Marine Biology

, Volume 161, Issue 1, pp 101–110

Impacts of near future sea surface pH and temperature conditions on fertilisation and embryonic development in Centrostephanus rodgersii from northern New Zealand and northern New South Wales, Australia

Authors

    • Department of Marine ScienceUniversity of Otago
    • Department for Earth, Environment and Life Sciences (DISTAV)University of Genoa
  • M. F. Barker
    • Department of Marine ScienceUniversity of Otago
  • S. A. Dworjanyn
    • National Marine Science CentreSouthern Cross University
  • M. Byrne
    • Schools of Medical and Biological Sciences, F13University of Sydney
  • M. D. Lamare
    • Department of Marine ScienceUniversity of Otago
Original Paper

DOI: 10.1007/s00227-013-2318-1

Cite this article as:
Pecorino, D., Barker, M.F., Dworjanyn, S.A. et al. Mar Biol (2014) 161: 101. doi:10.1007/s00227-013-2318-1

Abstract

Oceans are warming and becoming more acidic. While higher temperature and lower pH can have negative effects on fertilisation and development of marine invertebrates, warming may partially ameliorate the negative effect of lower pH. This study determined the effect of warming (3 °C) and decreased pH (0.3, 0.5, 1.1 units below ambient) on fertilisation and development in two populations of the sea urchin Centrostephanus rodgersii, one at its northern range limit (Coffs Harbour, New South Wales NSW, 30°27′S, 153°14′E) and the other one in New Zealand where the species may be a recent arrival (Mokohinau Islands, 35°56′S, 175°9′E). Both populations were sampled in August 2011. The two populations exhibited a differential response to temperature, while pH affected them similarly. Fertilisation was robust to pH levels forecast for 2100, and it was only slightly reduced at pH values forecast for 2300 (i.e. ≈5 and ≈8 % for the northern NSW and the New Zealand populations, respectively). Decreased pH (pH = 7.6) reduced the percentage of succeeding developmental stages. Progression through cleavage and hatching stages was faster at +3 °C in the New Zealand population but not in northern NSW urchins, while for the NSW population, there was a positive interaction between temperature and pH at hatching. Gastrulation was negatively affected by an extreme pH 7.0 treatment (60–80 % reduction) and least affected by increased temperature. The percentage of abnormal embryos at gastrulation increased significantly at +3 °C treatment in the northern NSW population. Predicted future increases in temperature may facilitate further expansion of the geographical range of C.rodgersii in New Zealand, with a minimal effect of concurrent reduced pH.

Introduction

Ocean warming and ocean acidification are two phenomena attributable to increased atmospheric CO2 (Caldeira and Wickett 2003; Feely et al. 2004, 2009; Orr et al. 2005; IPCC 2007; Halpern et al. 2008; Brierley and Kingsford 2009). Increased CO2 emissions since 1750 have already led to an average reduction in sea surface pH of 0.11 units (Haugan and Drange 1996). If present day releases of anthropogenic CO2 continue, the ocean’s CO2 concentration is expected to increase from ≈400 ppm to 700–1,000 ppm by 2100 (IS92 A2 scenario; IPCC 2007) and to 2,000 ppm by 2300 (Caldeira and Wickett 2003). This is forecast to result in a pH decrease in 0.14–0.41 units by 2100 and 0.30–0.7 units by 2300 (Caldeira and Wickett 2003; IPCC 2007; Doney et al. 2009). A concurrent increase in sea surface temperature (SST) is expected, with current models predicting regional increases in sea surface temperatures of 2.0–4.5 °C by 2100 (IPCC 2007).

Temperature and CO2 are known to affect physiological processes in marine invertebrates, especially in early life stages (review Byrne 2011). Temperature usually increases biochemical reaction rates, until a thermal threshold is reached beyond which physiological activity rapidly decreases. Higher ocean CO2 concentrations have several effects. By reducing the pH of seawater, ocean acidification reduces the concentration of carbonate ions and, as a consequence, their availability to marine calcifying organisms. Ocean acidification can increase organism pCO2 (hypercapnia) altering energetic requirements for acid–base regulation and other metabolic changes (Pörtner 2008; (Stumpp et al. 2011a, b; Melzner et al. 2009; Christensen et al. 2011).

