Ocean acidification but not warming alters sex determination in the Sydney rock oyster, Saccostrea glomerata

Abstract

Whether sex determination of marine organisms can be altered by ocean acidification and warming during this century remains a significant, unanswered question. Here, we show that exposure of the protandric hermaphrodite oyster, Saccostrea glomerata to ocean acidification, but not warming, alters sex determination resulting in changes in sex ratios. After just one reproductive cycle there were 16% more females than males. The rate of gametogenesis, gonad area, fecundity, shell length, extracellular pH and survival decreased in response to ocean acidification. Warming as a sole stressor slightly increased the rate of gametogenesis, gonad area and fecundity, but this increase was masked by the impact of ocean acidification at a level predicted for this century. Alterations to sex determination, sex ratios and reproductive capacity will have flow on effects to reduce larval supply and population size of oysters and potentially other marine organisms.

1. Background

Sex determination and the resulting sex ratios are critically important for the viability and success of populations [1–3]. A reduction in the number of females, for example, will directly reduce offspring supply and could affect recruitment into the population [4–6]. A reduction in the number of males may limit the number of mates, and a reduction in either sex may limit the genetic variability of the population leading to population bottlenecks and inability to respond to ecoevolutionary change [4,5,7,8]. In many marine organisms, sex is determined at or before conception and remains constant throughout life [9]. For other marine organisms, however, sex is not fixed and at critical stages during development can be influenced by the environment [9,10].

Whether sex determination of marine organisms can be altered by the combined effects of ocean acidification and warming during this century remains a significant, unanswered question. It is predicted that by 2100 surface ocean pH levels will decrease by 0.4 pH units and surface ocean temperatures will warm by 1.8–4°C [11,12]. Molluscs have already been shown to be particularly sensitive to ocean acidification and warming [13,14]. As a group, molluscs exhibit extreme diversity in their modes of sexual reproduction ranging from strict gonochorism, where individuals produce only one type of gamete (eggs or sperm) for their entire life, to hermaphroditism where individuals can produce both types of gametes, either simultaneously or sequentially. For many species of bivalves including oysters, protandric hermaphroditism is the norm, with juveniles maturing first as males and changing to females later in life [10]. Sex determination in these species is governed by an interaction between genetic and environmental factors [10,15]. In many species, a single locus model with a dominant maleness (M) allele and a protandric femaleness (F) allele is the primary determinant of sex [10,16]. MF genotypes are true males that do not change sex, whereas FF genotypes are protandric females, maturing first as males before changing sex later in life [10]. The rate at which these protandric females change sex is thought to be regulated by a combination of secondary genes and environmental factors such as temperature [17], salinity [18], food concentration [19], photoperiod [20], as well as chemical pollutants [10,20–24].

Questions have already been raised about whether ocean acidification may impact sex ratios [25]. Adults of the gastropod Hexaplex trunculus sampled from a naturally acidified field location in Levante Bay, Italy, were found to have a lower proportion of females (32.26%) compared to males (67.64%) [25]. Further, adults of the eastern oyster Crassostrea virginica were also found to have a higher proportion of males to females compared to the control group during a 5-week laboratory exposure to a severe elevated pCO₂ concentration of 18 480 µatm but not at 2260 µatm (micro-atmosphere) [26]. Whether sex determination can be altered in marine organisms by the combined effects of ocean acidification and warming predicted for the end of this century is yet to be determined and is critical for predicting how populations will respond over this century [27].

Here, we tested the hypothesis that ocean acidification and warming (pCO₂: 856 µatm; temperature: +4°C) will alter the sex determination of the Sydney rock oyster, Saccostrea glomerata, which is a protandric hermaphrodite species with a low frequency of simultaneous hermaphroditism (less than 1%) [28]. It forms the basis of a large aquaculture industry on the southeast coast of Australia which is a climate change hotspot [29] where in addition to ocean acidification, near-coastal sea surface temperatures are predicted to rise by 2–4°C by 2090 based under RCP8.5 relative to 1986–2005 temperatures [12]. We assessed the rate of gametogenesis, gonad area, fecundity, egg quality (total lipid content and size), extracellular pH_e, shell length and mortality, sex determination and the resulting sex ratio.

