedius industry in China (see Chang et al., 2004), thus we postulate here that near future OA will have a negative effect on commercial industry of S. intermedius. As previous studies reported that 8 h–24 h post-fertilization is the key phase in the development of sea urchin spicules (see Wilt and Ettendohn, 2007; Killian and Wilt, 2008), it is worth noting that results from spicule measurements, microscopic images and scanning electron microscope images at 70 h post-fertilization could be due to the accumulation of sustained CO2-driven OA treatment from the beginning of insemination. In addition, sea urchin larval ability to carry out normal functions is tightly coupled with their development and morphology. As documented before, changes in length and orientation of the ciliated arms of larval urchins would have effects on their ability to capture food, swim in still water, and be stable in flowing water (see Lamare and Barker, 1999). Although we did not get direct data on how CO2-driven OA affects the abilities of capture food and swimming in S. intermedius in the current study, morphological results (such as impaired larval symmetry, shortened and corroded larval spicules) observed here reflect an OA-induced reduce of metabolism and calcification efficiency in S. intermedius which might result in the decreasing of chance of larvae survival at 70 h postfertilization and recruitment to some extent. Therefore, we assumed that OA-treatment might reduce the chance of survival and population size in S. intermedius through reducing efficiency of metabolism and calcification, which also might be harmful to sustainable development of S. intermedius industry. Although with some limitations in our study, such as lacking the data on OA effects on other developmental stages (such as metamorphosis, juvenile and adult), our results still evidence the near future OA affect the sea urchin S. intermedius negatively and confirm the concern that the vulnerabilities of development of echinoderms to OA varies among species with some being more tolerant than others (Byrne and Przeslawski, 2013). Moreover, data observed in this study could enrich our understanding of the impact of CO2-driven OA on development in sea urchins. Well, further research is necessary to carry out to obtain not only information about the comprehensive effects on the entire life cycle in S. intermedius, but also the underlying bio-molecular mechanisms (such as alteration in gene expression, microRNAs and proteomics, etc.).

4.2. CO2 treatments and sea water chemistry According to the projected ocean pH for 2100 reported by IPCC (2013), one present pH group (Control) and three OA-experimental pHs (OA1 △pH = − 0.3 units; OA2 △pH = − 0.4 units; OA3 △pH = −0.5 units) were set up. Ambient air was bubbled into every rearing containers to maintain dissolved oxygen (DO) N 90%. Sea water of control group was filtered but not manipulated with ambient pH 8.00 ± 0.03 and temperature 20.06 ± 0.12 °C. pH of each experimental group was adjusted by injection of CO2 (99.999%, Dalian Special Gases Company) with a CO2-driven OA scenarios system described by Sheppard et al.(2010). Conditions of experimental seawater in each OA-treated groups were stably at OA1 (pH = 7.71 ± 0.01, △pH = − 0.3 units, 20.06 ± 0.12 °C), OA2 (pH = 7.60 ± 0.03, △pH = − 0.4 units, 20.06 ± 0.12 °C), OA3 (pH = 7.51 ± 0.01, △pH = −0.5 units, 20.06 ± 0.12 °C), respectively, that were monitored daily by using a pH meter (HI9124, HANNA) and water quality monitor (6920, YSI) during the entire course of incubation. The chemical parameters of the seawater in each experimental group are reported in Table 6 (statistical analysis in Table 7). Total alkalinity (TA) in seawater of each experimental group was determined by pH titration as described in the methods provide by the State Oceanic Administration People's Republic of China (SOA) (see GB/T 12763.42− 2007, 2007). pCO2, [HCO− 3 ], [CO3 ], calcite saturation (ΩCa) and aragonite saturation(ΩAr) values of each experimental group were calculated from salinity (Sal), temperature(T), pH and TA data by SWCO2 software (available at http://neon.otago.ac.nz/swco2). 4.3. Fertilization Spawning of S. intermedius was induced by coelomic injection of KCl (0.5 M, 1-2 ml per individual) after 1 h air exposure following standard methods (see Chang et al., 2012). Gametes of 3 males and 3 females were collected in order to avoid single male-female cross variations and have a homogeneous batch. Three individual experiments were conducted to improve the precision (n = 3). Before fertilization, eggs were collected in fresh filtered seawater (FSW, 0.22 μm) and sperm were collected dry and kept on ice in culture dishes before use following the method of Liu described (see Liu et al., 2005). Prior to gamete mixing, the quality of each gamete source was checked microscopically, eggs were checked for shape and appearance and sperm were checked for motility. The total numbers of eggs and sperm density for each individual experiment were measured by haemocytometer counts. In each individual experiment for fertilization, eggs were divided into 12 beakers

