Increased light availability enhances tolerance against ocean acidification-related stress in the calcifying macroalga Halimeda opuntia

1. Introduction

The atmospheric carbon dioxide (CO₂) concentration has been rising rapidly over the past 250 years owing to the large-scale use of fossil fuels and deforestation, and it is predicted to exceed 1 000 ppmv by the end of 2100 if anthropogenic CO₂ emissions are not effectively managed (Fabry et al., 2008; IPCC, 2013). Friedlingstein et al. (2019) estimated that the oceans have already absorbed CO₂ nearly (2.5±0.6) Gt/a from 2009 to 2018. This has resulted in a ~0.1 unit decrease in pH at the oceans’ surface layers and is projected to reach a mean of pH 7.8 by the end of this century (IPCC, 2013). This CO₂-induced ocean acidification (OA) has received much attention and may cause large-scale changes in many marine ecosystems owing to the increased bicarbonate (${\rm{HCO}}_3^- $) concentration, reduced pH level, and lower carbonate saturation (Ω) of surface seawaters (Orr et al., 2005; Doney et al., 2009; Cornwall et al., 2013). Hoegh-Guldberg et al. (2007) demonstrated that with the current rates of coral loss and bleaching of calcifying macroalgae caused by OA, seagrass and non-calcifying macroalgae may gradually become dominant communities on reefs and in other carbonate-based ecosystems. The effects of OA on marine calcifiers in a range of marine ecosystems, including tropical corals (Hoegh-Guldberg et al., 2007; Doo et al., 2019), calcifying macroalgae (Moya et al., 2012; Hofmann et al., 2014; Wei et al., 2020a), and a variety of benthic invertebrates (Dupont et al., 2008; Hall-Spencer et al., 2008), have been determined to be potentially deleterious on physiological performance, such as growth, calcification, reproduction, and photosynthetic processes.

The aragonite-depositing green algal genus Halimeda (Bryopsidales) is found widely in the tropical sea area (Wei et al., 2020b). This algal genus is composed of calcified green segments that play a major role as carbonate sediment producers (Payri, 1988; Sinutok et al., 2012; Wizemann et al., 2015). Halimeda species produce CaCO₃ an estimated 0.15–0.40 Gt/(m²·a), which accounts for approximately 8.0% of the total calcium produced by coral reef ecosystems (Milliman, 1993; Hillis, 1997). Thus, Halimeda species are considered a carbon sink for the long-term storage of atmospheric CO₂ emissions (Kinsey and Hopley, 1991; Rees et al., 2007). Peach et al. (2017) noted that photosynthesis, calcification rates (G_net), and aragonite crystal formation varied among six Halimeda species, but they were not obviously affected under elevated pCO₂ (1 300 ppmv) conditions. However, Wei et al. (2020a) found that elevated pCO₂ (1 600 ppmv) has an adverse influence on growth, G_net values, maximum quantum yields of photosystem II (F_v/F_m), and chlorophyll a (Chl a) accumulations in Halimeda cylindracea and Halimeda lacunalis. Although species-level variations in response to OA have been documented in G_net values and photosynthetic performance in field and laboratory experiments, significant gaps still exist in our knowledge of the metabolic responses to OA by these calcifiers owing to the pCO₂ concentrations used (e.g., 1 300 ppmv vs. 1 600 ppmv) and other potentially confounding factors, such as nutrient availability (Hofmann et al., 2014), seawater flow (Comeau et al., 2014a), temperature (Campbell et al., 2016), and irradiance (Zou and Gao, 2010; Vásquez-Elizondo and Enríquez, 2017; Wei et al., 2020a).

In the open ocean, light is another vital environmental factor for the growth and distribution of Halimeda species (Peach et al., 2017). Variations in light availability can affect algal photosynthesis, carbohydrate synthesis, and substance accumulation through the photo-energy absorbed by light-harvesting complexes (Porzio et al., 2011; Celis-Plá et al., 2017). Moderate increases in light intensity have positive influences on the physiological performance of fleshy and calcifying macroalgae (Teichberg et al., 2013; Bao et al., 2019). Teichberg et al. (2013) found that owing to differences in the incidence of underwater light, the growth, photosynthetic performance, and tissue composition (total organic carbon and nitrogen) of Halimeda opuntia at depths of 5 m were significantly greater than those at 15 m in Curacao, Netherlands Antilles. High sunlight levels in relatively shallow marine environments provide sufficient solar energy for calcifying macroalgae to uptake ${\rm{HCO}}_3^- $ (Borowitzka and Larkum, 1976; Koch et al., 2013). As CO₂ dissolves in seawater, the ${\rm{HCO}}_3^- $ concentration can increase, and this is used for the assimilation of biogenic calcium carbonate (Orr et al., 2005). Thus, it is essential to conduct experiments that combine OA conditions with other factors such as light availability (Hofmann et al., 2014).