2. Material and methods

(a). Collection and pCO₂ and temperature incubation

Two-year-old wild-caught adult Sydney rock oysters, S. glomerata (n = 1016; age determined from time of spat settlement) were collected from the Hastings River, New South Wales (NSW), Australia, in winter 2015 and were transferred to the hatchery at the Port Stephens Fisheries Institute (PSFI), Taylors Beach, NSW, Australia. Adults were selected when they were 2 years of age as this is the time when sex ratios are close to 50 : 50. Oysters were placed in damp hessian bags and transferred to the hatchery at the PSFI, Taylors Beach, NSW, Australia. Upon arrival at PSFI, a random subset of 28 oysters was shucked and a gonadal sample was collected for histological assessment and confirmation of reproductive quiescence. The remaining oysters were divided equally across 12 40 l trays supplied by continuously flowing, recirculating seawater from individual 750 l header tanks (flow rate 3 l min⁻¹). Oysters were acclimated in the trays at the field collection temperature of 18°C for 2 weeks (salinity 34.2). Seawater was collected from Little Beach (152°07′ E, 32°72′ S), Nelson Bay, NSW, Australia, and was filtered through 1 µm nominal filter bags before delivery into the hatchery. All pH was measured on the NBS (National Bureau of Standards) scale.

Two pCO₂ levels were used in the exposure: a current ambient atmospheric pCO₂ level of 392 µatm (control; pH_NBS 8.20) and an elevated atmospheric pCO₂ level predicted for 2100 of 856 µatm (pH_NBS 7.91). Further, two temperature levels were used in the experiment: the current optimal reproductive conditioning temperature of 24°C and an elevated temperature of 28°C representing a + 4°C warming. At the end of the 2-week acclimation period, the pCO₂ level in six of the 750 l header tanks was slowly increased (pH of seawater reduced by −0.05 pH units per day) until the desired pCO₂ level was reached. pCO₂ was obtained by negative feedback pH computers (Aqua Medic, Aqacenta Pty Ltd, Kingsgrove, NSW, Australia) which controlled the pCO₂ level in each tank by adjusting the pH. See Parker et al. [30,31] for further details. pH_NBS was measured daily in each of the tanks using a Wissenschaftlich-Technische-Werkstätten (WTW) combined Multi meter (3420) and combined electrode (SenTix940) and total alkalinity was measured in triplicate at each water change using Gran titration. To determine the pH value to set the tanks at, pH_NBS, total alkalinity (TA), temperature and salinity, and the desired elevated pCO₂ levels were entered into the CO₂sys calculation programme developed by Lewis and Wallace [32], using the dissociation constants of Mehrbach et al. [33]. Seawater physico-chemical properties can be found in electronic supplementary material, table S2.

The adults were allowed to acclimate to the elevated pCO₂ levels for 1 week. Following this acclimation, the temperature of the tanks was gradually increased from the acclimation temperature of 18°C to 24°C (control temperature) and 28°C (+4 warming) (0.5°C and 1°C increase per day for the control and warming temperature treatment, respectively), to trigger gonadal development. A second sample of gonadal tissue was taken prior to the temperature increase to ensure that no gonadal development occurred during the acclimation period. Temperature was controlled in each header tank by individual 1000 W aquarium heaters (Aquasonic, Australia). Adults were fed a combined algal diet of 50% Chaetoceros muelleri, 25% Pavlova lutheri and 25% Tisochrysis lutea twice daily at a concentration of 2 × 10⁹ cells oyster⁻¹ day⁻¹. The 40 l oyster trays were drained and cleaned daily and a complete water change of each header tank was made every second day. The oysters remained in the treatments for 8 weeks.