4. Materials and methods 4.1. Sea urchins and maintenance S. intermedius (average test diameter was 59.79 ± 1.84 mm) were transported from Dalian Haibao Fisheries Company to the Key

Table 5 One-way ANOVA results for the impact of OA on larval spicule basic elements in S. intermedius. SS Mean length of overall spicule

Mean length of body rod

Mean length of post oral arm

Mean distance between skeletal rods

Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total

6706.10 59.04 6765.14 282.45 106.52 388.97 4278.46 34.56 4313.01 236.04 158.48 394.52

df 3 8 11 3 8 11 3 8 11 3 8 11

MS 2235.37 7.38 – 94.15 13.32 – 1426.15 4.32 – 78.68 19.81 –

F 302.88 – – 7.07 – – 330.15 – – 3.97 – –

P

Tukey −8

1.42 × 10 – – 0.01 – – 1.01 × 10−8 – – 0.05 – –

Control N OA1 = OA2 N OA3 – – Control = OA1 = OA2 N OA3 – – Control N OA1 N OA2 N OA3 – – Control = OA1 = OA2 = OA3 – –

SS: sum of square; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. n = 3.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

Y. Zhan et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

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(1 l) at four pHs (Control = 8.00 ± 0.03; OA1 △pH = −0.3 units; OA2 △pH = − 0.4 units; OA3 △pH = − 0.5 units; 3 replicate beakers per pH-treatment) with a concentration of 50 eggs ml−1. The sperm concentration was 5 × 104 sperm ml−1. These gamete concentrations resulted in more than or equal to 85% fertilization and high rates of normal development (≥75%) in procedural controls (see Chang et al., 2004). Prior to fertilization, the eggs were pre-incubated in experimental FSW for 15 min and the sperm were placed in the experimental FSW for 1–2 s when added to the beakers containing eggs. After 15 min, the eggs were rinsed three times in experimental FSW to remove excess sperm and re-suspended in each fresh experimental FSW. Samples were collected from each pH condition at 5 min, 15 min, 30 min and 60 min after insemination, respectively, in order to calculate the fertilization rate of each OA-treated group at different time. Fertilization success was determined as the presence of a fertilization envelope or cleavage. 4.4. Embryonic development To study the effect of CO2-induced OA on the embryonic development in S. intermedius, early embryonic cleavage and the hatching rate of blastulae were determined. The culture conditions of each experimental group as shown in Table 6. Three individual experiments were conducted to improve the precision (n = 3). For every individual experiment, 100 S. intermedius embryos in each experimental group were collected randomly after 60 min, 90 min, 120 min, 150 min, 180 min and 210 min post-fertilization, respectively. The stage of each embryo at each time point in different experimental group was recorded under a microscope (50i, Nikon, Japan). Cleavage was defined as the presence of a minimum of 2 blastomeres. To calculate the hatching rate of blastulae, 1 ml of the culture water that had been incubated for 18 h were sampled randomly from breakers with an initial density of 50 eggs ml−1. The number of free swimming blastulae was recorded in plankton counting frame (1 ml) under a microscope (50i, Nikon, Japan) as described by Chang et al. (2004). The formula used to calculate the hatching rate of blastulae was: Percent hatched blastulae (%) = (numbers of free swimming blastulae at 18 h / initial density) × 100% 4.5. Larval development

Fig. 6. The impact of CO2-driven OA on larval spicule ultrastructure in S. intermedius visualized by SEM. A. Schematic of larval spicule structure. B. Surface and cross section SEM images of S. intermedius larval spicules cultured in each experimental group. Arrows indicate the corrosion regions.