Temperature and CO2 covary in the ocean, and increased temperature is expected to interact with reduced pH and hypercapnia, potentially counteracting the negative effect of acidification by increasing metabolic rate (Sheppard-Brennand et al. 2010; Arnberg et al. 2012). When considering the future effects of climate change, two-factor studies of both stressors are needed on the most vulnerable part of an organism’s life cycle, which, for marine species, is typically the gametes and developmental stages. If the effects of global change stressors on fertilisation, embryos or larvae are severe enough to produce a bottleneck in the population then the effect of these stressors on juvenile or adults becomes less relevant (Byrne 2011).

Previous two-factor studies on warming and acidification have examined six echinoderm species (Byrne et al. 2009, 2010a, b, 2011; Sheppard-Brennand et al. 2010; Foo et al. 2012, Nguyen et al. 2012, Wolfe et al. 2013), four bivalves (Parker et al. 2010; Talmage and Gobler 2011), one abalone (Byrne et al. 2010a), two barnacles (Findlay et al. 2010a, b) and one shrimp (Arnberg et al. 2012). In echinoderms and abalone, fertilisation was not impaired by predicted near future warming or lowered pH and no interaction between the two variables was detected (Byrne et al. 2009, 2010a, b). In the case of the two species of oyster, however, fertilisation decreased at the both suboptimal temperatures and lowered pH (Parker et al. 2010). Subsequent developmental stage responses to temperature and pH vary both among species and among developmental stage considered and may also be influenced by the developmental stage at which experiments are initiated (Byrne 2012). Low pH and increased temperature are often associated with a reduction in the developmental performances (Byrne et al. 2009; Sheppard-Brennand et al. 2010; Findlay et al. 2010a, b; Parker et al. 2010; Byrne et al. 2011a; Nguyen et al. 2012; Byrne and Andrew 2013), although an ameliorating effect of elevated temperature at lower pH conditions is reported for larval calcification (Sheppard-Brennand et al. 2010) and growth (Parker et al. 2010; Sheppard-Brennand et al. 2010; Byrne et al. 2011a).

We investigated the effect of increased temperature and acidification on fertilisation and development in two populations of the diademid sea urchin Centrostephanus rodgersii from the northern range edge of the species in eastern Australia in northern New South Wales at Coffs Harbour and a population from its south-eastern margin in New Zealand where the population may represent a recent extension of its range (Pecorino et al. 2013). The Coffs Harbour population is likely to be near the source of larvae for the populations in New Zealand (Pecorino et al. 2013). The responses of C. rodgersii developmental stages to changes in sea temperature are particularly relevant, because ocean warming has resulted in significant expansion of the species range in eastern Australia (Ling et al. 2009b).

Centrostephanus rodgersii was, until recently, restricted to the eastern coast of mainland Australia, but has expanded its geographical range to Tasmania, starting in the late-1960s (Ling et al. 2008). This sea urchin has an important ecological role, and it can cause phase shifts from macroalgal-dominated habitats to barrens devoid of foliose seaweeds (review Byrne and Andrew 2013). The lower thermal threshold for pluteus development for the Coffs Harbour and the New Zealand populations is ≈15 °C, and they also have a similar upper limit of 25–26 °C (Pecorino et al. 2013). The response of C. rodgersii developmental stages to temperature in the New Zealand and Australian populations may be especially relevant, as the thermal tolerance of the larvae appears to influence the distributions of the adult populations (Ling et al. 2009b; Pecorino et al. 2013), and because range shifts as a consequence of changing ocean climate are evident (Sagarin et al. 1999; Parmesan and Yohe 2003; Perry et al. 2005, Ling et al. 2009b).

Early development in marine invertebrates may be more sensitive to increased temperature than to acidification (review Byrne 2011; Arnberg et al. 2012), and this is investigated here with C. rodgersii. C. rodgersii has a very broad latitudinal range, spanning from northern New South Wales (latitude ≈30 °S; SST during spawning 20.4–21.2 °C; Byrne et al. 1998) to Tasmania (latitude ≈42 °S; SST during spawning 11–12 °C; Ling et al. 2008) and New Zealand (latitude ≈36 °S; SST during spawning 15.7–16.8 °C; Pecorino et al. 2012). Thus, this species is ideal for testing the hypothesis that individuals of the same species from different thermal environments differ in their response to the same environmental stressor. It is important to understand how the New Zealand population, and especially its early life stages, may be affected by predicted warmer and more acidic ocean conditions, compared to the Coffs Harbour population, near the likely source of propagules that reach New Zealand (Pecorino et al. 2013). These comparisons may shed light on the potential adaptive or acclimation responses of different populations of C. rodgersii and their capacity to further expand their range in New Zealand.

