Reference: Biol. Bull. 226: 1–7. (February 2014) © 2014 Marine Biological Laboratory
Present-Day Nearshore pH Differentially Depresses Fertilization in Congeneric Sea Urchins CHRISTINA A. FRIEDER*,† Scripps Institution of Oceanography, La Jolla, California 92093
Abstract. Ocean acidification impacts fertilization in some species of sea urchin, but whether sensitivity is great enough to be influenced by present-day pH variability has not been documented. In this study, fertilization in two congeneric sea urchins, Strongylocentrotus purpuratus and S. franciscanus, was found to be sensitive to reduced pH, ⬍7.50, but only within a range of sperm-egg ratios that was species-specific. By further testing fertilization across a broad range of pH, pH-fertilization curves were generated and revealed that S. purpuratus was largely robust to pH, while fertilization in S. franciscanus was sensitive to even modest reductions in pH. Combining the pH-fertilization response curves with pH data collected from these species’ habitat demonstrated that relative fertilization success remained high for S. purpuratus but could be as low as 79% for S. franciscanus during periods of naturally low pH. In order for S. franciscanus to maintain high fertilization success in the present and future, adequate adult densities, and thus sufficient sperm-egg ratios, will be required to negate the effects of low pH. In contrast, fertilization of S. purpuratus was robust to a broad range of pH, encompassing both present-day and future ocean acidification scenarios, even though the two congeners have similar habitats.
tion, and population densities are known to influence fertilization success (Levitan et al., 1992; Marshall, 2006; Riffell and Zimmer, 2007; Byrne, 2011). pH is an additional driver that has garnered recent attention due to concerns about pH reductions resulting from ocean acidification in shallow marine settings (Kurihara, 2008; Byrne, 2011, 2012). Evidence from multiple studies suggests that fertilization is sensitive to pH in some echinoderms, but not all. Of the 17 species studied to date, 10 exhibited reduced fertilization with decreased pH, but some of these results occurred at extremely low pH values (e.g., between 7.0 and 7.4), or only at low sperm-egg ratios (e.g., Kurihara and Shirayama, 2004; Gonzalez-Bernat et al., 2012). Some of the observed robustness of fertilization to pH could be reflecting speciesspecific adaptations to natural pH variability in shallowwater habitats. On the other hand, some of the observed sensitivity could be great enough that fertilization is depressed during present-day low pH conditions. Few fertilization assays published to date employ more than two or three pH treatment levels, but the realization that pH varies with space and time requires that fertilization be studied over a broad range of pH to elicit linear and nonlinear relationships and potential thresholds (Riebesell et al., 2010). The ensuing pH-fertilization response curves will improve our ability to predict when and where the effects of a rapidly changing ocean climate will impact the sustainability of natural populations. The underlying mechanism responsible for reduced fertilization at lower pH could be changes in gamete properties. For example, reduced pH has been observed to decrease sperm motility in a variety of marine invertebrates including corals, sea urchins, sea cucumbers, and sea stars (Havenhand et al., 2008; Morita et al., 2010; Schlegel et al., 2012; Uthicke et al., 2013; but see Caldwell et al., 2011). Intracellular pH in sperm is directly dependent on extracellular pH, and decreasing intracellular pH narcotizes sperm
Introduction Successful fertilization is vital for the persistence of populations. In the marine environment, broadcast spawners release their gametes into the water column, and the released gametes are exposed to the chemical and physical properties of the seawater. Temperature, turbulence, polluReceived 25 November 2013; accepted 31 January 2014. * To whom correspondence should be addressed. E-mail: ctanner@ucsd. edu † Current address: Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371. 1
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(Chia and Bickell, 1983; Christen et al., 1983; Vacquier, 1986; Brokaw, 1990). pH could also affect properties of the egg. Low intracellular egg pH may prevent fertilization and subsequent development as well as slow the time that the fast block to polyspermy is established (Desrosiers et al., 1996; Kurihara, 2008; Reuter et al., 2011a). In studies of the effects of pH on fertilization success, appropriate species-specific sperm-egg ratios should be utilized (Levitan et al., 1991; Levitan, 1993; Reuter et al., 2011a; Byrne, 2012). Studying a range of sperm-egg ratios is also important because, in the field, population densities of adult sea urchins influence the degree of sperm limitation (Levitan, 2004). When testing pH effects at multiple spermegg ratios, a fertilization kinetics model can be generated in which the probability of fertilization is a function of spermegg ratio (Vogel et al., 1982). Such an approach has been utilized to explore the effects of reduced pH on fertilization in Strongylocentrotus franciscanus and revealed a 97% decrease in fertilization efficiency from pHNBS 8.04 to 7.55 (Reuter et al., 2011a, b). Additional studies have revealed a 63% decrease in fertilization efficiency in S. fragilis from pHT 7.99 to 7.50, but no change in fertilization efficiency in Dendraster excentricus from pHT 8.06 to 7.53 (Frieder, 2013). In this study, I compare the influence of pH on fertilization success in two species of echinoids from temperate environments. Strongylocentrotus purpuratus and S. franciscanus, the purple and the red sea urchins, inhabit rocky reefs and kelp forests. S. purpuratus has an egg diameter of 70 – 80 m and a sperm velocity of 145 m s–1; S. franciscanus has an egg diameter of 120 –140 m and a sperm velocity of 130 m s–1 (Levitan, 1993). The larger egg size of S. franciscanus equates to higher fertilization rates at a given sperm-egg ratio because larger eggs present a larger target for sperm (Levitan, 1993). Despite differences in gamete traits between these species, I predicted that S. purpuratus and S. franciscanus would exhibit similar fertilization responses to reduced pH given that they are phylogenetically related (congeners) and have similar presentday pH-exposure histories. Fertilization experiments were carried out at ambient and low pH at multiple sperm-egg ratios. I then developed pH-fertilization response curves for both species across a broad range of pH at a sperm-egg ratio that was observed to be sensitive to pH change during the prior experiment. These response curves were combined with present-day pH data collected during these species’ spawning season to model relative fertilization output. Materials and Methods
where there is extensive overlap of adults (32.85°N, 117.29°W). Adults were maintained in flow-through aquaria at ambient sea-surface temperature, 15 °C. All sea urchins were fed assorted macroalgae, and all specimens were used within a month of collection. Fertilization assays Spawning was induced by injecting 0.55 mol l–1 KCl through the peristomial membrane. Oocytes were collected in dishes of filtered (0.22 m) seawater. The number of adults used in each experiment ranged from 2 to 5 females and 3 to 5 males. Sperm were collected dry from the gonopores of males, placed in a small vial, and kept on ice until use. A 0.1% dilution from dry sperm was made to verify sperm motility, and then preserved in 1% formalin. Sperm densities were calculated with a hemocytometer to determine the amount of diluted sperm required to attain desired sperm-egg ratios and sperm densities for each treatment. Oocytes from multiple females were mixed, and egg densities were determined by counting aliquots of 8 ⫻ 20 l. Eggs were added to experimental beakers at a density of 5 eggs ml–1 and were incubated in experimental conditions for 10 min before sperm were added. Dry sperm was diluted immediately before addition to experimental beakers. After sperm addition the seawater in each replicate was gently stirred. Fertilization proceeded for 20 min and was arrested by transferring the contents of each replicate to 1% formalin. The fertilization ratio, defined as the proportion of eggs fertilized, was determined by counting the frequency of occurrence of a fertilization envelope in at least 100 eggs per replicate. Two types of experiments were carried out in the Scripps experimental aquarium between February and April 2013, the reproductive season of both species, following the above protocol. Glass beakers (500 ml) were used, and there were three replicates per treatment level. The first experiment had two treatments: pH and sperm-egg ratio. The two pH levels were ambient pH, ⬎7.96, and low pH, ⬍7.53 (Table 1). The sperm-egg ratios varied between species and were based on preliminary experiments. There were eight S. purpuratus sperm-egg ratios ranging from 1:1 to 2 ⫻ 104:1, and eight S. franciscanus sperm-egg ratios ranging from 1:1 to 2 ⫻ 103:1. The second experiment tested nine pH levels ranging from 7.26 to 8.00 on fertilization in both species. The sperm-egg ratios used in the second experiment was 103:1 for S. purpuratus, and 2 ⫻ 103:1 for S. franciscanus; these were sperm-egg ratios at which fertilization was sensitive to pH as revealed by the prior experiment.