Elevated ocean pCO₂ has been shown to affect physiological processes in Halimeda, such as growth, calcification and skeletal minerology, with the later making them potentially more susceptible to herbivory (Hofmann et al., 2014; Wei et al., 2020a). Therefore, it is essential that these calcifying macroalgae make suitable modifications to address OA-related stress. Functional soluble organic osmolytes, such as proline, soluble carbohydrate (SC), soluble protein (SP), and free amino acids (FAAs), are secreted in abundance and protect the integrity of cellular structures, as well as other defense-related enzymes systems (Chen et al., 2017, 2018; Wei et al., 2020a). However, it is still not known whether these marine calcifying autotrophs will be able to tolerate future OA conditions, because the adverse effects of continued OA drive these calcifying species closer to their physiological tolerance limits (O’Donnell et al., 2010; Koch et al., 2013). Furthermore, some studies have indicated that a moderate increase in light availability may enhance the metabolic activities of Halimeda species in response to OA, indicating its potential role in mitigating adverse effects on these carbonate producers (Teichberg et al., 2013; Wei et al., 2020a).

The South China Sea is one of the most multifarious coral community habitats (Morton and Blackmore, 2001). The species of H. opuntia can be found widely distributed in coral reefs and lagoons, and it is most abundant at shallow depths (Wei et al., 2019, 2020b). At specific growth locations and over depth ranges, H. opuntia experiences varied sunlight irradiance conditions during the daytime (El-Manawy and Shafik, 2008; Peach et al., 2017). Although elevated light intensities have been shown to positively affect the physiological performance of Halimeda, the combined effects of OA and light intensity are unknown. Thus, to improve our knowledge of anti-stress responses in H. opuntia, a coupling experiment was conducted to examine growth, calcification, photosynthesis performance, soluble cell components, and metabolic enzyme-driven H. opuntia responses to CO₂ enrichment under moderately increased light availability. The resulting detailed physiological descriptions of OA provide an important scientific basis for predicting Halimeda spatial distributions in different seawater depths and for population protection during global climate changes.

2. Materials and methods

2.1 Sample collection

Samples of H. opuntia were collected by scuba diving in a back reef lagoon in the South China Sea (17.03°–17.07°N, 111.28°–111.32°E) at depths ranging from 12–15 m in July 2019. All the individuals were carefully detached from the sandy substrate. After collection, algal thalli were immediately incubated in aquaria in the ship having flow-through systems receiving ambient seawater and transported back to the marine research laboratory in Sanya, China.

For acclimation, the algae were reared in an aerated mesocosm tank (1 200 L) with a constant supply of fresh sandy-filtered seawater (~30 L/min) in the laboratory for 2 weeks. Ambient seawater conditions consisted of 27.0℃, 32 psu, pH 8.1, and 80 μmol photons/(m²·s) of photosynthetically active radiation with a 12:12 h day/night cycle in accordance with the method reported by Hofmann et al. (2014). After a 2-week acclimation period, thalli of H. opuntia displayed healthy green segments and no mortality was detected, indicating that subsequent experiments could be undertaken.

2.2 Experimental design and treatments

For the laboratory trial, healthy thalli of H. opuntia were randomly assigned to 2×3 factorial combination of two seawater pCO₂ levels and three light intensities (Table S1) to determine the mixed effects on the physiological performance of H. opuntia after 4 weeks (November 10 to December 8, 2019). For each treatment, there were 10 individuals (120–130 g fresh weight (FW) in total) in a 30-L transparent tank, and three biological replications were prepared per treatment. Filtered seawater was changed twice a week. The OA (elevated pCO₂) was achieved by bubbling pure CO₂ mixed with ambient air automatically into seawater in each tank (CE100C, Wuhan Ruihua Instrument and Equipment Ltd., Wuhan, China). High pCO₂ (HC) was set at 1 400 ppmv, which is the level predicted under extreme conditions by the end of this century (Caldeira and Wickett, 2005). The low CO₂ concentration (LC) was 400 ppmv, which is the current local average pCO₂ level. Light intensities were set at three levels, 30 (low, LL), 150 (middle, ML), and 240 (high, HL) μmol photons/(m²·s), that created using full-spectrum fluorescent aquarium lamps (Giesemann, Nettetal, Germany) and monitored by an optical quantum probe (ELDONET Terrestrial Spectro-radiometer, Frankfurt, Germany). These three light levels were set based on the saturation light (I_k) values of Halimeda species (Beach et al., 2003; Campbell et al., 2016). Although the light intensities selected in the present study were lower than at the sampling site (300–500 μmol photons/(m²·s)), previous research has revealed that I_k values in situ typically range from 30 μmol photons/(m²·s) to 250 μmol photons/(m²·s) (Beach et al., 2003; Hofmann et al., 2014; Campbell et al., 2016), which includes the light intensity ranges selected in the present study.