We investigated the effect of increased sea temperature and decreased pH, at levels forecast for 2100 and 2300, on fertilisation, cleavage, hatching, gastrulation and presence of abnormal embryos at gastrulation in the two populations. An extreme pH level (pH 7.0) was used to assess tolerance levels. We expected the percentage of fertilisation and survival to be reduced at lower pH, with a severe reduction at pH 7.0. For the New Zealand population, which is in an area with a lower temperature than its original distribution range, we expected to see an increased percentage of fertilisation, cleavage, hatching and gastrulation in the higher temperature treatment, potentially overriding the reduction caused by lowered pH.

Materials and methods

Specimen collection and spawning

Centrostephanus rodgersii were collected at the Mokohinau Islands, New Zealand (35°56′S, 175°9′E) (Fig. 1a), and Coffs Harbour, New South Wales (NSW), Australia (30°27′S, 153°14′E) (Fig. 1b), in August 2011 from depths of 1–2 and 2–5 m, respectively. Centrostephanus rodgersii collected at the Mokohinau Islands (hereafter “New Zealand population”; ambient sea temperature during collection was 14.3 °C, salinity was 35.5; Chang et al. 2003) were transported in a large bin filled with flowing seawater to the Leigh Marine Laboratory (University of Auckland) and placed in flow-through seawater tanks at ambient sea temperature. They were kept in the tank for 1 day under natural light conditions before spawning. C. rodgersii collected in northern New South Wales at Coffs Harbour (hereafter “northern New South Wales population” or “northern NSW population”; ambient sea temperature during collection was 21.0 °C, salinity was 35.6) were transported to the National Marine Science Centre (NMSC) Southern Cross University and used immediately for spawning.
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Fig. 1

a Location of Mokohinau Islands in New Zealand. Position of Centrostephanus rodgersii sampling site (35°56′S, 175°9′E) at Fanal Island indicated by the circle. b Position of Coffs Harbour in New South Wales (Australia) shown in small frame in lower left corner. Site of collection of C. rodgersii (30°27′S, 153°14′E) is marked by a circle in large frame

Spawning was induced by an intracoelomic injection of 1–2 ml of 0.5 M KCl. Sperm was collected “dry” from the aboral surface of males, placed in Petri dishes covered with Parafilm™ and kept chilled until needed. Eggs were collected by inverting females on beakers filled with fresh 0.22-μm filtered seawater (FSW; salinity was 35.6 for the northern NSW population and 35.5 for the New Zealand population), and the concentration of eggs in suspension estimated by counting the number of eggs in five 1-ml replicate subsamples. The eggs were then split into sealable 400-ml beakers filled with FSW at the experimental temperature and pH treatment (4 replicates each). The volume of suspension put into each beaker was calculated to achieve a concentration of ≈30 eggs ml−1. Gamete quality was checked microscopically for egg shape and sperm motility. Three males and three females were used, and the gametes were pooled prior to fertilisation. Multiple males and females were used to create a genetically diverse pool similar to natural spawning.

Experimental treatments

The effect of temperature and pH treatment on fertilisation, cleavage, hatching, gastrulation and abnormalities in the embryos at the gastrula stage was tested in a full-factorial design.

Two temperature treatments were applied, 14 and 17 °C for the New Zealand population and 21 and 24 °C for the northern NSW population, i.e. the temperature in the field at the time of collection, and a temperature 3 °C higher than that in each case, while four pH treatments (8.1, 7.8, 7.6 and 7.0) were applied to both populations. Temperature treatments were chosen so that the low temperature treatment was compatible with field conditions at the time of spawning, and the high temperature treatment was within the range of values predicted for the years 2070–2100 (IPCC 2007). The pH level 7.8 was chosen as representative of 2,100 seawater pH conditions according to the IS92 A2 scenario by IPCC (2007), while 7.6 was chosen as a conservative estimate for surface seawater pH for the year 2300 (Caldeira and Wickett 2003). An extreme value of pH (pH = 7.00) was used to test for tolerance levels.