Animal collection
Carbonate chemistry manipulation
Specimens of Strongylocentrotus purpuratus (Stimpson, 1857) and S. franciscanus (Agassiz, 1863) were collected at 15-m water depth by scuba from the La Jolla kelp forest
pH levels for each experiment were manipulated by mixing different proportions of ambient and high total dissolved inorganic carbon (CT) seawater. The high-CT reservoir was
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EFFECTS OF pH ON SEA URCHIN FERTILIZATION Table 1 Levels of pH treatment used during each experiment Expt.
Species
pH
AT
pCO2
1
Purple
7.96 7.37 7.99 7.50 7.95 7.88 7.80 7.72 7.62 7.54 7.46 7.38 7.26 8.00 7.94 7.84 7.78 7.69 7.61 7.54 7.44 7.36
2236 2239 2236 2239 2241 2240 2237 2238 2238 2239 2238 2236 2237 2235 2235 2235 2236 2236 2235 2235 2241 2237
495 2120 450 1578 501 607 736 920 1156 1427 1729 2081 2801 442 526 669 781 992 1203 1410 1797 2212
Red 2
Purple
Red
Experiment 1 tested fertilization success at high and low pH across multiple sperm-egg ratios. Experiment 2 developed fertilization response curves across a range of 9 pH values. pH is reported on the total scale, AT, total alkalinity in mol kg-1, pCO2 in atm calculated using CO2SYS software. Purple ⫽ Strongylocentrotus purpuratus; Red ⫽ S. franciscanus.
attained by bubbling a 3% pCO2 air mix into filtered seawater, and both reservoirs received flow-through filtered seawater. Experiment water conditions are provided in Table 1. All experiments were conducted at ambient seawater temperature during both species’ reproductive season, 15 °C, and temperature was maintained by submerging replicate containers within a common water bath. Discrete samples for determination of pH were taken for each treatment at the beginning of an experiment, and measured by a modified spectrophotometric method based on Dickson et al. (2007) using 1-cm cuvette cells and commercially available m-cresol dye with dye corrections based on Clayton and Byrne (1993). This method was calibrated with certified reference material from the Marine Physical Laboratory at Scripps Institution of Oceanography, and uncertainty of measurements was ⫾0.03 pH units. All pH values reported are at in situ temperature and on the total scale unless noted as pHNBS to indicate measurement on the NBS scale. Discrete samples were also taken for the determination of total alkalinity (AT) and salinity. AT for each treatment was determined using an open-cell, potentiometric titration with an accuracy of ⫾2 mol kg–1 (Dickson et al., 2007). Salinity was calculated from density measured at 20 °C on a density meter (Mettler Toledo DE45) with an
3
accuracy of ⫾0.05 salinity units. Salinity during the experiments ranged from 33.53 to 33.59. Although only one discrete sample for determination of pH and AT was taken per treatment, an independent measure of pH was taken with the use of Honeywell Durafet III pH sensors to determine pH variance among replicates. The standard deviation among replicates was less than 0.02, and there was no significant drift in pH during the experiments. pCO2 conditions were calculated from pH and AT using the MATLAB version of CO2SYS (van Heuven et al., 2011) with dissociation constants from Mehrbach et al. (1973) as refitted by Dickson and Millero (1987). The average propagated uncertainties based on uncertainty in pH and AT for pCO2 was ⫾85 atm. Data analysis and statistics To determine the overall effect of pH and sperm-egg ratio on fertilization success, I used a two-way analysis of variance (ANOVA; fixed factors pH and sperm-egg ratio). Percentage data were square-root-arcsine transformed. Vogel’s fertilization kinetics model (Vogel et al., 1982) was applied to the fertilization data to calculate fertilization rate as a function of sperm-egg ratio, and to test whether pH resulted in decreased fertilization efficiency. From this model the fertilization efficiency, /o, can be estimated. /o reflects changes in gamete performance at different pH levels; these include, for example, the fertilizable area of the egg, the proportion of sperm able to fertilize an egg, or the number of sperm contacts needed to allow for the penetration of a single spermatozoan. For each pH treatment this parameter was estimated iteratively, along with 95% confidence intervals, using the Marquardt method of nonlinear regression in MATLAB. /o between pH treatments was considered significantly different if the 95% confidence intervals did not overlap. An index of gamete performance was calculated as the sperm density required to fertilize 50% of the eggs in each pH treatment (f50). pH conditions in a natural habitat for both species Continuous pH data were collected near bottom (3 m above bottom) in the south La Jolla kelp forest (32.81°N, 117.29°W), and the data were used as an estimation of natural conditions during spawning events. pH data were converted to relative fertilization success from the laboratory-derived relationship of fertilization success and pH for both species. This model assumed constant sperm-egg ratios and temperature. The instrument package utilized was a SeapHOx, which includes a Honeywell Durafet III pH sensor and an SBE-37 MicroCAT CTD. Details of instrumentation, deployment, and calibration are provided in Frieder et al. (2012). Data were collected during the spawning season of S. purpuratus and S. franciscanus from 1 to 31 March, 2013.
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C. A. FRIEDER Table 2
ANOVA F-ratios for analyses of effects of pH and sperm-egg ratios on fertilization ratios and the fertilization efficiency of two species of Strongylocentrotus Species
pH (df)
Sperm:egg (df)
pH ⫻ Sperm:egg (df)
Ambient pH /o (CI)
Low pH /o (CI)
S. purpuratus S. franciscanus
286 (1,32) 351 (1,32)
776 (7,32) 167 (7,2)
51 (7,32) 25 (7,32)
0.47 (0.11) 0.16 (0.02)
0.12 (0.04) 0.02 (0.002)
/o ⫾ 95% confidence intervals (CI), estimated from fertilization kinetics model at ambient pH and low pH. F-ratio values in bold indicate P values ⬍ 0.0001. df, degrees of freedom. Root-mean-square error values for fit of data to the model at ambient and low pH, respectively, are 0.104 and 0.161 for S. purpuratus, and 0.62 and 0.36 for S. franciscanus.
Results
Relative fertilization output under natural pH conditions
Sperm-egg ratios sensitive to low pH and effects on fertilization efficiency Fertilization success was reduced at low pH for both species, but at different sperm-egg ratios (Table 2). Fertilization was significantly lower at sperm-egg ratios from 100 to 1000:1 in Strongylocentrotus purpuratus, and 50 to 2000:1 in S. franciscanus (Fig. 1). To maintain its index of gamete performance (f50), S. purpuratus required 1.5 times the sperm density (with constant egg density) with a pH decline from 7.96 to 7.37, and S. franiscanus required 16 times the sperm density with a pH decline from 7.99 to 7.50. Fertilization efficiency, /o, was significantly lower at low pH for both species (Table 2). S. franciscanus exhibited an 86% reduction in /o from 7.99 to 7.50, and S. purpuratus exhibited a 74% reduction in /o from 7.96 to 7.37. Shape of pH response curve Both linear and nonlinear responses of fertilization success to reduced pH were observed (Fig. 2). S. purpuratus fertilization success exhibited a nonlinear response to pH at a sperm-egg ratio of 1000:1. At pH values greater than 7.60, fertilization success was above 90%; from pH 7.60 to 7.44, fertilization began to slowly decrease, but a precipitous threshold existed below pH of 7.44. A modified MichaelisMenten equation was fit to the response (RMSE ⫽ 0.0238).
S. purpuratus ⫽ 共pH ⫺ 7.25兲/共0.0116 ⫹ pH ⫺ 7.25兲 (Eq. 1) In contrast, S. franciscanus fertilization success decreased from pH 8.00 to 7.36 at a sperm-egg ratio of 2000:1. The effect of pH on the fertilization ratio, , was well described by a negative linear relationship (F1,7 ⫽ 204, P ⬍ 0.0001, r2 ⫽ 0.97).