2.3 Monitoring experimental conditions

To ensure the consistency of seawater among treatments, seawater condition monitoring was conducted daily at 10:00 during the 4-week experimental period. Seawater temperature and salinity were monitored daily by a calibrated handle YSI meter (YSI Professional Plus, Yellow Springs, OH, USA). The pH (NBS scale) was determined using an S220 pH electrode (relative accuracy ±0.01 units, Thermo Scientific Orion, USA). Before each measurement, the pH electrode was calibrated using a standard buffer solution (pH 7 and 10). The seawater parameter (25.00 mL) for total alkalinity (TA) sampled from each aquarium was mixed with 8 μL of 50% saturated HgCl₂ solution and then temporarily stored in brown glass bottles at 4℃ under dark conditions until used. The TA was determined using potentiometric titrations of three replicate seawater samples and alkalinity reference materials (CRM; Andrew Dickson Lab, Scripps Institute of Oceanography) by the Gran titration method (Metrohm 877 Titrino Plus, Titrando Metrohm Inc., Herisau, Switzerland) (Dickson et al., 2007). All the measurements were conducted at 25.0℃ and completed within 24 h of sampling. The levels of dissolved inorganic carbon components (CO₂, ${\rm{CO}}_{\rm{3}}^{2-} $ and ${\rm{HCO}}_{3}^{-} $) and the aragonite saturation state (Ω_Arag) were calculated using salinity, temperature, pH, and TA with the Excel program CO2SYS (Pierrot et al., 2006).

2.4 Growth and ${\boldsymbol{G}}_{{\boldsymbol{net}}}$ measurements

The FWs (g) of H. opuntia were measured at the beginning and the end of the experiment. The relative growth rate (RGR; %/d) was calculated using the following formula,

where W_t1 and W_t0 represent the fresh weights obtained at times t1 and t0 days, respectively, and t1 and t0 represent the two sampling days.

At the end of the experiment, the instantaneous G_net was calculated using the TA anomaly technique in accordance with the description by Hofmann et al. (2014). Segments of H. opuntia (1.0 g FW) were sampled and incubated in 0.5-L transparent glass bottles containing filtered seawater under experimental conditions. The samples were constantly mixed with an electro-magnetic stirrer. The calculation of the G_net (μmol/(g·h)) was based on the following equation in which every mole of formed CaCO₃ reduces the TA by two equivalents,

where p represents the density of seawater (1.025 kg/L), TA₀ represents the initial TA of seawater, TA_n represents the TA of seawater after n hours of incubation (8 h in this study), V is the volume of the incubation chamber (0.5 L in this study), and t represents the incubation time (8 h). The algal FW in this study was 1.0 g.

2.5 Chlorophyll fluorescence and pigment contents

The photosynthetic performance of H. opuntia was measured at the end of the experiment using pulse amplitude modulated fluorometry by a DIVING-PAM II fluorometer with red light as the modulated light (Heinz Walz GmbH, Effeltrich, Germany). On the sampling date, segments of all the Halimeda thalli in each treatment were selected and adapted in darkness for nearly 20 min before measurements were taken. The F_v/F_m value was obtained using a saturation pulse (5 000 μmol photons /(m²·s), 0.6 s). The relative electron transport (rETR) versus incident irradiance curves (rapid light curves, RLCs), which reflect linear electron transport activity, were determined under different photosynthetically active photon flux levels (approximately 0, 45, 65, 89, 124, 189, 285, 421, 631, unit: μmol photons /(m²·s)) at 1-min intervals. From the RLCs, the maximum relative electron transport rate (rETR_max) was calculated using a nonlinear regression analysis with the following equation described by Eilers and Peeters (1988),

where a, b and c are constant parameters.

