Effects of CO2 and Seawater Acidification on the Early Stages of

Article pubs.acs.org/est

Effects of CO2 and Seawater Acidification on the Early Stages of Saccharina japonica Development Dong Xu,† Dongsheng Wang,‡ Bin Li,§ Xiao Fan,† Xiao W. Zhang,† Nai H. Ye,*,† Yitao Wang,‡ Shanli Mou,† and Zhimeng Zhuang† †

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, China College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong 266109, China § Shandong Marine Resource and Environment Research Institute, Yantai, Shandong 264006 China ‡

S Supporting Information *

ABSTRACT: In this paper, we demonstrated that ocean acidification (OA) had significant negative effects on the microscopic development of Saccharina japonica in a short-term exposure experiment under a range of light conditions. Under elevated CO2, the alga showed a significant reduction in meiospore germination, fecundity, and reproductive success. Larger female and male gametophytes were noted to occur under high CO2 conditions and high light magnified these positive effects. Under conditions of low light combined with high PCO2, the differentiation of gametophytes was delayed until the end of the experiment. In contrast, gametophytes were able to survive after having been subjected to a long-term acclimation period, of 105 days. Although the elevated PCO2 resulted in a significant increase in sporophyte length, the biomass abundance (expressed as individual density attached to the seed fiber) was reduced significantly. Further stress resistance experiments showed that, although the acidified samples had lower resistance to high light and high temperature conditions, they displayed higher acclimation to CO2-saturated seawater conditions compared with the control groups. These combined results indicate that OA has a severe negative effect on S. japonica, which may result in future shifts in species dominance and community structure.

■

INTRODUCTION

and UV stress in the unicellular chlorophyte Dunaliella tertiolecta.17 Brown algae comprise >70% of the biomass of cold temperate rocky seashores.18 Kelp species constitute significant ecological components of coastal ecosystems, forming widespread underwater forests and structuring the biodiversity on a regional scale. The effects of global climate change have however resulted in the disappearance of some kelp forests.19 There is much speculation relating to the potential effects of OA and the nature of such effects, particularly for species that have a heteromorphic life history style. Although numerous studies have been conducted on this topic, these have focused on the macroscopic life stages of seaweeds, and the potential effects of OA on the microscopic phases have been largely overlooked.20,21 A recent study by Gaitán-Espitia et al. reported that the combined effects of elevated temperature and PCO2 can significantly decrease germination rates and increase the mortality of spores during the early life-history stages of Macrocystis pyrifera.22 These observed responses are, however,

Increasing anthropogenic emission of CO2 has reduced the pH of the oceans and decreased the role of oceans in moderating climate change and alters carbonate regimes, known as ocean acidification (OA).1−5 The fundamental changes in ocean chemistry are likely to have measurable biological consequences on marine biodiversity and ecosystem functioning, as well as on the provision of ecosystem services.5−10 The predicted OA has been reported to facilitate growth and photosynthesis in noncalcifying seaweeds and seagrasses and is expected to result in a shift toward higher C:N ratios.11,12 The OA response was, however, species specific and varied among different developmental stages.13,14 Furthermore, OA could interact with other stressors, such as increasing light, warming water, decreasing oxygen concentration, overfishing, and eutrophication, and showed additive effect on these stressors.15 Gao et al. reported that the projected rising CO2 may reduce primary production of natural phytoplankton assemblages of the South China Sea while also magnify light stress.16 Olischläger and Wiencke documented that ocean acidification could alleviate low-temperature effects on growth and photosynthesis of the red alga Neosiphonia harveyi (Rhodophyta).14 Additionally, elevated CO2 of 100 pa was also proved to alleviate high PAR © XXXX American Chemical Society

Received: December 5, 2014 Revised: February 11, 2015 Accepted: February 19, 2015