Seawater chemistry

The desired pH was achieved by bubbling 100 % analytical grade CO2 into 10 l buckets of 0.22-μm FSW at an approximate flow rate of 5 l min−1 until the target pH was reached. Measurements were taken using a temperature-compensating pH meter (Eutech Instruments, P510) using the NIST scale. The pH meter was calibrated using two pH standards (pH 7.0 and 9.2; Labserv Pronalys, Biolab, New Zealand) kept at the same two temperatures of the treatments in each experiment. All the experimental jars were free of gas bubbles or air space and remained sealed for the duration of the experiments. To test the stability of the experimental conditions, seawater pH was checked again at the end of each experiment and was confirmed to be relatively stable, although a slight decrease was detected in all treatments (0.13 units maximum variation in the case of the New Zealand experiments at pH 7.00 at 14 °C; mean variation = 0.09 pH units; SD = 0.02 pH units).

Total alkalinity (TA) was measured via potentiometric titration for both sites, after adding HgCl2 to fix each water sample. These measurements were made at the Chemistry Department of the University of Otago for the New Zealand samples and at NMSC for the northern NSW samples. TA measurements were used, together with salinity, pH(NIST) and experimental temperature to calculate pCO2 and saturation state of calcite ΩC and aragonite ΩA (Table 1). These calculations were carried out using the CO2SYS Program (Lewis and Wallace 1998). In the case of the New Zealand experiment, at both 14 and 17 °C, calcite was undersaturated at pH 7.0 and aragonite was undersaturated at pH 7.6 and 7.0 (Table 1). For the northern NSW experiments, at 21 °C, aragonite and calcite were undersaturated at pH 7.0, and aragonite at pH 7.6, while at 24 °C, both minerals were only undersaturated at pH 7.0 (Table 1).
Table 1

Parameters of seawater carbonate system of experiments run on New Zealand and Australian populations of Centrostephanus rodgersi

 

T (°C)

pH

TA (mmol kg−1)

pCO2 (matm)

Ω Ca

Ω Ar

New Zealand

S = 35

14

8.10

2,243.7

468.270

3.257

2.087

14

7.80

2,243.7

1,010.555

1.765

1.131

14

7.60

2,243.7

1,651.575

1.149

0.736

14

7.04

2,243.7

6,237.613

0.329

0.211

17

8.10

2,243.7

477.823

3.546

2.288

17

7.80

2,243.7

1,038.311

1.936

1.249

17

7.60

2,243.7

1,701.772

1.263

0.815

17

7.04

2,243.7

6,451.499

0.363

0.234

Australia

S = 35.2

21

8.10

2,327.0

505.548

4.120

2.686

21

7.80

2,327.0

1,109.218

2.271

1.480

21

7.60

2,327.0

1,825.318

1.488

0.970

21

7.04

2,327.0

6,957.144

0.430

0.280

24

8.10

2,327.0

511.311

4.461

2.933

24

7.80

2,327.0

1,130.574

2.478

1.629

24

7.60

2,327.0

1,866.511

1.628

1.071

24

7.04

2,327.0

7,145.298

0.473

0.311

Salinity is shown below name of each site

Fertilisation assay

For the fertilisation assay, four 1-ml aliquots of egg suspension were taken from each beaker and placed in a well of a 12-well tissue culture dish. A volume of sperm solution to achieve a sperm/egg ratio of 100:1 was added to each well (sperm concentration 3,000 cell ml−1). By doing so, sperm were activated at each temperature/pH combination under which fertilisation took place. The plates were then covered with tight lids, sealed with Parafilm™ and kept at the experimental temperature by submerging them in temperature-controlled water baths. After 30 min, samples were fixed by adding a few drops of 7 % borax-buffered formalin. The % fertilisation in each of the four replicates for each treatment was evaluated by counting the proportion of eggs with a fertilisation envelope among the first 50 individuals examined microscopically.

Embryonic development

Spawned eggs were split into three 400-ml sealable beakers and fertilised by adding an appropriate volume of sperm solution to achieve a sperm/egg ratio of 100:1 (i.e. a sperm concentration of 3,000 cell ml−1). Temperatures in the treatments were kept constant by submerging the sealed beakers in water baths maintained at the appropriate experimental temperature. At each sampling time (Table 2), four 4-ml samples were taken from each beaker and fixed as above. The developmental stage of the first 50 embryos observed under a light microscope in each sample was assessed for the % cleavage, hatching, gastrulation and abnormal embryos at gastrulation. The average across the sub-samples was used to give a mean estimate for each replicate. Cleavage and gastrulation were assessed by looking at the morphology of embryos. Blastulae that did not have a fertilisation envelope were considered hatched and embryos with no internal cavity that had undergone exogastrulation or had protruding cellular masses were considered abnormal (Supplementary Fig. 1).
Table 2