The photosynthetic pigments present in segments of H. opuntia from each aquarium (n=3) were determined. A 100.0 mg FW per sample was finely ground in 10 mL of 90% acetone and then stored for 24 h at 4℃ in darkness for extraction. Subsequently, all the extracted solutions from samples were centrifuged at 5 000 r/min for 10 min at 4℃ (Eppendorf centrifuge 5810 R, Hamburg, Germany). Each supernatant was transferred to a quartz cuvette and its absorbance was measured at 664 nm, 647 nm and 470 nm using a spectrophotometer (UV 530 Beckman Coulter, Brea, CA, USA). Contents of Chl a and carotenoids (Car.) were calculated in accordance with the method reported by Wellburn (1994).

2.6 Determination of organic carbon and nitrogen content

Total tissue organic carbon (TC_org) and total nitrogen (TN) contents were measured after the 4-week experimental period. Whole–thallus samples were cleaned of epiphytes, detritus, and sediment by washing with filtered seawater 3–5 times. To determine TC_org, dry tissue samples were acidified with 1 mol/L HCl until no air bubbles were observed. Subsequently, all the samples were dried at 60℃ until no change in dry mass weight was detectable, and then, they were finely ground to powder using an automatic grinding mill (FSTPRP-24, Jingxin, Shanghai, China). Both TC_org and TN were measured using a CHN Elemental Analyzer (Flash EA300, Thermo Scientific, Milan, Italy).

2.7 Measurement of enzyme activity

At the end of the experiment, 200.0 mg H. opuntia segments from each aquarium (n=3) were sampled and frozen immediately in liquid nitrogen. All the samples were ground in 5.0 mL of 20 mol/L veronal buffer (pH 8.3) for the external carbonic anhydrase activity (eCAA) and nitrate reductase activity (NRA) determinations. The eCAA of H. opuntia was measured using the electrometric technique reported by Haglund et al. (1992), while NRA was measured using the method reported by Corzo and Niell (1991).

2.8 SC, SP and FAA contents

Approximately 200.0 mg FW H. opuntia segments were finely ground with a mortar and pestle in 10 mL distilled water. Afterwards, the extract was centrifuged at 5 000 r/min for 10 min (Eppendorf centrifuge 5810R, Hamburg, Germany) and then the supernatant was used to determine SC and SP contents using an ultraviolet spectrophotometer. The SC content was determined using the phenol-sulfuric acid method (Kochert, 1978a). The SP content was measured by Coomassie Brilliant Blue G-250 dye and standardized with bovine albumin (Kochert, 1978b).

To determine the FAA contents, 200.0 mg FW algae were sampled and finely ground in 4.5 mL of PBS buffer (pH 7.4). The homogenate was centrifuged at 5 000 r/min for 10 min. The supernatant was collected and preserved at 4.0℃. The FAA content was determined using an algal FAAs ELISA Kit (Mlbio, Shanghai, China) following the manufacturer’s instructions.

2.9 Statistical analysis

All results in this study are presented as a triplicate mean±standard deviation (SD). Origin 8.0 software was used to create the figures and data analyses were processed using Minitab 16.0 software. All the response variables measured during the experiment were analyzed using a two-way analysis of variance with pCO₂ and light intensities as the independent factors. Tukey’s Honestly Significant Difference was used to identify significant differences between treatments and P values were significant at the 95% confidence level.

3. Results

3.1 Seawater conditions

During the experimental period (Table S2), the average pH values within the HC (1 400 ppmv) and LC (400 ppmv) CO₂-treatments were 7.72 and 8.15, respectively. The mean TA values in LC and HC were (2 336±40) μmol/kg and (2 350±28) μmol/kg, respectively, which were not significantly different (P>0.05). The elevated pCO₂ significantly altered the carbon chemistry parameters in seawater (P<0.01). Compared with LC, the average CO₂ and ${\rm{HCO}}_{3}^{-} $ concentrations in HC increased from 11.66−12.81 μmol/kg to 39.10−39.81 μmol/kg and 1 782.9−1 828.8 μmol/kg to 2 138.0−2 165.3 μmol/kg, respectively, whereas the ${\rm{CO}}_{3}^{2-} $ concentration decreased from 197.2−206.0 μmol/kg to 86.8−95.9 μmol/kg. The daily calculated saturation state of aragonite (Ω_Arag) in HC ranged from 3.21 to 3.36 compared with 1.42 to 1.59 in LC (Table S2).