(b). Oyster sampling and measurement

(i). Histology—sex determination, stage of gametogenesis and gonad area

At 0, 3, 5, 7 and 8 weeks a gonadal sample was taken from seven oysters in each tank (21 oysters treatment⁻¹) for histology. The oysters selected were sacrificed and not returned to the tanks. There were 82 oysters per tank at the beginning of the experiment and 47 oysters per tank at the end of the experiment. A transverse cross-section of tissue was cut from the anterior of the sampled oysters through the gonad, stomach, digestive diverticular, intestine and labial palps at the point where the gills are intersected by the palps [16,34]. A second transverse cut was made 4–5 mm below the first. The resulting tissue was placed immediately in Davidson's fixative for 48 h, transferred into 50% ethanol for 2 h and stored in 70% ethanol before being embedded in paraffin. Of note, 5 µm sections of embedded tissue were cut and mounted on acid-washed glass and were stained with haematoxylin and eosin. Slides were examined under a compound microscope (Olympus × 40 to ×250) to determine oyster sex and stage of gonadal development.

Oyster sex and stage of gonadal development were determined according to Dinamani [28]. Briefly, seven stages of gonadal development are recognized which fall into five distinct phases. Phase I consists of the ripening period (G1–G3); Phase II identifies oysters that are fully ripe (G4); Phase III consists of the post-spawning stage with many follicles discharged and residual gametes present (G5); Phase IV is the early regressive stage, displaying collapsed follicles, large numbers of phagocytes, with occasional gametes (Gx); and Phase V is the main regressive stage consisting of gonadal cells that are indifferent.

Following microscopic examination of sex and gonadal development, the proportion of stomach area that was occupied by gonad tissue was determined by quantitative histology [34–36]. Each slide was scanned at a resolution of 2400 dots per inch (dpi) (Hewlett Packard Office Jet 7410) and uploaded into Adobe Photoshop CS6. Using the marquee property function the boundaries surrounding the visceral mass and the area occupied by gametes were selected. The number of picture elements in each selected section was determined by the histogram analytical tool and the proportion of the visceral mass that was occupied by gametes was used to determine the percentage gonad area [34].

(ii). Spawning—percentage spawning, total lipid content of eggs, egg size and fecundity

Following 8 weeks in the treatments (from the first day of temperature increase) the oysters were naturally induced to spawn. Briefly, 20 adults from each replicate were placed in shallow spawning tanks filled with filtered seawater (FSW) set at their conditioning temperature (24 or 28°C) and pCO₂ level (392 or 856 µatm). Using a submerged heater, the water in the tanks was gradually increased by 4°C and the oysters were left for 15 min. Freshwater was then added to each tank to reduce the salinity from 34.2 to 25 and adults were left for 15 min. Finally, the FSW was drained from the tanks and the oysters were left out of water for 15 min. This cycle was repeated a further three times (four spawning cycles in total). Spawning individuals were rinsed thoroughly and placed in individual 500 ml containers of FSW, set at the same temperature and pCO₂ level that the adult was conditioned at, to continue spawning. Once the adult had finished spawning it was immediately removed from the container. The total number of individuals that spawned in each replicate tank was recorded and expressed as spawning success.

Eggs from three females from each replicate (nine females treatment⁻¹) were rinsed with FSW through a 60 µm nylon mesh to remove debris. Three 0.5 ml well-homogenized subsamples from each batch of eggs were placed on separate Sedgwick Rafter slides and observed under a compound microscope (Olympus 100×). The total number of eggs on each slide was counted to determine the mean spawned fecundity of each female and the diameter of 30 eggs was measured to determine mean egg size.

Following the fecundity and egg size measurements, a minimum of 5000 eggs from each female (nine females treatment⁻¹) were collected, immediately frozen in liquid nitrogen and stored at −80°C awaiting analysis of total lipid content. Using methods modified from Bligh & Dyer [37], egg samples were quantitatively extracted overnight using one-phase methanol–chloroform–water extraction (2 : 1 : 0.8 v/v/v). The phases were separated by the addition of chloroform–water (final solvent ratio, 1 : 1 : 0.9 v/v/v methanol–chloroform–water). The total solvent extract (TSE) was concentrated using rotary evaporation at 40°C. Total lipid content was determined gravimetrically after evaporation of CHCl₃ under a stream of nitrogen until dry.