S. franciscanus ⫽ 0.602 ⫻ pH ⫺ 3.86
(Eq. 2)
pH data were collected during March 2013, which coincides with the spawning seasons of S. franciscanus and S. purpuratus (Fig. 3). Fertilization success for both species at a given in situ pH was modeled from the regressions in Eq. 1 and 2 and plotted as a function of relative output from ambient pH 8.0 (Fig. 3). During this month, pH at depth was affected by alternating periods of a well-mixed versus a well-stratified water column, reflecting pH alternating from approximately 7.95 to 7.70 over week–long time periods. The corresponding relative fertilization output of S. franciscanus ranged between 0.79 and 0.93, while relative fertilization output of S. purpuratus was maintained above 0.95. Discussion The two sea urchin species studied had differing pHfertilization response curves despite the fact that they were congeners and were collected from the same habitat and presumably had similar histories of pH exposure. The Strongylocentrotus purpuratus fertilization threshold to pH was well below 7.6, suggesting that fertilization in this species is rather robust to pH in the context of present-day variability regimes and regional acidification scenarios (Frieder et al., 2012; Hauri et al., 2013). In contrast, S. franciscanus fertilization was sensitive to modest reductions of 0.06 pH units from 8.00, and these values are known to occur regularly within its present habitat during its reproductive season (Fig. 3). Present-day low pH conditions are capable of depressing fertilization output of S. franciscanus by up to 20%, and this value will increase linearly as pH continues to decrease with ocean acidification. Similar decreases in fertilization success have been observed for S. franciscanus despite methodological differences that included gamete concentrations, test vessel volume, experimental temperature, and use of single male-female pairs (Reuter et al., 2011a). Reuter and colleagues found that at 10 °C, fertilization efficiency, /o, decreased by 97%, from 400 to 1800 ppm. Similar magnitudes of decrease in fertilization efficiency were observed during this study (Table 2).
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EFFECTS OF pH ON SEA URCHIN FERTILIZATION
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Figure 2. Fertilization response curve to pH for Strongylocentrotus purpuratus (circles) and S. franciscanus (triangles). Sperm density used was 5 sperm l–1 and 10 sperm l–1 for S. purpuratus and S. franciscanus, respectively. Each point represents the mean and standard deviation of three replicates. Relationship between fertilization success and pH was linear in S. franciscanus and nonlinear in S. purpuratus.
study. This reduction could be due to differential sensitivity of male gamete traits such as sperm motility. Decreases in sperm motility due to reductions in pH are known to decrease fertilization efficiency (e.g., Havenhand et al., 2008).
Figure 1. Fertilization success as a function of sperm-egg ratio in ambient pH (circles) and low pH (triangles) conditions in (a) Strongylocentrotus purpuratus, and (b) S. franciscanus. Error bars represent the standard deviation of three replicates. Ninety-five percent confidence intervals on fertilization curve per pH treatment indicated by shaded region for ambient pH (dark gray) and low pH (light gray). Root-mean-square error values for fit of data to each model are provided in Table 2.
Drastic differences in the shape of the pH-fertilization response curve between the two species could be due to differing effects of pH on gamete traits, which in turn could be a result of habitat differences between the two species. For example, S. purpuratus can live in shallow water, often in tide pools, while S. franciscanus is found subtidally. Tide-pool isolation can lead to extreme pH variations (Wootton et al., 2008), and so it is possible that S. purpuratus has evolved to be adapted to broader ranges of pH. S. purpuratus also occurs at higher population densities relative to S. franciscanus. Consequently, sperm limitation is considered to be a greater issue for S. franciscanus, and is suggested to have led to larger egg size in this species (Levitan, 1993). The advantage of larger eggs for S. franciscanus is reduced with decreases in pH, as seen in this
Figure 3. pH observations (upper panel) made in the south La Jolla kelp forest throughout March 2013 at 17-m water depth. Details of deployment and instrumentation are provided in Frieder et al. (2012). Model of relative fertilization (lower panel) in Strongylocentrotus purpuratus (dotted line) and S. franciscanus (solid line) from ambient pH of 8.00 to in situ pH conditions observed during spawning season. Relative fertilization was calculated from observed pH values and Eq. 1 & 2.
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Hence, zygote production is dependent on both egg size and the efficiency of sperm to fertilize that egg. Effects of pH on this dependency could lead to evolutionary pressure for a species to optimize egg size in response to reductions in sperm performance. For S. franciscanus, there could be pressure from ocean acidification to increase its egg size. For S. purpuratus, the strong threshold in the fertilization response curve at pH ⬍7.60 could represent failure of a particular gamete trait to fulfill its role in fertilization. For example, this could represent a block in the fertilization potential of the egg, or a failure in the activation of the sperm or the acrosomal reaction. However, given these environmentally low ranges of pH at which fertilization is hindered, there is likely no evolutionary pressure from pH reduction on gamete traits of S. purpuratus. The role of sperm-egg ratios in regulating the magnitude of the pH effect on fertilization has been increasingly recognized. Both species under study exhibited reduced fertilization success, but only within species-specific sperm-egg ranges. This phenomenon has been documented for S. franciscanus by Reuter et al. (2011a), as well as for other sea urchins. For example, the Antarctic sea urchin Sterechinus neumayeri has robust fertilization down to pHNBS 7.30, and at these low levels the effect of pH on fertilization was observed only at a low sperm-egg ratio of 50:1 (Ericson et al., 2010; Ho et al., 2013). In another temperate sea urchin, Heliocidaris erythrogramma, no effect of pHNBS down to 7.6 on fertilization was observed even across a broad range of sperm-egg ratios (Byrne et al., 2010). Although those studies, and this one, conducted fertilization assays with gametes originating from many males and females (except Reuter et al., 2011a), the effect of pH across sperm-egg ratios can also depend upon male-female pairs (Sewell et al., 2014). For pH to depress fertilization during natural spawning events, sperm densities need to be limiting. Field experiments of induced spawning of S. franciscanus revealed fertilization success varying between 0% and 82% (Levitan et al., 1992). The degree to which sperm limitation occurs depends on the biotic and abiotic conditions under which spawning actually takes place (Levitan and Sewell, 1998; Yund, 2000). Adult sea urchins of the two species under study do not aggregate during natural spawning, and direct observations have witnessed 30% to 44% of individuals spawning at a time, with the majority spawning, 88%, being male (Levitan, 2002). Additionally, 98% of females of S. franciscanus produce embryos sired by multiple males (Levitan, 2004). Population density, especially male density, has also been shown to be a major determinant of sperm limitation (Levitan et al., 1992). The reduction of population densities of S. franciscanus by human exploitation could enhance sperm limitation during natural spawning events (Pfister and Bradbury, 1996), and would exacerbate reduced pH effects on fertilization success and result in
decreased population densities, an Allee effect. Given how important sperm-egg ratios are in regulating the magnitude of the pH effect and how human exploitation is reducing the levels of S. franciscanus, there is a need for fishery management to consider the interplay of present-day pH conditions, ocean acidification, and population densities. These factors could influence sustainable harvest levels. This study highlights the importance of sperm-egg ratios in modulating the effects of pH on fertilization success in echinoids, and demonstrates that two congeners coexisting in nature can differ greatly in their fertilization response to ocean acidification. This finding complicates the validity of generalized extrapolations about winners and losers based on shared characteristics of taxa. Given the degree of fertilization sensitivity to reduced pH in S. franciscanus, it is possible to identify time periods when present-day pH conditions could limit reproductive output during natural spawning events. In a more acidic ocean, maintaining fertilization output of this species will require adequate adult densities. Acknowledgments I thank A. Dickson for providing access to his laboratory facilities to run AT and salinity samples. Specimen collections were assisted by P. Zerofski and E. Kelly. I thank L. Levin, D. Holway, and J. Leichter for comments on earlier drafts of this manuscript, and V. Vaquier and A. Hamdoun for insightful discussions. This research was supported by NSF-OCE Award No. 0927445. Literature Cited Brokaw, C. J. 1990. The sea urchin spermatozoon. BioEssays 12: 449 – 452. Byrne, M. 2011. Impact of ocean warming and ocean acidification on marine invertebrate life history stages: vulnerabilities and potential for persistence in a changing ocean. Oceanogr. Mar. Biol. Annu. Rev. 49: 1– 42. Byrne, M. 2012. Global change ecotoxicology: identification of early life history bottlenecks in marine invertebrates, variable species responses and variable experimental approaches. Mar. Environ. Res. 76: 3–15. Byrne, M., N. Soars, P. Selvakumaraswamy, S. A. Dworjanyn, and A. R. Davis. 2010. Sea urchin fertilization in a warm, acidified and high pCO2 ocean across a range of sperm densities. Mar. Environ. Res. 69: 234 –239. Caldwell, G. S., S. Fitzer, C. S. Gillespie, G. Pickavance, E. Turnbull, and M. G. Bentley. 2011. Ocean acidification takes sperm back in time. Invertebr. Reprod. Dev. 55: 217–221. Chia, F. S., and L. R. Bickell. 1983. Echinodermata. Pp. 545– 620 in Reproductive Biology of Invertebrates, Vol. 2, K. G. Adiyodi and R. G. Adiyodi, eds. Wiley, New York. Christen, R., R. W. Schackmann, and B. M. Shapiro. 1983. Metabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration. J. Biol. Chem. 258: 5392–5399. Clayton, T. D., and R. H. Byrne. 1993. Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results. Deep-Sea Res. 40: 2115–2129.