3.2 Growth and calcification rates

After 4 weeks of exposure, elevated pCO₂ and light intensities had significant effects on the RGR and G_net of H. opuntia (Fig. 1; Table S3). The increased light intensity stimulated growth rates at the two CO₂-concentration levels. In contrast to under LL conditions, the RGR increased by 19.28%−33.32% and 26.64%−50.42% under ML and HL conditions, respectively (Fig.1). However, the RGR of H. opuntia under HC conditions significantly declined by 13.14%−41.29% compared with under LC conditions. The G_net decreased by nearly three-fold under HC-LL conditions ((0.218±0.035) μmol/(g·h)) compared with under LC-LL conditions ((0.625±0.042) μmol/(g·h)). Calcification rates for H. opuntia increased along with light intensity, as shown in Fig. 1, regardless of the pCO₂ level.

Figure  1.  Relative growth rates (RGR, %/d) and net calcification rates (G_net, μmol/(g·h)) in Halimeda opuntia (mean±SD, n=3) after six pCO₂–light treatments. Significant differences in RGR and G_net among the treatments are indicated by different uppercase and lowercase letters, respectively (Tukey’s test, P<0.05).

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3.3 Photosynthetic performance and pigment contents

Rapid light curves indicated there were significant effects of HC on the rETR of H. opuntia under HL conditions, but not under ML or LL conditions (Fig. 2). Notably, H. opuntia displayed a significant correlation between F_v/F_m and G_net (Fig. 3). The F_v/F_m values ranged from 0.665 to 0.704 and 0.696 to 0.711 after HC and LC treatments, respectively, regardless of the three light levels (Fig. 4a; Table S3). Conversely, the rETR_max was only significantly affected by light availability (P<0.01). The rETR_max was greater in HL (6.262−6.584 μmol e⁻/(m²·s)) than in ML (5.357−5.694 μmol e⁻/(m²·s)) and LL (5.157−5.674 μmol e⁻/(m²·s)) (Fig. 4a).

Figure  2.  Rapid light curves of the electron transport rate (rETR) plotted against the photosynthetic active radiation (PAR) of Halimeda opuntia incubated under six pCO₂–light conditions.

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Figure  3.  Correlation between photosynthetic maximum quantum yield (F_v/F_m) and calcification rate (G_net, μmol/(g·h)) of Halimeda opuntia.

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Figure  4.  Variations in photosynthetic maximum quantum yields (F_v/F_m) and maximum relative electron transport rates (rETR_max) (a), and in Chl a (μg/g FW) and carotenoid (Car., μg/g FW) contents (b) of Halimeda opuntia (mean±SD, n=3) under six pCO₂–light conditions. Significant differences among the treatments are indicated by different uppercase and lowercase letters, respectively (Tukey’s test, P<0.05).

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There was a significant interaction effect on the Chl a content in H. opuntia (Table 3S). Under HC conditions, the highest Chl a content was obtained in HL treatments ((139.1±15.9) μg/g FW), whereas under LC conditions, the highest Chl a content was measured in LL treatments ((168.6±6.6) μg/g FW) (Fig. 4b). The carotenoids (Car.) content was independently affected by HC and light intensities (P<0.01) (Table 3S). In HC treatments, the Car. contents ranged from (26.36±2.1) μg/mg FW to (36.15±3.6) μg/mg FW, which was 17.75%−40.01% higher than in LC treatments (18.65−26.11 μg/mg FW). Compared with under HL and ML conditions, the Car. contents under LL conditions increased by 32.61%−38.45% and 13.87%−41.34% with HC and LC treatments, respectively (Fig. 4b).

3.4 Tissue TC_org and TN contents

Both CO₂ enrichment and increasing light availability resulted in tissue TC_org accumulation in H. opuntia over the 4-week experiment (Fig. 5; Table 3S). Under HC conditions, the TC_org content significantly increased by 13.78%−17.91% compared with under LC conditions (Fig. 5). The highest TC_org content [17.64% ±1.02% dry weight (DW)] occurred during the HC-HL treatments, and the lowest content (13.28% ±1.34% DW) occurred during the LC-LL treatment. The TN content exhibited a similar variation pattern, because the elevated pCO₂ significantly stimulated nitrogen accumulation (P=0.041) (Table S3). However, light variation had no effect on the TN content in H. opuntia as shown in Table S3 (P=0.168).