In order to investigate the impact of CO2-driven OA on larval development in S. intermedius, we mainly focused on variations on larvae survival at 70 h post-fertilization, morphology, basic spicule elements and larval spicule ultra-structure. Three individual experiments were conducted to improve the precision (n = 3). Embryos were reared to four-armed larval stage at four-pH conditions as shown in Table 6. Since studies have reported that sea urchin embryos can survive up to 8 days after fertilization without suffering from malnutrition (see Sheppard Brennand et al., 2010; Todgham and Hofmann, 2009; Moulin et al., 2011), we did not feed the larvae during incubation and chosen 70 h after insemination as the endpoint to calculate the survival

Table 6 Seawater parameters for the four experimental groups. Carbonate system

Control

OA1

OA2

OA3

pH TA (mmol/l) Sal (‰) T (°C) pCO2 (μtam) −1 [HCO− ) 3 ](μmol l −1 [CO2– ) 3 ](μmol l ΩCa ΩAr

8.00 ± 0.03 2.35 ± 0.04 31.23 ± 0.13 20.06 ± 0.12 693.53 ± 54.18 2039.96 ± 18.60 128.59 ± 7.71 3.16 ± 0.19 2.04 ± 0.12

7.71 ± 0.01 2.35 ± 0.04 31.23 ± 0.13 20.06 ± 0.12 1442.7 ± 35.62 2180.30 ± 3.63 70.41 ± 1.50 1.73 ± 0.04 1.12 ± 0.03

7.62 ± 0.01 2.35 ± 0.04 31.23 ± 0.13 20.06 ± 0.12 1799.24 ± 43.89 2210.19 ± 3.03 58.02 ± 1.26 1.42 ± 0.04 0.92 ± 0.02

7.51 ± 0.01 2.35 ± 0.04 31.23 ± 0.13 20.06 ± 0.12 2349.15 ± 56.62 2240.02 ± 2.42 45.64 ± 1.00 1.12 ± ±0.02 0.72 ± 0.02

2− Values of pCO2, [HCO− 3 ], [CO3 ], ΩCa and ΩAr were determined as “Materials and methods” described.

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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Table 7 One-way ANOVA results for seawater parameters for the four experimental groups. Carbonate system pH

TA (mmol/l)

Sal (‰)

T (°C)

pCO2 (μtam)

−1 [HCO− ) 3 ](μmol l

−1 [CO2– ) 3 ](μmol l

ΩCa

ΩAr

Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total Between groups Within groups Total

SS

df

MS

F

P

Tukey

0.40 0.00 0.40 0.00 0.01 0.01 0.00 0.14 0.14 0.00 0.12 0.12 4,571,248.15 4594.95 4,575,843.10 70,534.11 748.01 71,282.12 10,923.17 37.88 10,961.05 7.33 0.08 7.41 3.04 0.03 3.07

3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11 3 8 11

0.13 3.00 × 10−4 – 0.00 0.00 – 0.00 0.02 – 0.00 0.01 – 1,523,749.38 574.37 – 23,511.37 93.50 – 3641.06 4.74 – 2.44 0.01 – 1.01 0.00 –

440.67 – – 0.00 – – 0.00 – – 0.00 – – 2652.91 – – 251.46 – – 768.96 – – 244.73 – – 257.98 – –

3.21 × 10−9 – – 1.00 – – 1.00 – – 1.00 – – 2.50 × 10−12 – – 2.97 × 10−8 – – 3.51 × 10−10 – – 3.31 × 10−8 – – 2.69 × 10−8 – –

Control N OA1 N OA2 N OA3 – – Control = OA1 = OA2 = OA3 – – Control = OA1 = OA2 = OA3 – – Control = OA1 = OA2 = OA3 – – OA3 N OA2 N OA1 N Control – – OA3 N OA2 N OA1 N Control – – Control N OA1 N OA2 N OA3 – – Control N OA1 N OA2 N OA3 – – Control N OA1 N OA2 N OA3 – –

SS: sum of square; df: degree of freedom; MS: mean square; F: F-value; Tukey: Tukey test. n = 3.