This content downloaded from 132.234.251.230 on September 10, 2017 17:47:46 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
EFFECTS OF pH ON SEA URCHIN FERTILIZATION Desrosiers, R., J. De´silets, and F. Dube´. 1996. Early developmental events following fertilization in the giant scallop Placopecten magellanicus. Can. J. Fish. Aquat. Sci. 53: 1382–1392. Dickson, A. G., and F. J. Millero. 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 34: 1733–1743. Dickson, A. G., C. L. Sabine, and J. R. Christian, eds. 2007. Guide to best practices for ocean CO2 measurements. PICES Special Publication 3. Sidney, British Columbia. Ericson, J. A., M. D. Lamare, S. A. Morley, and M. F. Barker. 2010. The response of two ecologically important Antarctic invertebrates (Sterechinus neumayeri and Parborlasia corrugatus) to reduced seawater pH: effects on fertilisation and embryonic development. Mar. Biol. 157: 2689 –2702. Frieder, C. A. 2013. Evaluating low oxygen and pH variation and its effects on invertebrate early life stages on upwelling margins. Ph.D. dissertation, University of California, San Diego. Frieder, C. A., S. H. Nam, T. R. Martz, and L. A. Levin. 2012. High temporal and spatial variability of dissolved oxygen and pH in a nearshore California kelp forest. Biogeosciences 9: 3917–3930. Gonzalez-Bernat, M. J., M. D. Lamare, and M. Barker. 2012. Effects of reduced seawater pH on fertilisation, embryogenesis and larval development in the Antarctic seastar Odontaster validus. Polar Biol. 36: 235–247. Hauri, C., N. Gruber, M. Vogt, S. C. Doney, R. A. Feely, Z. Lachkar, A. Leinweber, A. M. P. McDonnell, M. Munnich, and G.-K. Plattner. 2013. Spatiotemporal variability and long-term trends of ocean acidification in the California Current System. Biogeosciences 10: 193–216. Havenhand, J. N., F.-R. Buttler, M. C. Thorndyke, and J. E. Williamson. 2008. Near-future levels of ocean acidification reduce fertilization success in a sea urchin. Curr. Biol. 18: R651–R652. Ho, M. A., C. Price, C. K. King, P. Virtue, and M. Byrne. 2013. Effects of ocean warming and acidification on fertilization in the Antarctic echinoid Sterechinus neumayeri across a range of sperm concentrations. Mar. Environ. Res. 90: 136 –141. Kurihara, H. 2008. Effects of CO2-driven ocean acidification on the early developmental stages of invertebrates. Mar. Ecol. Prog. Ser. 373: 275–284. Kurihara, H., and Y. Shirayama. 2004. Effects of increased atmospheric CO2 on sea urchin early development. Mar. Ecol. Prog. Ser. 274: 161–169. Levitan, D. R. 1993. The importance of sperm limitation to the evolution of egg size in marine invertebrates. Am. Nat. 141: 517–536. Levitan, D. R. 2002. Density-dependent selection on gamete traits in three congeneric sea urchins. Ecology 83: 464 – 479. Levitan, D. R. 2004. Density-dependent sexual selection in external fertilizers: variances in male and female fertilization success along the continuum from sperm limitation to sexual conflict in the sea urchin Strongylocentrotus franciscanus. Am. Nat. 164: 298 –309. Levitan, D. R., and M. A. Sewell. 1998. Fertilization success in freespawning marine invertebrates: review of the evidence and fisheries implications. Can. Spec. Publ. Fish. Aquat. Sci. 125: 159 –164. Levitan, D. R., M. A. Sewell, and F.-S. Chia. 1991. Kinetics of fertilization in the sea urchin Strongylocentrotus franciscanus: Interaction of gamete dilution, age, and contact time. Biol. Bull. 181: 371–378.
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Levitan, D. R., M. A. Sewell, and F.-S. Chia. 1992. How distribution and abundance influence fertilization success in the sea urchin Strongylocentrotus franciscanus. Ecology 73: 248 –254. Marshall, D. J. 2006. Reliably estimating the effect of toxicants on fertilization success in marine broadcast spawners. Mar. Pollut. Bull. 52: 734 –738. Mehrbach, C., C. H. Culberson, J. E. Hawley, and R. N. Pytkowicz. 1973. Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol. Oceanogr. 18: 897– 907. Morita, M., R. Suwa, A. Iguchi, M. Nakamura, K. Shimada, K. Sakai, and A. Suzuki. 2010. Ocean acidification reduces sperm flagellar motility in broadcast spawning reef invertebrates. Zygote 18: 103–107. Pfister, C. A., and A. Bradbury. 1996. Harvesting red sea urchins: recent effects and future predictions. Ecol. Appl. 6: 298 –310. Reuter, K. E., K. E. Lotterhos, R. N. Crim, C. A. Thompson, and C. D. G. Harley. 2011a. Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus. Glob. Change Biol. 17: 163–171. Reuter, K. E., K. E. Lotterhos, R. N. Crim, C. A. Thompson, and C. D. G. Harley. 2011b. Corrigendum: Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus. Glob. Change Biol. 17: 2512–2512. Riebesell, U., V. J. Fabry, L. Hansson, and J.-P. Gattuso, eds. 2010. Guide to Best Practices for Ocean Acidification Research and Data Reporting. Publications Office of the European Union, Luxembourg. Riffell, J. A., and R. K. Zimmer. 2007. Sex and flow: the consequences of fluid shear for sperm-egg interactions. J. Exp. Biol. 210: 3644 –3660. Schlegel, P., J. N. Havenhand, M. R. Gillings, and J. E. Williamson. 2012. Individual variability in reproductive success determines winners and losers under ocean acidification: a case study with sea urchins. PLoS ONE 7: e53118. Sewell, M. A., R. B. Millar, P. C. Yu, L. Kapsenberg, and G. E. Hofmann. 2014. Ocean acidification and fertilization in the Antarctic sea urchin Sterechinus neumayeri: the importance of polyspermy. Environ. Sci. Technol. 48: 713–722. Uthicke, S., D. Pecorino, R. Albright, A. P. Negri, N. Cantin, M. Liddy, S. Dworjanyn, P. Kamya, M. Byrne, and M. Lamare. 2013. Impacts of ocean acidification on early life-history stages and settlement of the coral-eating sea star Acanthaster planci. PLoS ONE 8: e82938. Vacquier, V. D. 1986. Activation of sea urchin spermatozoa during fertilization. Trends Biochem. Sci. 11: 77– 81. van Heuven, S. D., J. W. B. Pierrot, R. E. Lewis, and D. W. R. Wallace. 2011. MATLAB Program Developed for CO2 System Calculations, ORNL/CDIAC-105b. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. Vogel, H., G. Czihak, P. Chang, and W. Wolf. 1982. Fertilization kinetics of sea urchin eggs. Math. Biosci. 58: 189 –216. Wooton, J. T., C. A. Pfister, and J. D. Forester. 2008. Dynamic patterns and ecological impacts of declining ocean pH in a highresolution multi-year dataset. Proc, Natl. Acad. Sci. 105: 18848 – 18853. Yund, P. 2000. How severe is sperm limitation in natural populations of marine free-spawners? Trends Ecol. Evol. 15: 10 –13.