A

DOI: 10.1021/es5058924 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

■

based mainly on short-term experiments, which may have underestimated the long-term sensitivity of species to climate change.23 The recovery mechanism of the community after long-term acclimation also remains unknown.9 The important macroalgal genera Saccharina dominate rocky shores of cold-temperate regions, and their distribution is constrained by abiotic factors, such as light and temperature.24 The alga has a life cycle that involves alternation between two multicellular generations, a sporophyte and a gametophyte.25 It is known that the microscopic stage is more susceptible to environmental stressors compared with the macroscopic stage.21 A previous study showed that the adaptation potential of a population was related to its life history, which is the highest in species with short generation times and large population sizes.23 However, there is limited information available on how a further shift in ocean carbonate chemistry will affect noncalcifying photosynthetic kelps with a heteromorphic life history. Redmond found that lower pH values (7.6−7.7) resulted in slightly larger gametophytes of Saccharina latissima compared with pH 7.9 at 16 and 19 °C.26 Another study on Laminaria hyperborean indicated that female gametogenesis and vegetative growth of sporophytes were both significantly enhanced under the expected future PCO2 of 70 Pa.27 As an ecologically and economically important blown macroalga, Saccharina japonica is a major component of productive beds in the northwest coast of Pacific ocean and has been widely used as food and raw industrial materials.24,28 Although numerous studies have pointed out that abiotic stresses such as light, temperature, and nutrients are essential for S. japonica development and physiological performance, there is no data available on how further ocean acidification will affect its physiological characteristics.24 Additionally, it was also indicated that adaptive ecological−physiological differences exist between life history phases.29 Unfortunately, most OA studies of seaweeds have overlooked the microscopic phases.20,21 However, the successful survival in microscopic phases from stress conditions was a prerequisite to the subsequent colonization of kelp beds as a macroscopic sporophyte. Therefore, we first investigated the combined effects of OA and light on its microscopic development from meiospore germination to sporophyte formation in a short-term experiment. As long as the sporophyte formed, they were further cultured under different PCO2’s until juvenile sporophyte formed in another relative long-term experiment, where physiological charateristics were compared among treatments. Finally, we compared stress resistance of juvenile sporophytes grown in acidified water with that of a control group in the last experiment. We hypothesized that (i) elevated PCO2 would have a negative effect on microscopic development, whereas the corresponding increase or decline in light would present an interactive effect with PCO2; (ii) although extreme low pH would decrease meiospore germination and growth significantly in the short term, some individuals would be able to survive over the long-term at a cost of a reduction in abundance; and (iii) acidified algal samples would be less able to adjust to stressful conditions compared with control groups.