(iii). Shell length, extracellular pH and mortality

To determine whether elevated pCO₂ and temperature had effects on physiological processes other than reproduction, shell length, extracellular pH (pH_e) and mortality of the oysters was determined after 8 weeks. Prior to spawning, the shell length (antero-posterior measurement) of the oysters remaining in each tank was measured. Two oysters were shucked from each replicate tank (6 oysters treatment⁻¹) and 0.2 ml of haemolymph was extracted from the pericardial cavity using a 1 ml needle syringe. Following the extraction the samples were transferred immediately into a 0.5 ml Eppendorf tube and the pH_e of the sample was measured using a micro pH probe (Metrohm 827 biotrode; calibrated with NBS standards). Mortality was recorded daily throughout the experiment.

(iv). Data analysis

To ensure that differences in the ratios of female to male oysters under elevated pCO₂ and temperature occurred due to differential sex determination and not differential mortality between males and females, a three-factor generalized linear mixed model (GLMM) was run using data from the ratio of females to males obtained from histology slides and a second GLMM was run with this data combined with the cumulative mortalities that occurred during the 8-week exposure to the treatments. Since the sex of the mortalities could not be determined for every oyster, any unknown sex was treated as female for the control group and as a male for all other treatment groups. This was done to reflect the ‘worst’ possible combinations. Sex ratio data were analysed by GLMM using a binomial distribution and a logit function; ‘Temperature’ and ‘pCO₂’ were fixed and orthogonal factors and ‘Tank’ was random and nested in the other two factors. The random ‘Tank’ factor was found to explain little variance (Var < 0.001). The ratio of oysters that spawned compared to those that didn't spawn were analysed using a two factor generalized linear model (GLM) with ‘Temperature’ and ‘pCO₂’ as fixed factors also using a binomial distribution and a logit function. The ‘ANOVA’ function (CAR package) was then used to conduct Wald (type II, χ²) tests of analysis of deviance on linear models to determine p values. Parson's residual plots were used to check the normality of data and the residual deviance and residual degrees of freedom were used to check for over-dispersion (Resid.Dev/Resid.Df for all analyses was < 1.5). All binomial analyses were done using the LMERTEST package in R software version 3.4.0 (R Development Core Team 2015) following the methods outlined by Wilson and Hardy [38].

To determine differences in stages of gonadal development the ‘multinom’ function in the NNET package on R software version 3.4.0 (R Development Core Team 2015) was used to conduct multinomial logistic regression with the factors ‘Week’, ‘Temperature’ and ‘pCO₂’. Once multinomial regression models were created, the ‘ANOVA’ function (STATS package) was used to conduct likelihood ratio tests among multinomial models to determine differences among factors. Multinomial models were created and analysed following the methods of Agresti [39] outlined for categorical data analysis.

Differences between elevated pCO₂ and temperature on the size and total lipid content of eggs, fecundity, pH_e, shell length, and mortality, were determined using a two-factor Analysis of Variance (ANOVA). Differences between elevated pCO₂ and temperature on the gonad area data were analysed at each time point (0, 3, 5, 7, 8 weeks) using a three-factor ANOVA. The mean value for each tank was used as the observation, with a resulting sample size of n = 3. Cochran's test was used to test for heterogeneity of variances in the data and the Student Newman Keuls (SNK) test was used to detect differences among means [40] using the software GMAV5 [41], for all ANOVA analyses.