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Present-Day Nearshore pH Differentially Depresses Fertilization in Congeneric Sea Urchins CHRISTINA A. FRIEDER*,† Scripps Institution of Oceanography, La Jolla, California 92093
Abstract. Ocean acidification impacts fertilization in some species of sea urchin, but whether sensitivity is great enough to be influenced by present-day pH variability has not been documented. In this study, fertilization in two congeneric sea urchins, Strongylocentrotus purpuratus and S. franciscanus, was found to be sensitive to reduced pH, ⬍7.50, but only within a range of sperm-egg ratios that was species-specific. By further testing fertilization across a broad range of pH, pH-fertilization curves were generated and revealed that S. purpuratus was largely robust to pH, while fertilization in S. franciscanus was sensitive to even modest reductions in pH. Combining the pH-fertilization response curves with pH data collected from these species’ habitat demonstrated that relative fertilization success remained high for S. purpuratus but could be as low as 79% for S. franciscanus during periods of naturally low pH. In order for S. franciscanus to maintain high fertilization success in the present and future, adequate adult densities, and thus sufficient sperm-egg ratios, will be required to negate the effects of low pH. In contrast, fertilization of S. purpuratus was robust to a broad range of pH, encompassing both present-day and future ocean acidification scenarios, even though the two congeners have similar habitats.
tion, and population densities are known to influence fertilization success (Levitan et al., 1992; Marshall, 2006; Riffell and Zimmer, 2007; Byrne, 2011). pH is an additional driver that has garnered recent attention due to concerns about pH reductions resulting from ocean acidification in shallow marine settings (Kurihara, 2008; Byrne, 2011, 2012). Evidence from multiple studies suggests that fertilization is sensitive to pH in some echinoderms, but not all. Of the 17 species studied to date, 10 exhibited reduced fertilization with decreased pH, but some of these results occurred at extremely low pH values (e.g., between 7.0 and 7.4), or only at low sperm-egg ratios (e.g., Kurihara and Shirayama, 2004; Gonzalez-Bernat et al., 2012). Some of the observed robustness of fertilization to pH could be reflecting speciesspecific adaptations to natural pH variability in shallowwater habitats. On the other hand, some of the observed sensitivity could be great enough that fertilization is depressed during present-day low pH conditions. Few fertilization assays published to date employ more than two or three pH treatment levels, but the realization that pH varies with space and time requires that fertilization be studied over a broad range of pH to elicit linear and nonlinear relationships and potential thresholds (Riebesell et al., 2010). The ensuing pH-fertilization response curves will improve our ability to predict when and where the effects of a rapidly changing ocean climate will impact the sustainability of natural populations. The underlying mechanism responsible for reduced fertilization at lower pH could be changes in gamete properties. For example, reduced pH has been observed to decrease sperm motility in a variety of marine invertebrates including corals, sea urchins, sea cucumbers, and sea stars (Havenhand et al., 2008; Morita et al., 2010; Schlegel et al., 2012; Uthicke et al., 2013; but see Caldwell et al., 2011). Intracellular pH in sperm is directly dependent on extracellular pH, and decreasing intracellular pH narcotizes sperm
Introduction Successful fertilization is vital for the persistence of populations. In the marine environment, broadcast spawners release their gametes into the water column, and the released gametes are exposed to the chemical and physical properties of the seawater. Temperature, turbulence, polluReceived 25 November 2013; accepted 31 January 2014. * To whom correspondence should be addressed. E-mail: ctanner@ucsd. edu † Current address: Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371. 1
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(Chia and Bickell, 1983; Christen et al., 1983; Vacquier, 1986; Brokaw, 1990). pH could also affect properties of the egg. Low intracellular egg pH may prevent fertilization and subsequent development as well as slow the time that the fast block to polyspermy is established (Desrosiers et al., 1996; Kurihara, 2008; Reuter et al., 2011a). In studies of the effects of pH on fertilization success, appropriate species-specific sperm-egg ratios should be utilized (Levitan et al., 1991; Levitan, 1993; Reuter et al., 2011a; Byrne, 2012). Studying a range of sperm-egg ratios is also important because, in the field, population densities of adult sea urchins influence the degree of sperm limitation (Levitan, 2004). When testing pH effects at multiple spermegg ratios, a fertilization kinetics model can be generated in which the probability of fertilization is a function of spermegg ratio (Vogel et al., 1982). Such an approach has been utilized to explore the effects of reduced pH on fertilization in Strongylocentrotus franciscanus and revealed a 97% decrease in fertilization efficiency from pHNBS 8.04 to 7.55 (Reuter et al., 2011a, b). Additional studies have revealed a 63% decrease in fertilization efficiency in S. fragilis from pHT 7.99 to 7.50, but no change in fertilization efficiency in Dendraster excentricus from pHT 8.06 to 7.53 (Frieder, 2013). In this study, I compare the influence of pH on fertilization success in two species of echinoids from temperate environments. Strongylocentrotus purpuratus and S. franciscanus, the purple and the red sea urchins, inhabit rocky reefs and kelp forests. S. purpuratus has an egg diameter of 70 – 80 m and a sperm velocity of 145 m s–1; S. franciscanus has an egg diameter of 120 –140 m and a sperm velocity of 130 m s–1 (Levitan, 1993). The larger egg size of S. franciscanus equates to higher fertilization rates at a given sperm-egg ratio because larger eggs present a larger target for sperm (Levitan, 1993). Despite differences in gamete traits between these species, I predicted that S. purpuratus and S. franciscanus would exhibit similar fertilization responses to reduced pH given that they are phylogenetically related (congeners) and have similar presentday pH-exposure histories. Fertilization experiments were carried out at ambient and low pH at multiple sperm-egg ratios. I then developed pH-fertilization response curves for both species across a broad range of pH at a sperm-egg ratio that was observed to be sensitive to pH change during the prior experiment. These response curves were combined with present-day pH data collected during these species’ spawning season to model relative fertilization output. Materials and Methods
where there is extensive overlap of adults (32.85°N, 117.29°W). Adults were maintained in flow-through aquaria at ambient sea-surface temperature, 15 °C. All sea urchins were fed assorted macroalgae, and all specimens were used within a month of collection. Fertilization assays Spawning was induced by injecting 0.55 mol l–1 KCl through the peristomial membrane. Oocytes were collected in dishes of filtered (0.22 m) seawater. The number of adults used in each experiment ranged from 2 to 5 females and 3 to 5 males. Sperm were collected dry from the gonopores of males, placed in a small vial, and kept on ice until use. A 0.1% dilution from dry sperm was made to verify sperm motility, and then preserved in 1% formalin. Sperm densities were calculated with a hemocytometer to determine the amount of diluted sperm required to attain desired sperm-egg ratios and sperm densities for each treatment. Oocytes from multiple females were mixed, and egg densities were determined by counting aliquots of 8 ⫻ 20 l. Eggs were added to experimental beakers at a density of 5 eggs ml–1 and were incubated in experimental conditions for 10 min before sperm were added. Dry sperm was diluted immediately before addition to experimental beakers. After sperm addition the seawater in each replicate was gently stirred. Fertilization proceeded for 20 min and was arrested by transferring the contents of each replicate to 1% formalin. The fertilization ratio, defined as the proportion of eggs fertilized, was determined by counting the frequency of occurrence of a fertilization envelope in at least 100 eggs per replicate. Two types of experiments were carried out in the Scripps experimental aquarium between February and April 2013, the reproductive season of both species, following the above protocol. Glass beakers (500 ml) were used, and there were three replicates per treatment level. The first experiment had two treatments: pH and sperm-egg ratio. The two pH levels were ambient pH, ⬎7.96, and low pH, ⬍7.53 (Table 1). The sperm-egg ratios varied between species and were based on preliminary experiments. There were eight S. purpuratus sperm-egg ratios ranging from 1:1 to 2 ⫻ 104:1, and eight S. franciscanus sperm-egg ratios ranging from 1:1 to 2 ⫻ 103:1. The second experiment tested nine pH levels ranging from 7.26 to 8.00 on fertilization in both species. The sperm-egg ratios used in the second experiment was 103:1 for S. purpuratus, and 2 ⫻ 103:1 for S. franciscanus; these were sperm-egg ratios at which fertilization was sensitive to pH as revealed by the prior experiment.