Sampling time for fertilisation and different developmental stages considered for Centrostephanus rodgersii in New Zealand and Australia

Stage

Sampling time (hours after fertilisation)

New Zealand

Australia

Fertilisation

0.5

0.5

Cleavage

4

2

Hatching

24

21

Gastrulation

44

40

Abnormal

44

40

Sampling times chosen according to Huggett et al. (2005) and previous trials on this species

Statistical analyses

Results were analysed by two-way ANOVA using temperature and pH as fixed orthogonal factors and % fertilisation, cleavage, hatching, gastrulation and abnormal embryos as response variables in the different analyses. All data were arcsine-square root transformed, normality of the distribution was visually checked using normal quantile–quantile plots and homoscedasticity confirmed by Levene’s test. When significant differences were present, a Tukey’s Honestly Significant Difference (HSD) test was used for post hoc analyses. All statistical analyses were performed, and graphs were done using the computer software R (R Development Core Team 2010).

Results

For the New Zealand population, the % fertilisation was ≥95.5 % at pH 8.1 and pH 7.8, it was ≥78 and ≥87.5 % at pH 7.6 (at 14 and 17 °C, respectively), and it was ≤30 % at pH 7.0 at both temperature treatments (Fig. 2). There was no significant effect of temperature on % fertilisation (ANOVA, F(1, 24) = 7.762, p = 0.192) and a significant effect of pH (ANOVA, F(3, 24) = 366.596, p < 0.001), while the interaction between the two factors was not significant (ANOVA, F(3, 24) = 2.603, p = 0.075; Supplementary Table 1a, Fig. 2a). The % fertilisation of the northern NSW population at both temperatures at pH 8.1 and 7.8 was ≥80 %. It decreased to 67.5 and 69.5 % at pH 7.6 (at 21 and 24 °C, respectively) and was ≤7.5 % at pH 7.0 (Fig. 2a). Temperature did not have a significant effect (ANOVA, F(1, 24) = 3.143, p = 0.089) on  % fertilisation, while pH did (ANOVA, F(3, 24) = 765.899, p < 0.001). There was no significant interaction between the two factors (ANOVA, F(3, 24) = 0.587, p = 0.629; Supplementary Table 1a). The % fertilisation at pH 8.1 and 7.8 was significantly higher than at pH 7.6 and 7.0 at both temperatures.
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Fig. 2

Percentage of a fertilisation, b cleavage and c hatching (mean ± s.e.; 50 scored eggs for each replicate and 4 replicates each treatment) at two temperatures and four pH treatments for the New Zealand and Australian populations of Centrostephanus rodgersii. Temperature treatments are 14 °C White bar and 17 °C Grey bar for C. rodgersii in New Zealand; and 21 °C White bar and 24 °C Grey bar for C. rodgersii in Australia. For each developmental stage and each population, different letters represent significant differences

The % cleavage for the New Zealand population (Fig. 2b) was consistently higher at 17 °C than at 14 °C, and it decreased from pH 8.1 to pH 7.0. An ANOVA indicated that there was a significant effect of temperature (ANOVA, F(1, 88) = 7.762, p ≤ 0.01) and of pH (ANOVA, F(3, 88) = 355.596, p < 0.001) on  % cleavage, but no significant interaction (ANOVA, F(3, 88) = 1.059, p = 0.075) between the two factors (Supplementary Table 1b). For the northern NSW population, at both temperatures, the % cleavage was ≥74.8 % at pH 8.1 and pH 7.8 (Fig. 2b), was 39.2 and 43.3 % at pH 7.6 (at 21 and 24 °C, respectively, and was ≤2.3 % at pH 7.00. There was a significant effect of temperature (ANOVA, F(1, 88) = 7.092, p < 0.01) and pH (ANOVA, F(3, 88) = 1,038.091, p < 0.001) on % cleavage with no significant interaction between the two variables (ANOVA, F(3, 88) = 0.198, p = 0.898; Supplementary Table 1b).