3. Results

(a). Sex determination, stage of gametogenesis and gonad area

Adult sex determination was influenced by elevated pCO₂, but not temperature (table 1; figure 1). Our generalized mixed model found elevated pCO₂ was positively related to an increased ratio of female oysters (Z = 2.16, p = 0.023), however, temperature had no significant effect on sex ratios (p = 0.398) and the treatments did not interact (p = 0.429). The percentage of females in the ambient pCO₂ and temperature treatment was 61.73 ± 6.53%. This proportion significantly increased in the elevated pCO₂ treatments to 77.54 ± 5.58% and 77.78 ± 3.70% at 24°C and 28°C, respectively. When data were analysed including mortalities assumed in a ‘worst-case scenario’, we found that pCO₂ was still positively related to an increase in female oysters (Z = 1.77, p = 0.072; electronic supplementary material, table S1, and figure S1).

Table 1.

Sex determination, reproductive capacity and physiology of the Sydney rock oyster, S. glomerata following 8 weeks of exposure to ambient (392 µatm; 24°C) and elevated (856 µatm; 28°C) pCO₂ and temperature during reproductive conditioning; salinity = 34.5; n = 3; ±s.e. Asterisks indicate a significant difference. For all data in the table there were significant effects of elevated pCO₂ and/or temperature but no interaction between factors.

	24°C		28°C
	392 µatm	856 µatm	392 µatm	856 µatm
females (%)	61.73 ± 6.53	77.54 ± 5.58*	70.24 ± 1.19	77.78 ± 3.70*
individuals that spawned (%)	78.33 ± 4.41	31.67 ± 8.82***	75.00 ± 7.64	43.33 ± 12.02***
fecundity (×106)	6.53 ± 1.28	2.71 ± 0.73**	12.30 ± 1.25***	9.03 ± 0.14***
egg diameter (µm)	50.92 ± 0.39	49.71 ± 0.45	48.06 ± 0.53***	47.44 ± 0.10***
total lipid content of eggs (ng)	6.79 ± 0.72	6.14 ± 1.01	4.48 ± 0.34**	2.76 ± 0.69**
total spawned lipid content gonad−1 (mg)	45.19 ± 11.07	15.66 ± 1.99**	55.34 ± 8.11	25.03 ± 6.43**
pHe w8	7.46 ± 0.02	7.01 ± 0.05*	7.42 ± 0.01	7.00 ± 0.04*
shell length (µm)	52.24 ± 0.36	51.85 ± 0.05*	49.50 ± 0.84***	46.76 s ± 0.71*
mortality (%)	0.43 ± 0.43	2.99 ± 0.43**	3.42 ± 0.85**	4.70 ± 0.43**

*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 1.

The percentage of S. glomerata adults that were female and male following exposure to ambient (392 µatm; 24°C) and elevated (856 µatm; 28°C) pCO₂ and temperature during reproductive conditioning; error bars = s.e.m.; n = 3.

There was a significant interaction effect between ‘week × temperature’ (LR = 24.3, p = 0.059) and ‘pCO₂ × temperature’ (LR = 11.2, p = 0.047) on the stage of gametogenic development (electronic supplementary material, table S3). Generally, under ambient pCO₂ the rate of gonadal development was faster in the 28°C treatment compared with the 24°C treatment, with more oysters observed to be at the ripe stage (G4) at 28°C (figure 2). By week 8, however, this effect of temperature was no longer apparent with the number of oysters in each stage of gonadal development being similar across the temperature treatments (figure 2). Under elevated pCO₂ the rate of gonadal development was similar at 24 and 28°C across each week. Finally, the rate of gonadal development was faster at ambient compared with elevated pCO₂ (figure 2). Histological analysis at week 8 showed that the appearance of fully gravid female gonads differed across the CO₂ and temperature treatments (figure 3).

Figure 2.

The percentage of S. glomerata adults at each gonadal stage following exposure to the ambient (392 µatm; 24°C) and elevated (856 µatm; 28°C) pCO₂ and temperature treatments during reproductive conditioning. Gonadal samples taken at 0, 3, 5, 7 and 8 weeks; n = 3. Stage I consists of the ripening period (G1–G3); Stage II identifies oysters that are fully ripe (G4); Stage III consists of the post-spawning stage with many follicles discharged and residual gametes present (G5); Stage IV is the early regressive stage, displaying collapsed follicles, large numbers of phagocytes, with occasional gametes (Gx); and Stage V is the main regressive stage consisting of gonadal cells that are indifferent.