Animal collection
Carbonate chemistry manipulation
Specimens of Strongylocentrotus purpuratus (Stimpson, 1857) and S. franciscanus (Agassiz, 1863) were collected at 15-m water depth by scuba from the La Jolla kelp forest
pH levels for each experiment were manipulated by mixing different proportions of ambient and high total dissolved inorganic carbon (CT) seawater. The high-CT reservoir was
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EFFECTS OF pH ON SEA URCHIN FERTILIZATION Table 1 Levels of pH treatment used during each experiment Expt.
Species
pH
AT
pCO2
1
Purple
7.96 7.37 7.99 7.50 7.95 7.88 7.80 7.72 7.62 7.54 7.46 7.38 7.26 8.00 7.94 7.84 7.78 7.69 7.61 7.54 7.44 7.36
2236 2239 2236 2239 2241 2240 2237 2238 2238 2239 2238 2236 2237 2235 2235 2235 2236 2236 2235 2235 2241 2237
495 2120 450 1578 501 607 736 920 1156 1427 1729 2081 2801 442 526 669 781 992 1203 1410 1797 2212
Red 2
Purple
Red
Experiment 1 tested fertilization success at high and low pH across multiple sperm-egg ratios. Experiment 2 developed fertilization response curves across a range of 9 pH values. pH is reported on the total scale, AT, total alkalinity in mol kg-1, pCO2 in atm calculated using CO2SYS software. Purple ⫽ Strongylocentrotus purpuratus; Red ⫽ S. franciscanus.
attained by bubbling a 3% pCO2 air mix into filtered seawater, and both reservoirs received flow-through filtered seawater. Experiment water conditions are provided in Table 1. All experiments were conducted at ambient seawater temperature during both species’ reproductive season, 15 °C, and temperature was maintained by submerging replicate containers within a common water bath. Discrete samples for determination of pH were taken for each treatment at the beginning of an experiment, and measured by a modified spectrophotometric method based on Dickson et al. (2007) using 1-cm cuvette cells and commercially available m-cresol dye with dye corrections based on Clayton and Byrne (1993). This method was calibrated with certified reference material from the Marine Physical Laboratory at Scripps Institution of Oceanography, and uncertainty of measurements was ⫾0.03 pH units. All pH values reported are at in situ temperature and on the total scale unless noted as pHNBS to indicate measurement on the NBS scale. Discrete samples were also taken for the determination of total alkalinity (AT) and salinity. AT for each treatment was determined using an open-cell, potentiometric titration with an accuracy of ⫾2 mol kg–1 (Dickson et al., 2007). Salinity was calculated from density measured at 20 °C on a density meter (Mettler Toledo DE45) with an
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accuracy of ⫾0.05 salinity units. Salinity during the experiments ranged from 33.53 to 33.59. Although only one discrete sample for determination of pH and AT was taken per treatment, an independent measure of pH was taken with the use of Honeywell Durafet III pH sensors to determine pH variance among replicates. The standard deviation among replicates was less than 0.02, and there was no significant drift in pH during the experiments. pCO2 conditions were calculated from pH and AT using the MATLAB version of CO2SYS (van Heuven et al., 2011) with dissociation constants from Mehrbach et al. (1973) as refitted by Dickson and Millero (1987). The average propagated uncertainties based on uncertainty in pH and AT for pCO2 was ⫾85 atm. Data analysis and statistics To determine the overall effect of pH and sperm-egg ratio on fertilization success, I used a two-way analysis of variance (ANOVA; fixed factors pH and sperm-egg ratio). Percentage data were square-root-arcsine transformed. Vogel’s fertilization kinetics model (Vogel et al., 1982) was applied to the fertilization data to calculate fertilization rate as a function of sperm-egg ratio, and to test whether pH resulted in decreased fertilization efficiency. From this model the fertilization efficiency, /o, can be estimated. /o reflects changes in gamete performance at different pH levels; these include, for example, the fertilizable area of the egg, the proportion of sperm able to fertilize an egg, or the number of sperm contacts needed to allow for the penetration of a single spermatozoan. For each pH treatment this parameter was estimated iteratively, along with 95% confidence intervals, using the Marquardt method of nonlinear regression in MATLAB. /o between pH treatments was considered significantly different if the 95% confidence intervals did not overlap. An index of gamete performance was calculated as the sperm density required to fertilize 50% of the eggs in each pH treatment (f50). pH conditions in a natural habitat for both species Continuous pH data were collected near bottom (3 m above bottom) in the south La Jolla kelp forest (32.81°N, 117.29°W), and the data were used as an estimation of natural conditions during spawning events. pH data were converted to relative fertilization success from the laboratory-derived relationship of fertilization success and pH for both species. This model assumed constant sperm-egg ratios and temperature. The instrument package utilized was a SeapHOx, which includes a Honeywell Durafet III pH sensor and an SBE-37 MicroCAT CTD. Details of instrumentation, deployment, and calibration are provided in Frieder et al. (2012). Data were collected during the spawning season of S. purpuratus and S. franciscanus from 1 to 31 March, 2013.
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C. A. FRIEDER Table 2
ANOVA F-ratios for analyses of effects of pH and sperm-egg ratios on fertilization ratios and the fertilization efficiency of two species of Strongylocentrotus Species
pH (df)
Sperm:egg (df)
pH ⫻ Sperm:egg (df)
Ambient pH /o (CI)
Low pH /o (CI)
S. purpuratus S. franciscanus
286 (1,32) 351 (1,32)
776 (7,32) 167 (7,2)
51 (7,32) 25 (7,32)
0.47 (0.11) 0.16 (0.02)
0.12 (0.04) 0.02 (0.002)
/o ⫾ 95% confidence intervals (CI), estimated from fertilization kinetics model at ambient pH and low pH. F-ratio values in bold indicate P values ⬍ 0.0001. df, degrees of freedom. Root-mean-square error values for fit of data to the model at ambient and low pH, respectively, are 0.104 and 0.161 for S. purpuratus, and 0.62 and 0.36 for S. franciscanus.
Results
Relative fertilization output under natural pH conditions
Sperm-egg ratios sensitive to low pH and effects on fertilization efficiency Fertilization success was reduced at low pH for both species, but at different sperm-egg ratios (Table 2). Fertilization was significantly lower at sperm-egg ratios from 100 to 1000:1 in Strongylocentrotus purpuratus, and 50 to 2000:1 in S. franciscanus (Fig. 1). To maintain its index of gamete performance (f50), S. purpuratus required 1.5 times the sperm density (with constant egg density) with a pH decline from 7.96 to 7.37, and S. franiscanus required 16 times the sperm density with a pH decline from 7.99 to 7.50. Fertilization efficiency, /o, was significantly lower at low pH for both species (Table 2). S. franciscanus exhibited an 86% reduction in /o from 7.99 to 7.50, and S. purpuratus exhibited a 74% reduction in /o from 7.96 to 7.37. Shape of pH response curve Both linear and nonlinear responses of fertilization success to reduced pH were observed (Fig. 2). S. purpuratus fertilization success exhibited a nonlinear response to pH at a sperm-egg ratio of 1000:1. At pH values greater than 7.60, fertilization success was above 90%; from pH 7.60 to 7.44, fertilization began to slowly decrease, but a precipitous threshold existed below pH of 7.44. A modified MichaelisMenten equation was fit to the response (RMSE ⫽ 0.0238).
S. purpuratus ⫽ 共pH ⫺ 7.25兲/共0.0116 ⫹ pH ⫺ 7.25兲 (Eq. 1) In contrast, S. franciscanus fertilization success decreased from pH 8.00 to 7.36 at a sperm-egg ratio of 2000:1. The effect of pH on the fertilization ratio, , was well described by a negative linear relationship (F1,7 ⫽ 204, P ⬍ 0.0001, r2 ⫽ 0.97).