For the New Zealand population, the % hatching was significantly higher at 17 °C across all pH treatments and decreased when pH was reduced (Fig. 2c). The interaction of temperature and pH was significant (ANOVA, F(3, 88) = 10.011, p < 0.001; Supplementary Table 1c). The % hatching for the northern NSW population decreased from ≈80 % at pH 8.1 to ≈5 % at pH 7.0. At pH 7.6, it was significantly lower at 21 °C than 24 °C (Fig. 2c). There was a statistically significant interaction between temperature and pH (ANOVA, F(3, 88) = 9.631, p < 0.001; Supplementary Table 1c).

The % gastrulation of the New Zealand population (Fig. 3a) decreased with reducing pH. At 17 °C, it was 81.2 % at pH 8.1 and 21.7 % at pH 7.0. At 14 °C, it decreased from 76.7 % at pH 8.1–19.7 % at pH 7.0. The effect of both temperature (ANOVA, F(1, 88) = 6.696, p < 0.05) and pH (ANOVA, F(3, 88) = 451.659, p < 0.001) was significant (Supplementary Table 1d). The interaction between temperature and pH was not significant (ANOVA, F(3, 88) = 0.078, p = 0.972). In the northern NSW population, the % gastrulation decreased with pH (Fig. 3a). There was no significant difference between temperatures across pH treatments 8.1, 7.8 and 7.0, while the % gastrulation was significantly lower at 24 °C than at 21 °C at pH 7.6. An ANOVA showed that the effects of temperature and pH were significant (ANOVA, F(1, 88) = 10.043, p < 0.01; ANOVA, F(3, 88) = 1,730.456, p < 0.001) and there was a significant interaction between them (ANOVA, F(3, 88) = 4.038, p < 0.01; Supplementary Table 1d).
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Fig. 3

Percentage of a gastrulation and b abnormal embryos (mean ± s.e.; 50 scored larvae for each replicate and 3 replicates each treatment) at two temperature and four pH treatments for the New Zealand and Australian populations of Centrostephanus rodgersii. Temperature treatments are 14 °C White bar and 17 °C for C. rodgersii in New Zealand; and 21 °C White bar and 24 °C for C. rodgersii in Australia. For each developmental stage and each population, different letters represent significant differences

The % abnormal embryos for the New Zealand population (Fig. 3b) increased from pH 8.1 to pH 7.0 and was marginally higher at 17 °C than at 14 °C across all pH treatments. There was a significant effect of both temperature (ANOVA, F(1, 88) = 12.903, p < 0.001) and pH (ANOVA, F(3, 88) = 44.510, p < 0.001), but no significant interaction between the two variables (Supplementary Table 1e). The % abnormal embryos in the northern NSW population (Fig. 3b) increased from pH 8.1 to pH 7.0 at both temperatures, and there was a significant effect of both temperature (ANOVA, F(1, 88) = 41.905, p < 0.001) and pH (ANOVA, F(3, 88) = 109.681, p < 0.001). No significant interaction was present (ANOVA, F(3, 88) = 1.864, p = 0.228; Supplementary Table 1e).

Discussion

Centrostephanus rodgersii has expanded its range from mainland Australia to Tasmania as the result of regional increases in sea temperatures that have favoured larval development and transport (Ling et al. 2008, 2009a, b). Therefore, understanding the present day distribution of C. rodgersii and the potential for future range expansions requires information on the biology of its early life stages. This knowledge is especially relevant when considering that this sea urchin can radically change well-vegetated subtidal habitats to barren grounds (review Byrne and Andrew 2013).

In New Zealand, C. rodgersii occurs at the southern margin of its lower thermal tolerance threshold for larval development, which is ≈15 °C for these individuals (Pecorino et al. 2013), and so a stimulatory effect of increased temperature on development, as found here, was expected. The combined effect of temperature and pH was also expected to differ from the NSW range-edge population, where this species lives near the optimum thermal tolerance window for development (≈17–25 °C, Pecorino et al. 2013).

The % fertilisation of the New Zealand population of C. rodgersii was not affected by pH treatments at both temperatures, suggesting that fertilisation in the New Zealand population is robust to future predicted warming and acidification. Fertilisation in the northern NSW population was not significantly affected by increased temperature, and it was still >80 %, showing that this process was relatively robust to pH changes. The reduction in % fertilisation and development at pH 7.00 shows that this level of acidification is toxic to gametes and embryos. Byrne et al. (2010a) examined the effect of warming and acidification on fertilisation in C. rodgersii and five other invertebrate species along the SE Australian coast, also showing that fertilisation was robust to these stressors. We did note a significant decline (≈8 %) in fertilisation at pH 7.6, although this difference to the Byrne et al. (2010a) result may be due to different methods used (see Byrne 2012).