Article

EXPERIMENTAL DETAILS

Target Species and Biological Material. On 22 October 2013, mature wild sporophytes S. japonica were collected in Sungo Bay (37°01′−37°09′ N, 122°24′−122°35′ E), a semienclosed bay located on the northwestern coast of the Yellow Sea, China. Within 3 h, the sporophytes were transported to the laboratory in a tank of cold seawater. Reproductive sorus tissue was excised and cleaned of any visible fouling organisms with filtered seawater, blotted dry, and kept in the dark at 10 ± 1 °C overnight. To induce spore release, sporophylls were immersed in 10 °C sterilized seawater for approximately 30 min. The resulting spore slurry was filtered through a 20 μm mesh. One hundred cut-glass microscope slides (20 × 20 mm) were sterilized and divided into groups, with a total of three slides in each group. Each slide was inoculated by pipetting 1 mL of the spore solution into Petri dishes containing 60 mL of pHadjusted f/2 medium. Thirty seed fiber plates (coir fiber rope, 130 cm in length, 0.6 cm in diameter) were also sterilized and introduced into a 1000 mL glass beaker and inoculated by pipetting 10 mL of the spore solution into Petri dishes containing 600 mL of pH-adjusted f/2 medium. The spores were allowed to settle undisturbed and germinate on the glass or seed fiber plate for 24 h at 10 °C. Experimental Design. The experiment was conducted in three parts. To assess the short-term combined effects of PCO2 and light on the microscopic reproductive process, the shortterm experiment (STE) lasted for 32 days. To assess algal phenotypic plasticity, a longer-term experiment (LTE) was set up that lasted for 120 days when juvenile sporophyte formed. To further assess the stress resistance of acidified algal samples, a third resistance experiment (RE) was conducted. Short-Term Experiment (STE). In the STE, conducted to explore the combined effects of PCO2 and lighton microscopic development, three levels of PCO2 (39 Pa, current CO2 levels; 100 Pa, predicted CO2 levels in 2100; and 200 Pa, predicted CO2 levels in 2300) combined with three light levels (10, 20, and 30 μmol photons m−2 s−1) were set up. The corresponding pH of the adjusted culture medium under each PCO2 was 8.1, 7.8, and 7.5, respectively, which was monitored using a pH meter (Orion ROSS, Thermo Electron). The light source was provided by a 17 W, cool-white lamp. Three levels of light, including low light (LL, 10 μmol photons m−2 s−1), medium light (ML, 20 μmol photons m−2 s−1), and high light (HL, 30 μmol photons m−2 s−1) under each PCO2 were set based on the distance to the light source and measured by a LI-COR-250 light meter (LI-COR, Nebraska). Individual Petri dishes were cultured inside in a CO2 chamber (HP1000G-D), which was programmed to supply 39, 100, and 200 Pa CO2 by bubbling at 10 °C, with a 12 h/12 h light/dark photoperiod. The pHadjusted culture medium was replaced every 3 days to maintain a stable pH level. Variation of parameters in the carbonate system was calculated using the CO2SYS Package based on pH, temperature, salinity, and total alkalinity (TA). TA was measured using the 848 Titrino PLUS automatic titrator (Metrohm) on 100 mL samples filtered with Glass Microfibre Filters (GF/F). The STE was run for 32 days, at which time numerous juvenile sporophytes had formed in the control samples. During this period, the spore germination rate, gametophyte size and sex ratio, fertility, and sporophyte production were monitored for each treatment under a Nikon Eclipse 80i microscope (Nikon). The development B


Article


amplitude-modulated method on a Dual Pulse-AmplitudeModulation (PAM)100 (Walz) system that was connected to a PC, based on WinControl software. PSII photosynthetic parameters, including the optimal PSII quantum yield (Fv:Fm), effective PSII quantum yield (YII), and relative electron transport rate of PSII (rETR), were calculated according to Hiriart-Baer et al.30 The rapid fluorescence lightresponse curve (LRC) was also generated by Dual-PAM-100, from which the light utilization efficiency (α) was obtained automatically. Fresh sporophyte tissue was homogenized and extracted by 90% acetone. After centrifugation, the concentrations of chlorophyll a (Chl a) and chlorophyll c (Chl c) were spectrophotometrically determined following the method of Jeffrey and Humphrey.31 Element Analysis of Tissue Carbon and Nitrogen Content. To determine the tissue C and N content, the algal fragments were dried at 60 °C for 24 h. The dried samples were further homogenized in liquid nitrogen and the C and N content (%) was then determined by using a Vario EL III automatic elemental analyzer (Elementar Analysensysteme); the C/N ratio was determined on the basis of Gutow et al.32 Resistance Experiment (RE). In the laboratory, juvenile sporophytes in seed fibers that had been cultured for 105 days under moderate pH 7.8 and control pH 8.1 in the LTE were prepared and used in three stress resistance experiments. In the stress resistance experiment of high light, three fibers from each pH-exposed group were cultured in f/2 medium at 10 °C with a 12 h/12 h light/dark photoperiod but exposed to light of 400 μmol photons m−2 s−1 for 2 h. In the stress resistance experiment of high temperature, three fibers from each pHexposed group were cultured in f/2 medium at 20 μmol photons m−2 s−1 with a 12 h/12 h light/dark photoperiod but treated to a high temperature of 25 °C for 48 h. In the stress resistance experiment of low pH, three fibers from each pHexposed group were cultured in f/2 medium at 10 °C with 20 μmol photons m−2 s−1 and a 12 h/12 h light/dark photoperiod but treated to saturated CO2-adjusted seawater (pH 5.0) for 12 h. PSII photosynthetic performance was monitored at the initial and end of each treatment. Statistical Analysis. A two-way ANOVA was used to detect the combined effects of PCO2 (three levels, 39, 100, and 200 Pa) and light (10, 20, and 30 μmol photons m−2 s−1) on the microscopic development of S. japonica from meiospore germination to sporophyte formation in the STE using Statistical Program for Social Sciences 17.0 (SPSS, Chicago, IL, USA). A one-way ANOVA was used to analyze the effect of PCO2 on the physiological responses of juvenile sporophytes in the LTE and RE. Post hoc tests were examined using Tukey’s test for two-way ANOVA and Dunnett’s Test for multivariate ANOVA. The assumptions of homogeneity of variance and normality were assessed by scatter plots of residuals and normal curves of residuals, respectively. The significance level was set at 0.05 for all tests, unless otherwise stated.