rate of larvae at 70 h post-fertilization. This avoids the potential confound influence induced by exogenous food (i.e. algae). To calculate larvae survival rate, 1 ml of the culture water that had been incubated for 70 h after fertilization were sampled randomly from breakers with an initial density of 50 eggs ml−1. The number of free swimming larvae with normal stomachs was recorded in plankton counting frame (1 ml) under a microscope (50i, Nikon, Japan). The following formula was used to calculate larvae survival rate at 70 h post-fertilization: Percent survival larvae (%) = (Larval density at 70 h / initial density) × 100%. As four-armed larvae had well developed calcified arms (spicules) at 70 h post-fertilization (Fig. 7), basic spicule elements, such as PO arm and BR, were good for measurement at this stage. For each individual experiment, 50 larvae sampled randomly from each experimental group were fixed following the method described by Mann et al. (2010) and photographed for further morphology

observation under microscope (50i, Nikon, Japan). The mean length of overall spicules, BRs, PO arms and SD of each pluteus were measured to indicate larval development rate and calcification by using DN-2 software (Dalian Zhonghe Company). Larval symmetry showed in Supplementary Fig. S1 and the asymmetry degree (Ad) was calculated by following formula: Ad ð%Þ ¼

OLL −OLS  100% OLL

To observe the ultrastructure of larval calcified endoskeleton (spicules), spicules of four-armed larvae were isolated as described by Mann et al. (2010). The surface and cross sections of larval spicules from specimens in each experimental group were observed by using a scanning electron microscope (Quanta200, FEI). 4.6. Data analysis

Fig. 7. Four-armed larva morphology and schematic figure of four-armed larva of S. intermedius. A. Four-armed larva morphology, scale bar: 50 μm. B. Schematic figure of four-armed larva. BR, body rod; PO, post oral arm.

All data are expressed as mean values ± standard deviation (Mean ± s.d.). Statistical differences on fertilization success, early embryonic cleavage, hatching rate of blastulae, four-armed larvae survival rate at 70 h post-fertilization, larval spicule element measurements(OL, LPO, LBR and SD) and larval asymmetry degree between control and each OA-treated group were investigated by SPSS software (version 16.0). Grand mean values for each three experiments were used to do one-way ANOVA analysis (pH as factor, n = 3). Before analysis, all data are expressed as mean values ± standard deviation (Mean ± s.d.). Statistical differences on fertilization success, early embryonic cleavage, percent hatched blastulae, larvae survival rate at 70 h post-fertilization, larval spicule element measurements (OL, L PO, L BR and SD) and larval asymmetry degree between control and each OA-treated group were investigated using a one-way ANOVA (pH as factor) by SPSS software (version 16.0). Before analysis, Shapiro-Wilk and Levene's test was used to indicate normality and homogeneity of variance. Post-hoc analyses of fertilization success, early embryonic cleavage, hatching rate of blastulae, larvae survival rate at 70 h post-fertilization, larval spicule element

Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003

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measurements (OL, LPO, LBR and SD), and larval asymmetry degree were done by Tukey test. For the data analysis for fertilization success (60 min post-fertilization) and early embryonic cleavage (60 min 2 cell fert− 1, 90 min 4 cell fert− 1, 8 cell fert− 1 (120 min, 150 min), 210 min 16 cell fert− 1 ) were not equal variances, Tamhane's T2-test was employed for statistical analysis. The level of significance was considered as p b 0.05. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2016.08.003. Authors' contributions YYZ and YQC conceived and designed the experiments. Performed the experiments: WBH, MBL, LZD and XYH. Analyzed the data: WBH, WJZ, YYZ, YQC and CL. Wrote the paper: YYZ and WBH. All authors read and approved the manuscript. Competing financial interest The authors declare that they have no competing financial interest. Acknowledgements We are greatly indebted to Dr. John Lawrence for revising the English text. We are grateful to anonymous reviewers for their helpful comments to improve the manuscript. This work was supported by the National Natural Science Foundation of China (NSFC No. 41206128), the Program for Liaoning Excellent Talents in University (LNET No. LJQ2013079) and the National High-tech R&D Program (863 Program, No. 2012AA10A412). References Andrew, N.L., Agatsuma, Y., Ballesteros, E., Bazhin, A.G., Creaser, E.P., Barnes, D.K.A., Botsford, L.W., Bradury, A., Campbell, A., Dixon, J.D., Einarsson, S., Gerring, P.K., Hebert, K., Hunter, M., Hur, S.B., Johnson, C.R., Juinio-Menez, M.A., Kalvass, P., Miller, R.J., Moreno, C.A., Palleiro, J.S., Riva, D., Roinson, S.M.L., Schroeter, S.C., Steneck, R.S., Vadas, R.L., Woodby, D.A., Xiaoqi, Z., 2003. Status and management of world sea urchin fisheries. Oceanogr. Mar. Biol. Annu. Rev. 40, 343–425. Byrne, M., Przeslawski, R., 2013. Multistressor impacts of warming and acidification of the ocean on marine invertebrates' life histories. Integr. Comp. Biol. 53, 582–596. Byrne, M., Gonzalez-Bernat, M., Doo, S., Foo, S., Soars, N., Lamare, M., 2013a. Effects of ocean warming and acidification on embryos and non-calcifying larvae of the invasive sea star Patiriella regularis. Mar. Ecol. Prog. Ser. 473, 235–246. Byrne, M., Lamare, M., Winter, D., Dworjanyn, S.A., Uthicke, S., 2013b. The stunting effect of a high CO2 ocean on calcification and development in sea urchin larvae, a synthesis from the tropics to the poles. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 368, 20120439. Calosi, P., Rastrick, S.P., Graziano, M., Thomas, S.C., Baggini, C., Carter, H.A., Hall-Spencer, J.M., Milazzo, M., Spicer, J.I., 2013. Distribution of sea urchins living near shallow water CO2 vents is dependent upon species acid-base and ion-regulatory abilities. Mar. Pollut. Bull. 73, 470–484. Chang, Y.Q., Ding, J., Song, J., Yang, W., 2004. The Research of Biology and Aquaculture in Sea Cucumber and Sea Urchins. China Ocean Press, Beijing in Chinese. Chang, Y.Q., Zhang, W.J., Zhao, C., Song, J., 2012. Estimates of heritabilities and genetic correlations for growth and gonad traits in the sea urchin Strongylocentrotus intermedius. Aquac. Res. 43, 271–280. Collins, S., Rost, B., Rynearson, T.A., 2014. Evolutionary potential of marine phytoplankton under ocean acidification. Evol. Appl. 7, 140–155. Comeau, S., Jeffree, R., Teyssié, J.L., Gattuso, J.P., 2015. Response of the Arctic pteropod Limacina helicina to projected future environmental conditions. PLoS One 5, 1–7. Cooley, S.R., Donry, S.C., 2009. Anticipating ocean acidification's economic consequences for commercial fisheries. Environ. Res. Lett. 4, 024007. Cripps, G., Lindeque, P., Flynn, K.J., 2014. Have we been underestimating the effects of ocean acidification in zooplankton? Glob. Chang. Biol. 20, 3377–3385. Dam, H.G., 2013. Evolutionary adaptation of marine zooplankton to global change. Ann. Rev. Mar. Sci. 5, 349–370. Doney, S., 2009. The consequences of human-driven ocean acidification for marine life. Biol. Reprod. 1, 36. Dupont, S.T., Thorndyke, M.S., 2013. Direct impacts of near-future ocean acidification on sea urchins. In: Fernández-Palacios, J.M., de Nascimento, L., Hernández, J.C., Clemente, S., González, A., Díaz-González, J.P. (Eds.), ClimateChange Perspective from the Atlantic: PastPresent and Future. Servicio de Publicaciones, Universidad de La Laguna, pp. 461–485. Dupont, S., Ortega-Martínez, O., Thorndyke, M., 2010. Impact of near-future ocean acidification on echinoderms. Ecotoxicology 19, 449–462.

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Please cite this article as: Zhan, Y., et al., The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius..., Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.08.003