Figure 3.

Histological images of the transverse cross-section of the S. glomerata female oyster gonad showing differences in the appearance of the gonad at the fully ripe stage (stage G4) following exposure to (a) ambient pCO₂ and temperature (392 µatm; 24°C), (b) elevated pCO₂ and ambient temperature (856 µatm; 24°C), (c) ambient pCO₂ and elevated temperature (392 µatm; 28°C), and (d) elevated pCO₂ and elevated temperature (856 µatm; 28°C) for 8 weeks during reproductive conditioning 100×. Scale bar = 100 µm. In figures a, b and d follicles contain less eggs than figure c and are surrounded by a greater amount of connective tissue.

The gonad area of S. glomerata also differed with elevated pCO₂ × temperature × week (F_4,40 = 4.88, p = 0.0027; figure 4). Elevated pCO₂ decreased the gonad area at both experimental temperatures (24°C by week 7; 28°C by week 5). Gonad area was greater in oysters reared at elevated temperature at weeks 5–7, but by week 8 this difference in gonad area was no longer present.

Figure 4.

The percentage gonad area in S. glomerata adults following exposure to ambient (395 µatm; 24°C) and elevated (856 µatm; 28°C) pCO₂ and temperature treatments during reproductive conditioning. Gonadal samples taken at 0, 3, 5, 7 and 8 weeks; error bars = s.e.m.; n = 3.

(b). Percentage spawning, total lipid content of eggs, egg size and fecundity

The percentage of individuals which spawned was negatively correlated with the elevated pCO₂ treatment (Z = −4.908, p < 0.001). We found that 77% of oysters spawned in the ambient pCO₂ treatments compared with 38% in the elevated pCO₂ treatments (table 1). There were no effects of temperature (p = 0.47) and the treatments did not interact (p = 0.233). The percentage of individuals that spawned closely matched the proportion of oysters observed during histological analysis to be at the ripe stage (G4 stage; table 1; figure 2).

The size and total lipid content of eggs was significantly lower at 28°C compared with 24°C (egg size: F_1,8 = 34.06, p = 0.0042; total lipid content: F_1,8 = 15.33, p = 0.0044; table 1) but was not affected by elevated pCO₂. Calculation of the total lipid content of all spawned eggs (total lipid content per egg × fecundity) showed that there was a significant effect of elevated CO₂ on the total spawned lipid content per female oyster but no effect of temperature (F_1,8 = 15.32, p = 0.0045; table 1). Overall, the total lipid content of eggs was 57% lower at elevated pCO₂ and temperature compared to the ambient treatments (table 1).

While egg size and total lipid content decreased at elevated temperature, fecundity increased (F_1,8 = 40.77, p = 0.0002; table 1). On average, the spawned fecundity of oysters reared at an elevated temperature (28°C) was 40% greater than that of oysters reared at the control temperature (24°C). Fecundity was also significantly greater in oysters reared at ambient compared with elevated pCO₂ (F_1,8 = 14.05, p = 0.0056; table 1). The total lipid content of all spawned eggs (total lipid content per egg × fecundity) was greater at ambient compared with elevated pCO₂ (F_1,8 = 15.32, p = 0.0045; table 1).

(c). Shell length, extracellular pH, and mortality

There was an effect of elevated pCO₂ and temperature but no interaction on the shell length and mortality of adult oysters. Shell length was greater at ambient compared with elevated pCO₂ (F_1,8 = 7.28, p = 0.0272; table 1) and at 24°C compared with 28°C (F_1,8 = 45.81, p = 0.0001; table 1). Mortality of oysters was relatively low throughout the 8-week exposure with a maximum mortality of 4.70 ± 0.43% (28°C, 856 µatm). Mortality was increased at elevated compared with ambient pCO₂ (F_1,8 = 11.57, p = 0.0093; table 1) and at 28°C compared with 24°C (F_1,8 = 17.29, p = 0.0032; table 1). Finally, extracellular pH (pH_e) was decreased at elevated compared with ambient pCO₂ (F_1,8 = 167.48, p = 0.0000; table 1). No effect of temperature was observed.