S. franciscanus ⫽ 0.602 ⫻ pH ⫺ 3.86
(Eq. 2)
pH data were collected during March 2013, which coincides with the spawning seasons of S. franciscanus and S. purpuratus (Fig. 3). Fertilization success for both species at a given in situ pH was modeled from the regressions in Eq. 1 and 2 and plotted as a function of relative output from ambient pH 8.0 (Fig. 3). During this month, pH at depth was affected by alternating periods of a well-mixed versus a well-stratified water column, reflecting pH alternating from approximately 7.95 to 7.70 over week–long time periods. The corresponding relative fertilization output of S. franciscanus ranged between 0.79 and 0.93, while relative fertilization output of S. purpuratus was maintained above 0.95. Discussion The two sea urchin species studied had differing pHfertilization response curves despite the fact that they were congeners and were collected from the same habitat and presumably had similar histories of pH exposure. The Strongylocentrotus purpuratus fertilization threshold to pH was well below 7.6, suggesting that fertilization in this species is rather robust to pH in the context of present-day variability regimes and regional acidification scenarios (Frieder et al., 2012; Hauri et al., 2013). In contrast, S. franciscanus fertilization was sensitive to modest reductions of 0.06 pH units from 8.00, and these values are known to occur regularly within its present habitat during its reproductive season (Fig. 3). Present-day low pH conditions are capable of depressing fertilization output of S. franciscanus by up to 20%, and this value will increase linearly as pH continues to decrease with ocean acidification. Similar decreases in fertilization success have been observed for S. franciscanus despite methodological differences that included gamete concentrations, test vessel volume, experimental temperature, and use of single male-female pairs (Reuter et al., 2011a). Reuter and colleagues found that at 10 °C, fertilization efficiency, /o, decreased by 97%, from 400 to 1800 ppm. Similar magnitudes of decrease in fertilization efficiency were observed during this study (Table 2).
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EFFECTS OF pH ON SEA URCHIN FERTILIZATION
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Figure 2. Fertilization response curve to pH for Strongylocentrotus purpuratus (circles) and S. franciscanus (triangles). Sperm density used was 5 sperm l–1 and 10 sperm l–1 for S. purpuratus and S. franciscanus, respectively. Each point represents the mean and standard deviation of three replicates. Relationship between fertilization success and pH was linear in S. franciscanus and nonlinear in S. purpuratus.
study. This reduction could be due to differential sensitivity of male gamete traits such as sperm motility. Decreases in sperm motility due to reductions in pH are known to decrease fertilization efficiency (e.g., Havenhand et al., 2008).
Figure 1. Fertilization success as a function of sperm-egg ratio in ambient pH (circles) and low pH (triangles) conditions in (a) Strongylocentrotus purpuratus, and (b) S. franciscanus. Error bars represent the standard deviation of three replicates. Ninety-five percent confidence intervals on fertilization curve per pH treatment indicated by shaded region for ambient pH (dark gray) and low pH (light gray). Root-mean-square error values for fit of data to each model are provided in Table 2.
Drastic differences in the shape of the pH-fertilization response curve between the two species could be due to differing effects of pH on gamete traits, which in turn could be a result of habitat differences between the two species. For example, S. purpuratus can live in shallow water, often in tide pools, while S. franciscanus is found subtidally. Tide-pool isolation can lead to extreme pH variations (Wootton et al., 2008), and so it is possible that S. purpuratus has evolved to be adapted to broader ranges of pH. S. purpuratus also occurs at higher population densities relative to S. franciscanus. Consequently, sperm limitation is considered to be a greater issue for S. franciscanus, and is suggested to have led to larger egg size in this species (Levitan, 1993). The advantage of larger eggs for S. franciscanus is reduced with decreases in pH, as seen in this
Figure 3. pH observations (upper panel) made in the south La Jolla kelp forest throughout March 2013 at 17-m water depth. Details of deployment and instrumentation are provided in Frieder et al. (2012). Model of relative fertilization (lower panel) in Strongylocentrotus purpuratus (dotted line) and S. franciscanus (solid line) from ambient pH of 8.00 to in situ pH conditions observed during spawning season. Relative fertilization was calculated from observed pH values and Eq. 1 & 2.
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C. A. FRIEDER
Hence, zygote production is dependent on both egg size and the efficiency of sperm to fertilize that egg. Effects of pH on this dependency could lead to evolutionary pressure for a species to optimize egg size in response to reductions in sperm performance. For S. franciscanus, there could be pressure from ocean acidification to increase its egg size. For S. purpuratus, the strong threshold in the fertilization response curve at pH ⬍7.60 could represent failure of a particular gamete trait to fulfill its role in fertilization. For example, this could represent a block in the fertilization potential of the egg, or a failure in the activation of the sperm or the acrosomal reaction. However, given these environmentally low ranges of pH at which fertilization is hindered, there is likely no evolutionary pressure from pH reduction on gamete traits of S. purpuratus. The role of sperm-egg ratios in regulating the magnitude of the pH effect on fertilization has been increasingly recognized. Both species under study exhibited reduced fertilization success, but only within species-specific sperm-egg ranges. This phenomenon has been documented for S. franciscanus by Reuter et al. (2011a), as well as for other sea urchins. For example, the Antarctic sea urchin Sterechinus neumayeri has robust fertilization down to pHNBS 7.30, and at these low levels the effect of pH on fertilization was observed only at a low sperm-egg ratio of 50:1 (Ericson et al., 2010; Ho et al., 2013). In another temperate sea urchin, Heliocidaris erythrogramma, no effect of pHNBS down to 7.6 on fertilization was observed even across a broad range of sperm-egg ratios (Byrne et al., 2010). Although those studies, and this one, conducted fertilization assays with gametes originating from many males and females (except Reuter et al., 2011a), the effect of pH across sperm-egg ratios can also depend upon male-female pairs (Sewell et al., 2014). For pH to depress fertilization during natural spawning events, sperm densities need to be limiting. Field experiments of induced spawning of S. franciscanus revealed fertilization success varying between 0% and 82% (Levitan et al., 1992). The degree to which sperm limitation occurs depends on the biotic and abiotic conditions under which spawning actually takes place (Levitan and Sewell, 1998; Yund, 2000). Adult sea urchins of the two species under study do not aggregate during natural spawning, and direct observations have witnessed 30% to 44% of individuals spawning at a time, with the majority spawning, 88%, being male (Levitan, 2002). Additionally, 98% of females of S. franciscanus produce embryos sired by multiple males (Levitan, 2004). Population density, especially male density, has also been shown to be a major determinant of sperm limitation (Levitan et al., 1992). The reduction of population densities of S. franciscanus by human exploitation could enhance sperm limitation during natural spawning events (Pfister and Bradbury, 1996), and would exacerbate reduced pH effects on fertilization success and result in
decreased population densities, an Allee effect. Given how important sperm-egg ratios are in regulating the magnitude of the pH effect and how human exploitation is reducing the levels of S. franciscanus, there is a need for fishery management to consider the interplay of present-day pH conditions, ocean acidification, and population densities. These factors could influence sustainable harvest levels. This study highlights the importance of sperm-egg ratios in modulating the effects of pH on fertilization success in echinoids, and demonstrates that two congeners coexisting in nature can differ greatly in their fertilization response to ocean acidification. This finding complicates the validity of generalized extrapolations about winners and losers based on shared characteristics of taxa. Given the degree of fertilization sensitivity to reduced pH in S. franciscanus, it is possible to identify time periods when present-day pH conditions could limit reproductive output during natural spawning events. In a more acidic ocean, maintaining fertilization output of this species will require adequate adult densities. Acknowledgments I thank A. Dickson for providing access to his laboratory facilities to run AT and salinity samples. Specimen collections were assisted by P. Zerofski and E. Kelly. I thank L. Levin, D. Holway, and J. Leichter for comments on earlier drafts of this manuscript, and V. Vaquier and A. Hamdoun for insightful discussions. This research was supported by NSF-OCE Award No. 0927445. Literature Cited Brokaw, C. J. 1990. The sea urchin spermatozoon. BioEssays 12: 449 – 452. Byrne, M. 2011. Impact of ocean warming and ocean acidification on marine invertebrate life history stages: vulnerabilities and potential for persistence in a changing ocean. Oceanogr. Mar. Biol. Annu. Rev. 49: 1– 42. Byrne, M. 2012. Global change ecotoxicology: identification of early life history bottlenecks in marine invertebrates, variable species responses and variable experimental approaches. Mar. Environ. Res. 76: 3–15. Byrne, M., N. Soars, P. Selvakumaraswamy, S. A. Dworjanyn, and A. R. Davis. 2010. Sea urchin fertilization in a warm, acidified and high pCO2 ocean across a range of sperm densities. Mar. Environ. Res. 69: 234 –239. Caldwell, G. S., S. Fitzer, C. S. Gillespie, G. Pickavance, E. Turnbull, and M. G. Bentley. 2011. Ocean acidification takes sperm back in time. Invertebr. Reprod. Dev. 55: 217–221. Chia, F. S., and L. R. Bickell. 1983. Echinodermata. Pp. 545– 620 in Reproductive Biology of Invertebrates, Vol. 2, K. G. Adiyodi and R. G. Adiyodi, eds. Wiley, New York. Christen, R., R. W. Schackmann, and B. M. Shapiro. 1983. Metabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration. J. Biol. Chem. 258: 5392–5399. Clayton, T. D., and R. H. Byrne. 1993. Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results. Deep-Sea Res. 40: 2115–2129.