Figure  5.  Variations in tissue total organic carbon (TC_org, % DW) and nitrogen (TN, % DW) from the tissues of Halimeda opuntia (mean±SD, n=3) under six pCO₂–light conditions. Significant differences in TC_org and TN among the treatments are indicated by different uppercase and lowercase letters, respectively (Tukey’s test, P<0.05).

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3.5 eCAA and NRA levels

The eCAA was significantly higher under HC conditions than under LC conditions (Fig. 6; Table S3). Under the former, the eCAA of H. opuntia was 0.455−0.518 IU/mg FW, increasing by 13.35%−22.31% compared with under the latter. Increased light availability also played a positive role in regulating eCAA (P<0.01) (Table S3). The NRA was affected by significant interactions between HC and the light intensities (Table S3). The NRA increased under HC conditions, producing values of 0.553−0.698 pg/mg FW, compared with values of 0.361−0.526 pg/mg FW under LC conditions (Fig. 6). Moderately increasing the light intensity positively enhanced the NRA levels by 7.78%−26.22% and 30.75%−45.71% under HC and LC conditions, respectively (Fig. 6).

Figure  6.  Mean±SD (n=3) external carbonic anhydrase activity (eCAA, IU/mg FW) and nitrate reductase activity (NRA, pg/mg FW) levels during six pCO₂–light treatments. Significant differences in eCAA and NRA among the treatments are indicated by different uppercase and lowercase letters, respectively (Tukey’s test, P<0.05).

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3.6 SC, SP, and FAA contents

The SC contents measured in H. opuntia at the end of the experiment were notably affected by interactions between HC and the light intensities (P<0.01) (Table S3). Under HC conditions, the SC content ranged from (0.432±0.021) μg/mg FW to (0.668±0.014) μg/mg FW, which was 12.21%−51.47% higher than under LC conditions (0.385−0.441 μg/mg FW). Compared with LL exposure, the SC content under ML to HL exposure (150−240 μmol photons/(m²·s)) increased by 23.38%−54.63% and 12.73%−14.55% under HC and LC conditions, respectively (Fig. 7a). Similar trends were documented for the SP content in H. opuntia. The SP content under HC conditions (0.347−0.547 μg/mg FW) was 11.58%−15.08% higher than under LC conditions (0.311−0.478 μg/mg FW) (Fig. 7b). In addition, there was a positive effect on light availability on SP accumulation, resulting in increases of 25.65%−57.64% and 21.86%−53.70% under HC and LC conditions, respectively (Fig. 7b, Table S3). The SC/SP ratios at HC levels were remarkably greater than at LC levels, and the lowest SC/SP ratio (0.923) occurred during the LC-HL treatment (Fig. 7d). The FAA content in H. opuntia was notably impacted by interactions, as shown in Table S3 (P<0.01), indicating that pCO₂ had a strong light level-dependent effects. The HC conditions resulted in a significant increase in the FAA content, which was 33.41%−54.74% higher than in thalli grown under LC conditions. Compared with LL conditions, moderately increasing the light intensity (ML to HL) positively enhanced the FAA contents by 2.19%−11.22% and 12.11%−17.37% under HC and LC conditions, respectively (Fig. 7c).

Figure  7.  Variations in soluble carbohydrate (SC, μg/mg FW) (a), soluble protein (SP, μg/mg FW) (b), free amino acids (FAA, ng/mg FW) (c), and SC/SP (d) ratio of Halimeda opuntia (mean±SD, n=3) under six pCO₂–light conditions. Significant differences among the treatments are indicated by different lowercase letters (Tukey’s test, P<0.05).

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4. Discussion

The effects of CO₂-induced OA have caused growing concerns regarding potential stresses on marine calcifying organisms, biological communities, and coral reef ecosystems (Moodley et al., 2000; Feely et al., 2004; Zeebe et al., 2008). As shown in previous studies (Hofmann et al., 2014; Campbell et al., 2016; Wei et al., 2020a), our data suggest that OA has deleterious impacts on growth rates, as well as calcification and photosynthetic processes, in the calcifying macroalga H. opuntia (Figs 1 and 2). However, this study further revealed that photosynthetic performance was positively increased by a higher light availability level, resulting in the accumulation of photosynthetic products (such as SC) and the presence of sufficient energy and carbon skeletons to meet nitrogen requirements (Chen et al., 2017; Wei et al., 2021). Contents of soluble cell components (such as SP and FAA) also increased notably under HC and ML to HL conditions. Moreover, the observed accumulations of TC_org and TN are likely associated with increased metabolic enzyme-driven activities under HC and ML to HL conditions. Thus, moderately increasing the light availability enhanced H. opuntia tolerance to OA. This conclusion is consistent with previous studies reported by Wei et al. (2019, 2020a), in which OA and increased light influenced the photosynthesis and calcification performances of two Halimeda species (H. cylindracea and H. lacunalis) in opposing directions, and the positive effects of moderate increases in light availability offset the adverse effects of the decline in pH caused by elevated pCO₂.