4. Discussion

(a). Altered sex determination

Here, we found that ocean acidification, but not warming altered sex determination in S. glomerata, leading to a significant change in the population sex ratio. Following 8 weeks of exposure to elevated pCO₂ (at both 24 and 28°C) there were 16% more females compared to the control group (392 µatm; 24°C). This result is in contrast to the only other study to consider the impact of ocean acidification on sex determination in an oyster species to date [26]. When adults of the oyster, C. virginica were reared at a severe level of ocean acidification (18 480 µatm) for 5 weeks they displayed a greater proportion of males to females compared to the control group. This change in sex ratio, however, was based on the response of only five oysters as no other oysters showed evidence of gonadal development in this severe acidification level. When C. virginica were exposed to an ocean acidification level projected for the end of the century, no change in sex ratios were observed.

In a wide range of marine organisms the cost of producing eggs is greater than the cost to produce sperm [27]. For this reason, it is hypothesized that suboptimal environmental conditions would favour males because they required less energy. Indeed in many oyster species studied to date males have been found to predominate when energetic reserves are low [42–44]. Honkoop et al. [45] found, however, that for S. glomerata the cost of reproduction does not differ between males and females. As a result it is unlikely that an energetic cost hypothesis influenced sex determination of S .glomerata in this study.

Burkenroad [46] found sex determination in an individual oyster to be influenced by chemical cues released from surrounding oysters. Populations of oysters in close proximity (less than 40 mm apart) had a female to male sex ratio of 1 : 1, whereas, isolated oysters had a sex ratio of 3.9 : 1. The oysters in our study were held in close proximity, yet had a skewed sex ratio of 2.3 : 1 when exposed to elevated pCO₂. Perhaps these oysters lost the ability to detect/respond to the chemical cues released from surrounding oysters leading to altered sex determination. An inability to respond to chemical cues during exposure to ocean acidification has previously been reported in other marine organisms, including fish and molluscs [47–48].

Finally, altered sex determination of S. glomerata in this study may have occurred due to a sex-specific difference in response to ocean acidification. The response of an organism to ocean acidification can differ significantly between males and females [49–51]. For example, females of the tropical sea urchin Echinometra mathaei are impacted less by ocean acidification than males, with females having a higher gonad index and ability to spawn [49]. Further, in molluscs, exposure to ocean acidification resulted in sex-based differences in the metabolome of the mussel Mytilus edulis [50] and biochemical composition of the limpet Nacella concinna gonad [51]. Female S. glomerata may respond better to ocean acidification than males (e.g. better maintenance of pH_e and/or metabolic rate), therefore, making it more beneficial to be female. Such sex-specific differences in the response of S. glomerata to ocean acidification require further investigation.

(b). Reduced reproductive capacity

Following 8 weeks of conditioning, there was a significant delay in the rate of gametogenesis with fewer oysters reaching the ripe stage and successfully spawning in the elevated pCO₂ treatments compared with the ambient pCO₂ controls (at both 24 and 28°C). Delayed gametogenesis was observed in the oyster C. virginica during exposure to severe elevated pCO₂ concentrations of 5584 and 18 480 µatm but not at 2260 µatm [26]. A delay in the rate of gametogenesis, and thus delayed spawning dates, may result in a mismatch in the timing of larval release and peak phytoplankton blooms, and also reduce the number of reproductive cycles possible in a given season [52]. As a result, both larval food availability and larval supply may be reduced. A delay in the rate of gametogenesis will also reduce the marketability of S. glomerata, which is dependent on reproductive condition [16]. Exposure to ocean acidification had no impact on the energy which was invested per egg (measured via egg size and total lipid content), however, fecundity was reduced by up to 58%, suggesting that less energy was invested per gonad. Similar reductions in fecundity have been reported in marine organisms other than oysters during exposure to ocean acidification [53]. This is likely the result of a reallocation of energy away from reproduction to help maintain acid–base balance, growth and survival [54], all of which were significantly reduced in S. glomerata following exposure to elevated pCO₂.