For Paracentrotus lividus, the response of fertilisation to decreased pH is influenced by tide pool of origin (Moulin et al. 2011), with the authors suggesting that spermatozoa already subjected to greater acidified conditions in the testes could have a greater concentration of, or more effective, proteins for transmembrane transport of H+ involved in the regulating of internal pH that, in turn, affects sperm motility (i.e. Christen et al. 1982). This difference is interpreted as evidence for acclimatisation to a more variable pH regime potentially mediated by the presence of more, or more effective, Na+/K+-ATPase pumps and Na+/H+ transporters that are actively involved in the regulation of the internal pH of spermatozoa (Gatti and Christen 1985).

The response of fertilisation to decreased pH varies among studies, including research within the same species. These later differences may, in part, be due to the use of both single male–female crosse or multiple parents (Byrne and Andrew 2013). As shown for the sea urchin Heliocidaris erythrogramma, single male–female crosses result in a much more variable response to decreased pH (Schlegel et al. 2012) than fertilisations involving multiple parents (Byrne et al. 2010b). For example, Reuter et al. (2011) reported a 72 % reduction in fertilisation efficiency at pH 7.81 in single male–female crosses with Strongylocentrotus franciscanus. Polyandry is known to increase fertilisation success in sea urchins due to the strong influence of gamete binding compatibility and the large difference in fertilisation success that can occur among male–female pairs, as is the case for C. rodgersii (Palumbi 1999; Evans and Marshall 2005; Foo et al. 2012). The present study agrees with previous reports on the synergistic effects of temperature and pH on the fertilisation of sea urchins (Byrne et al. 2009a, b) with no interaction between the two stressors found. The hypothesis that increased temperature would buffer the negative effect of pH is, therefore, not supported.

Cleavage, hatching and gastrulation in Centrostephanus rodgersii in New Zealand were robust to the most extreme seawater pH and temperature conditions predicted for 2100. The same is true for C. rodgersii in northern NSW, apart from gastrulation, which, at increased temperature, occurred at a significantly lower percentage at pH 0.3 units lower than ambient. At ambient temperature, no significant difference was present.

A strong effect of pH was also found on the % abnormal embryos. For the New Zealand population of C. rodgersii, a decrease in 0.3 pH units from ambient to pH 7.8 was sufficient to cause a significant increase in the number of abnormal embryos. A significant increase in abnormality was also present from pH 7.8–7.6, while further pH decrease did not cause significant additional change. This response is similar to that recorded for the embryos and larvae of C. rodgersii in previous studies (Doo et al. 2012; Foo et al. 2012) and the juveniles of H. erythrogramma (Byrne et al. 2011). For the northern NSW population of C. rodgersii, abnormal embryos were present at larger numbers at near future levels of acidification (Doo et al. 2012). This negative effect was increased, in the present study, by higher temperature: at ambient pH and 24 °C, in fact, the number of abnormal embryos is comparable to that found at pH 7.8 and 21 °C. This is similar for the 24 °C and pH 7.8 treatment and the 21 °C and pH 7.6 treatment. This shows that increased temperature is additional to the negative effect of lowered pH and causes a disproportionately higher % of abnormal embryos at a given pH.

For the northern NSW population of C. rodgersii, there was no difference between hatching at pH 8.1, 7.8 and 7.6 in the increased temperature treatments, while it was significantly lower at pH 7.6 at ambient temperature. This may be evidence for increased temperature buffering the negative effect of pH on development. Rahman et al. (2009) found similar results in the tropical sea urchin Tripneustesgratilla, where increased temperature led to an increased hatching rate, within temperatures found in the field throughout a whole year, before dropping from ≈90 to ≈50 % between 29 and 31 °C.

Hatching is mediated by the hatching enzyme (Ishida 1936; Rahman et al. 2009) and an increase in the hatching rate in the New Zealand C. rodgersii may be influenced by the increased activity of such enzyme at higher temperature, as the +3 °C treatment is closer to the thermal optimum determined for development of this population, than to the temperatures at which the New Zealand population is presently spawning, ≈15 °C (Pecorino et al. 2013). The increase in hatching at this level of warming may compensate somewhat for the negative effect of pH. In contrast, the increase in hatching rate of the northern NSW population was less pronounced and could possibly be caused by the hatching enzyme of these urchins already being near its thermal optimum. Consequently, hatching rate is already at a very high level. A 3 °C temperature increase, therefore, may not cause substantial increases, apart at pH 7.6, where it partially compensated for the reduction caused by low pH.