Many previous reports on Halimeda species (Teichberg et al., 2013; Hofmann et al., 2014; Campbell et al., 2016; Peach et al., 2017) and other marine calcifiers (Hofmann et al., 2008; Manzello, 2010; O’Donnell et al., 2010) exposed to OA have focused on reduced growth and calcification. In this study, a lower G_net for H. opuntia was demonstrated under HC conditions compared with LC conditions. Although biological process of growth and calcification drew down TA, the frequent seawater changes (twice a week) intended to counteract such fluctuations in TA, which resulted in TA in LC just a bit lower than in HC. With CO₂ enrichment, the pH values and Ω_Arag drastically decreased (Table S2), which may reduce the formation rate of calcium carbonate in algal tissues (Orr et al., 2005; Andersson et al., 2009; Ries et al., 2009; 2010; Hofmann et al., 2014). A similar conclusion was reported by Comeau et al. (2014b), who demonstrated that the G_net of Halimeda minima declined by 11.0% when the pCO₂ level was double that of ambient conditions, and they suggested that decreased carbonate ion availability inhibits CaCO₃ deposition. As observed in other Halimeda species exposed to OA (Vásquez-Elizondo and Enríquez, 2016; Campbell et al., 2016), the calcification data provided evidence that increased light compensated for the potential negative effects of HC conditions through the up-regulation of photosynthesis (Fig. 3). This may be because algal calcification is energetically dependent on photosynthesis and can provide a large fraction of the energy required by the biomineralization process (Chisholm, 2000, 2003; Meyer et al., 2016). Peach et al. (2017) hypothesized that increased photosynthesis compensates for the adverse effects of OA on algal calcification processes. Photosynthesis promotes algal calcification by removing CO₂ or bicarbonate from the calcification site, which increases the local pH and, thus, facilitates CaCO₃ precipitation (Borowitzka and Larkum, 1976; Campbell et al., 2016).

The HC treatment had adverse effects on thallus photosynthesis in H. opuntia, and the severity of these effects was significant under LL conditions (30 μmol photons/(m²·s)), resulting in declines in F_v/F_m and rETR (Figs 2 and 4a). Similar negative effects of elevated pCO₂ on algal photosynthesis have been reported previously in other Halimeda species (Price et al., 2011; Sinutok et al., 2012; Campbell et al., 2016; Meyer et al., 2016). For instance, Price et al. (2011) reported carbonate dissolution and a photosynthetic capacity reduction in H. opuntia after 2 weeks of exposure to pCO₂ at (900±90) ppmv, and proposed that this resulted from the depressed expression of CO₂ concentrating mechanisms (CCMs). Thus, elevated pCO₂ caused a decrease in CCM activity, forcing algal photosynthesis to rely on passive CO₂ diffusion, and thereby critically limiting the efficiency of photosynthetic carbon assimilation (Prins et al., 1982; Elzenga and Prins, 1989; Miedema and Prins, 1991; Price et al., 2011). According to the results of present study, the excess dissolved CO₂ compromised both photophysiology and Ω_Arag at the site of calcification in H. opuntia, indicating that OA has adverse effects on this calcifying species. Concurrently, H. opuntia resists OA stress by regulating pigment synthesis. A significant decline in the Chl a content under HC conditions (Fig. 4b) suggested pigment degradation and increased HL-associated stress (Sinutok et al., 2012). However, the Car. contents, especially under LL conditions, accumulated markedly, which supports a natural photoacclimation response of algae to shade adaptation (Boardman, 1977; Teichberg et al., 2013). Based on photosynthetic performance (Fig. 4a), although there was no statistically significant differences between HC-LL and HC-ML, or between HC-ML and HC-HL, a positive correlation between increasing light intensity and F_v/F_m was observed after a 4-week in HC treatments, which indicated that the effects of a moderate increase in light availability could offset the severe impacts of HC-induced low pH (Wei et al., 2020a).