Elevated temperature (+4°C) as a sole stressor, had a slightly positive impact on the reproductive capacity of S. glomerata. Under ambient pCO₂, oysters that were conditioned at 28°C had a faster rate of gametogenesis with 57% of oysters reaching the gravid stage by week 5 compared with 0% in the 24°C control. Further, the fecundity of the oysters conditioned at 28°C was nearly double that of the control oysters. This was accompanied, however, by a significant reduction in the size and total lipid content of each egg, suggesting that in response to warming the oysters reallocated energy to produce more eggs with smaller size/lower nutritional content. Reduced egg size is a common response of marine organisms during exposure to elevated temperature [52] believed to be driven by higher energetic demands and thus lower energy available for reproduction. Increased fecundity, however, is not a common response, with most studies reporting reduced egg production during exposure to elevated temperature [53]. Such an opposing response of S. glomerata in this study may improve their chances of population success in a warming ocean. Previous studies have suggested that the optimal larval ‘strategy’ for planktotrophic species under stress is to produce the maximum number of eggs, each having minimal energy reserves [55,56]. Conversely, however reduced egg size has a negative effect on growth and survival in the resulting offspring [57]. Further research is needed to assess the impact of reduced egg size and total lipid content on the response of S. glomerata larvae.

5. Conclusion

Successful reproduction is essential for the sustainability of marine populations, ensuring the replenishment of loses that result from mortality [8]. Here, we show that sex determination in the ecologically and economically important Sydney rock oyster is altered under predicted levels of future ocean acidification, resulting in more females. Exposure of S. glomerata to elevated pCO₂ during reproductive conditioning also caused delayed gametogenesis and reduced fecundity, both of which have the capacity to lower larval supply and cause population decline. The female biased sex ratios found in this study during exposure to elevated pCO₂, may partially alleviate these impacts, with a higher number of females linked to increased larval supply and recruitment into the population [4,5]. If the number of females becomes too high, however, there may no longer be enough males available to stimulate spawning or to fertilize adequate numbers of eggs [7]. Further, a reduction in the number of males will limit the effective population size and number of individuals which can contribute to the population [8]. As a result, oyster populations may experience bottlenecks and an inability to respond to change. Under ambient pCO₂, elevated temperature had a slightly positive impact on reproductive capacity. This positive impact was masked, however, when elevated temperature was combined with elevated pCO₂—reflecting the real-world scenario predicted for marine organisms over this century. Further research is needed to test whether exposure to ocean acidification over multiple reproductive cycles and multiple generations will change the impacts on sex determination and reproductive capacity. Previous studies to date have shown that for some marine species negative impacts of ocean acidification on reproductive capacity are improved following transgenerational exposure [58], whereas for others it is not [59]. We have no understanding, however, of the transgenerational response of sex determination. Elucidating such impacts is critical to understanding population dynamics in an acidifying, warming ocean.

Ethics

Research was carried according to the University of Sydney animal ethics guidelines.

Data accessibility

The datasets supporting this article have been uploaded as part of the electronic supplementary material.

Authors' contributions

L.M.P., W.A.O., M.B., M.D., R.A.C., H.O.P. and P.M.R. designed the experiment, L.M.P. ran the experiment, M.B. prepared histology slides, L.M.P. and M.D. analysed histology slides, P.V. and M.G. determined total lipid content of eggs, L.M.P. and E.S. analysed the data, L.M.P. and P.M.R. led the writing of the manuscript.

Competing interests

We declare we have no competing interests.

Funding

This research was supported by an Australian Research Council Discovery Indigenous grant to L.M.P., R.C., P.M.R. and P.V. (grant number IN140100025).