This content downloaded from 132.234.251.230 on September 10, 2017 17:47:46 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
EFFECTS OF pH ON SEA URCHIN FERTILIZATION Desrosiers, R., J. De´silets, and F. Dube´. 1996. Early developmental events following fertilization in the giant scallop Placopecten magellanicus. Can. J. Fish. Aquat. Sci. 53: 1382–1392. Dickson, A. G., and F. J. Millero. 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 34: 1733–1743. Dickson, A. G., C. L. Sabine, and J. R. Christian, eds. 2007. Guide to best practices for ocean CO2 measurements. PICES Special Publication 3. Sidney, British Columbia. Ericson, J. A., M. D. Lamare, S. A. Morley, and M. F. Barker. 2010. The response of two ecologically important Antarctic invertebrates (Sterechinus neumayeri and Parborlasia corrugatus) to reduced seawater pH: effects on fertilisation and embryonic development. Mar. Biol. 157: 2689 –2702. Frieder, C. A. 2013. Evaluating low oxygen and pH variation and its effects on invertebrate early life stages on upwelling margins. Ph.D. dissertation, University of California, San Diego. Frieder, C. A., S. H. Nam, T. R. Martz, and L. A. Levin. 2012. High temporal and spatial variability of dissolved oxygen and pH in a nearshore California kelp forest. Biogeosciences 9: 3917–3930. Gonzalez-Bernat, M. J., M. D. Lamare, and M. Barker. 2012. Effects of reduced seawater pH on fertilisation, embryogenesis and larval development in the Antarctic seastar Odontaster validus. Polar Biol. 36: 235–247. Hauri, C., N. Gruber, M. Vogt, S. C. Doney, R. A. Feely, Z. Lachkar, A. Leinweber, A. M. P. McDonnell, M. Munnich, and G.-K. Plattner. 2013. Spatiotemporal variability and long-term trends of ocean acidification in the California Current System. Biogeosciences 10: 193–216. Havenhand, J. N., F.-R. Buttler, M. C. Thorndyke, and J. E. Williamson. 2008. Near-future levels of ocean acidification reduce fertilization success in a sea urchin. Curr. Biol. 18: R651–R652. Ho, M. A., C. Price, C. K. King, P. Virtue, and M. Byrne. 2013. Effects of ocean warming and acidification on fertilization in the Antarctic echinoid Sterechinus neumayeri across a range of sperm concentrations. Mar. Environ. Res. 90: 136 –141. Kurihara, H. 2008. Effects of CO2-driven ocean acidification on the early developmental stages of invertebrates. Mar. Ecol. Prog. Ser. 373: 275–284. Kurihara, H., and Y. Shirayama. 2004. Effects of increased atmospheric CO2 on sea urchin early development. Mar. Ecol. Prog. Ser. 274: 161–169. Levitan, D. R. 1993. The importance of sperm limitation to the evolution of egg size in marine invertebrates. Am. Nat. 141: 517–536. Levitan, D. R. 2002. Density-dependent selection on gamete traits in three congeneric sea urchins. Ecology 83: 464 – 479. Levitan, D. R. 2004. Density-dependent sexual selection in external fertilizers: variances in male and female fertilization success along the continuum from sperm limitation to sexual conflict in the sea urchin Strongylocentrotus franciscanus. Am. Nat. 164: 298 –309. Levitan, D. R., and M. A. Sewell. 1998. Fertilization success in freespawning marine invertebrates: review of the evidence and fisheries implications. Can. Spec. Publ. Fish. Aquat. Sci. 125: 159 –164. Levitan, D. R., M. A. Sewell, and F.-S. Chia. 1991. Kinetics of fertilization in the sea urchin Strongylocentrotus franciscanus: Interaction of gamete dilution, age, and contact time. Biol. Bull. 181: 371–378.
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Levitan, D. R., M. A. Sewell, and F.-S. Chia. 1992. How distribution and abundance influence fertilization success in the sea urchin Strongylocentrotus franciscanus. Ecology 73: 248 –254. Marshall, D. J. 2006. Reliably estimating the effect of toxicants on fertilization success in marine broadcast spawners. Mar. Pollut. Bull. 52: 734 –738. Mehrbach, C., C. H. Culberson, J. E. Hawley, and R. N. Pytkowicz. 1973. Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol. Oceanogr. 18: 897– 907. Morita, M., R. Suwa, A. Iguchi, M. Nakamura, K. Shimada, K. Sakai, and A. Suzuki. 2010. Ocean acidification reduces sperm flagellar motility in broadcast spawning reef invertebrates. Zygote 18: 103–107. Pfister, C. A., and A. Bradbury. 1996. Harvesting red sea urchins: recent effects and future predictions. Ecol. Appl. 6: 298 –310. Reuter, K. E., K. E. Lotterhos, R. N. Crim, C. A. Thompson, and C. D. G. Harley. 2011a. Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus. Glob. Change Biol. 17: 163–171. Reuter, K. E., K. E. Lotterhos, R. N. Crim, C. A. Thompson, and C. D. G. Harley. 2011b. Corrigendum: Elevated pCO2 increases sperm limitation and risk of polyspermy in the red sea urchin Strongylocentrotus franciscanus. Glob. Change Biol. 17: 2512–2512. Riebesell, U., V. J. Fabry, L. Hansson, and J.-P. Gattuso, eds. 2010. Guide to Best Practices for Ocean Acidification Research and Data Reporting. Publications Office of the European Union, Luxembourg. Riffell, J. A., and R. K. Zimmer. 2007. Sex and flow: the consequences of fluid shear for sperm-egg interactions. J. Exp. Biol. 210: 3644 –3660. Schlegel, P., J. N. Havenhand, M. R. Gillings, and J. E. Williamson. 2012. Individual variability in reproductive success determines winners and losers under ocean acidification: a case study with sea urchins. PLoS ONE 7: e53118. Sewell, M. A., R. B. Millar, P. C. Yu, L. Kapsenberg, and G. E. Hofmann. 2014. Ocean acidification and fertilization in the Antarctic sea urchin Sterechinus neumayeri: the importance of polyspermy. Environ. Sci. Technol. 48: 713–722. Uthicke, S., D. Pecorino, R. Albright, A. P. Negri, N. Cantin, M. Liddy, S. Dworjanyn, P. Kamya, M. Byrne, and M. Lamare. 2013. Impacts of ocean acidification on early life-history stages and settlement of the coral-eating sea star Acanthaster planci. PLoS ONE 8: e82938. Vacquier, V. D. 1986. Activation of sea urchin spermatozoa during fertilization. Trends Biochem. Sci. 11: 77– 81. van Heuven, S. D., J. W. B. Pierrot, R. E. Lewis, and D. W. R. Wallace. 2011. MATLAB Program Developed for CO2 System Calculations, ORNL/CDIAC-105b. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. Vogel, H., G. Czihak, P. Chang, and W. Wolf. 1982. Fertilization kinetics of sea urchin eggs. Math. Biosci. 58: 189 –216. Wooton, J. T., C. A. Pfister, and J. D. Forester. 2008. Dynamic patterns and ecological impacts of declining ocean pH in a highresolution multi-year dataset. Proc, Natl. Acad. Sci. 105: 18848 – 18853. Yund, P. 2000. How severe is sperm limitation in natural populations of marine free-spawners? Trends Ecol. Evol. 15: 10 –13.
This content downloaded from 132.234.251.230 on September 10, 2017 17:47:46 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