It is more difficult to attribute the reduced % gastrulation at lowered pH in C. rodgersii to a specific cause, as no single factor controlling it has yet been identified. This lower developmental success could be due to metabolic stress, and the consequences of the energy demands associated with additional acid–base balance. This reallocation of energy to homoeostasis and away from growth and development (i.e. the increased transcription of acid–base regulating proteins) has been implicated in slowed development of Strongylocentrotus purpuratus larvae reared under low pH (Stumpp et al. 2011a, b).

An adaptive capacity in early development of C. rodgersii was highlighted by Foo et al. (2012). The authors tested the effect of pH and temperature on multiple male–female crosses and found a high diversity of responses to both variables. While some of the crosses showed a low tolerance to pH and thermal stress (i.e. ≈20 % of embryos cleaved in the case of one male genotype at pH 7.6 and ≈40 % at +4 °C above ambient temperature), others showed a high tolerance of the same stressors (>80 % of embryos cleaved in the case of one male genotype at both pH 7.6 and +4 °C above ambient temperature). The contribution of tolerant genotypes could therefore be important in assuring the persistence of a species in future warmer and more acidic scenarios, and it could be the basis of the positive response of the New Zealand population to climate change stressors that we found in the course of the present study.

Studies performed on the embryonic development of sea urchins show mixed results, although most report no effect of decreased pH on the percentage of early developmental stages (Kurihara and Shirayama 2004; Byrne et al. 2009; Ericson et al. 2012), and only one case of reduced cleavage and gastrulation at increased temperature (+4 °C; Byrne et al. 2009. Havenhand et al. (2008), conversely, found lowered cleavage in H. erythrogramma at pH levels within those forecast for 2100. Overall, early (pre-larval) development in sea urchins exhibits high tolerance to near future projected ocean pH and temperatures (Byrne 2011, 2012), potentially reflecting the conditions that sea urchins that live in shallow waters naturally encounter.

A recent study on the thermal tolerance of the early life stages of C. rodgersii (Pecorino et al. 2013) suggests that future scenarios of warmer seas could lead to range extension of this sea urchin in northern New Zealand. This would be favoured by a shift southward of the 15 °C isotherm, which has been identified as the lower thermal threshold for the development of the local population. The present study further supports this speculation, since it demonstrates that the 0.3 pH units reduction forecast to take place by 2100, concurrent with a 3 °C temperature increase, may not negatively impact fertilisation and early development in the New Zealand C. rodgersii population, thus not hindering its potential range extension. A pH reduction has, furthermore, already taken place worldwide (Haugan and Drange 1996), and C. rodgersii has, during the same time frame, expanded its geographical to New Zealand (Pecorino et al. 2013) and Tasmania (Ling et al. 2009a, b), thus showing that this phenomenon is possible under acidifying conditions.

This study highlights the strong influence of temperature on development of C. rodgersii. In New Zealand, this species occurs at temperatures suboptimal for development. The positive effect of temperature on development may override the small negative effect of acidification. The NSW population at their northern (warm) range edge at Coffs Harbour may already be at the upper end of its thermal tolerance and therefore might not be favoured by a temperature increase.

Acknowledgments

The authors wish to thank the staff of the Leigh Marine Laboratory of the University of Auckland and the staff of NMSC at Coffs Harbour for their help during the collection of the sea urchins and while running the experiments. This research was supported by a New Zealand Marine Sciences Society Student Research Grant and by a Grant of the Australian Research Council (MB, SD) that supported the construction of the facility in Coffs Harbour.

Supplementary material

227_2013_2318_MOESM1_ESM.docx (21 kb)
Supplementary material 1 (DOCX 21 kb)
227_2013_2318_MOESM2_ESM.tiff (898 kb)
Photomicrographs of a representative fertilised egg and subsequent developmental stages of Centrostephanus rodgersii. From left to right: fertilised egg, cleaved embryo, hatched blastula, gastrula and abnormal embryo. Scale bars represent 100 μm. (TIFF 897 kb)

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© Springer-Verlag Berlin Heidelberg 2013