The HC level in seawater modulated TC_org and TN accumulations in algal tissues (Fig. 5), which may result from metabolic enzymatic-driven activities, especially eCAA and NRA (Hofmann et al., 2014; Chen et al., 2018; Wei et al., 2021). In the present study, CO₂ enrichment, as well as increased light intensities positively enhanced the eCAA of H. opuntia, which contributed to CO₂ and $ {\rm{HCO}}_3^- $ catalytic interconversion (Moya et al., 2012; Vidal-Dupiol et al., 2013; Hofmann et al., 2014). The SC synthesis in H. opuntia was enhanced by the positive effects of eCAA and photosynthetic performance (Fig. 7a), which resulted in an improvement in the irradiance-harvesting capacity to absorb light and the up-regulation of the photosynthetic electron-transport chain (Van Oijen et al., 2004; Chen et al., 2017). Moreover, the HC treatment in this study significantly increased both the NRA and the TN accumulation in H. opuntia (Figs 5 and 6), which is consistent with the conclusions reported by Chen et al. (2016, 2017) and Wei et al. (2020a, 2021). Indeed, a positive correlation between increased light availability and higher TN content was observed during the experimental period, even though it was not statistically significant (P=0.168) (Table S3). These results indicated that increased light may cause a greater photosynthetic performance (F_v/F_m and RLCs) and soluble organic osmolytes (such as SC) modifications in H. opuntia to mitigate OA stress.

Soluble organic molecules may play roles in buffering metabolic depression to cope with OA-related challenges (Xiong et al., 2002; Sun et al., 2013; Chen et al., 2017, 2018; Wei et al., 2020a). Light-mediated changes in cellular biochemical composition are associated with the acclimation of algal growth and photosynthesis (Hemm et al., 2004; Zou and Gao, 2013). Under HC conditions, the SC and SP contents were remarkably greater than those of H. opuntia tissues under LC conditions (Fig. 7). Notably, higher light intensities and eCAA may promote photosynthetic processes, which in turn increases photosynthetic product and SC accumulations to improve flexibility and plasticity in response to OA. These modifications may contribute to membrane integrity and ensure the function of cellular activities, and they may provide the physiological basis for resistance to adverse external environments by algal cells (Sun et al., 2013; Chen et al., 2017; Wei et al., 2020a). Such shifts in metabolic performance may result from the enhancement of internal and external eCAA levels that facilitate the conversion of CO₂ and $ {\rm{HCO}}_3^- $ to satisfy the photosynthetic carbon demand (De Beer and Larkum, 2001). The SP content is another important indicator of plant metabolism (Sun et al., 2015). Here (Fig. 7b), an obvious increase in the SP content was observed during HC treatments. Yang et al. (2009) revealed that a greater SP accumulation increases key enzyme activities involved in plant photosynthesis and accelerates substance and energy metabolism. Furthermore, consistent with previous studies (Rizhsky et al., 2004; Fougère et al., 1991; Wei et al., 2020a), the increase in the FAAs content might increase the anti-oxidative capacity of H. opuntia. Wei et al. (2020a) demonstrated that under elevated pCO₂ conditions (1 600 ppmv), proline contents, an FAA in both H. cylindracea and H. lacunalis, were two- to four-fold higher than under ambient pCO₂ conditions (400 ppmv), and they hypothesized that the FAA contents were largely secreted and function in protecting cellular structures and scavenging reactive oxygen species, as well as in other related enzyme systems, to alleviate OA stress (Verma, 1999; Xiong et al., 2002).

In summary, deteriorating marine conditions caused by human activities threaten the diversity levels, distributions and biomasses of coral reef ecosystems, including calcifying macroalgae. The results of the current study indicated that OA has adverse effects on growth, calcification, photosynthesis, and other physiological performances of H. opuntia. However, our results also indicated that increased light availability enhanced anti-stress capabilities through the accumulation of soluble organic molecules, especially SC, SP, and FAA that help alleviate OA stress. Moreover, the up-regulation of eCAA and NRA was significantly promoted by increased CO₂ levels and/or light availability, which favored inorganic carbon and nitrogen uptake and assimilation (Losada and Guerrero, 1979; Syrett, 1981). These results increase our understanding of whether marine autotrophic calcifiers have the physiological plasticity (species-specific tolerance) to compensate for the effects of OA and whether they will continue to build carbon skeletons under future CO₂ conditions.