process was recorded by means of photographs taken with a Nikon CCD DS-file digital camera (Nikon) coupled to a computer and viewed using imaging software: Nikon Image System-Elements (NIS) and Nikon Instech. Percent germination (%) was monitored on day 7 on the basis of germ tube production per 100 randomly selected spores under the 10× objective of a light microscope. Dishes were further cultivated for another 7 days, whereupon the gametophyte sex ratio was determined using the recorded photographs under a 4× objective (>200 gametophytes per replicate). Sex ratio was expressed as the males/(males + females). The female and male gametophyte size was measured with NIS-elements BR software (Nikon). Fecundity is a measure of the number of reproductive females that release eggs that then begin to fertilize. The fecundity ratio was calculated as the number of fertile female gametophytes/total females presented on the 26th day. Moreover, the average number of oogonia presented per female gametophyte was monitored for 50 female gametophytes. The number of fertile female gametophytes and the total number of females were record randomly under the 4× objective of a light microscope. We terminated the fertilization phase on the 32th day, when the reproductive success was determined. This was expressed as the ratio of sporophytes to the total number of female gametophytes. Over the following days, the sporophyte cells divided horizontally and subsequently vertically. Additionally, on the 14th day, when gametophyte differentiation had occurred and been processed, female and male gametophytes were isolated by a glass pipet and cultured separately in f/2 medium for 1 month. During this period, gametophyte clones were propagated vegetatively and then cultured under the same treatments as described above for 1 week. At the end of the experiment, photosystem II (PSII) photosynthetic parameters were determined and analyzed in Figure S1, Supporting Information. Long-Term Experiment (LTE). The LTE, conducted to explore the effects of different PCO2’s (of 39, 100, and 200 Pa) on the physiological response of juvenile sporophytes, was carried out in triplicate in 1000 mL glass beakers using seed fiber plate as a substrate at 10 °C with 20 μmol photons m−2 s−1 and a 12 h/12 h light/dark photoperiod. The same protocol was used to manipulate the pH of the medium as described above (at three levels, 8.1, 7.8, and 7.5). The experiment lasted for 4 months. During the first 30 days, microscopic development was examined using a light microscope until formation of juvenile sporophytes. Then, the seed fiber plates were transferred to an aquarium with 10 L of seawater supplemented with f/2 medium, which was bubbled with various levels of CO2-mixed air to maintain a stable pH and replaced every 5 days to ensure a certain level of nutrients. The growth of juvenile sporophytes was recorded on the 35th, 75th, 90th, and 105th days by means of photographs taken with a Nikon charge-coupled device (CCD) digital camera (Nikon), and the size was quantified using imaging software (NIS-Elements, Nikon Instech). Moreover, the algal abundance (expressed as the average number of individual juvenile sporophytes attached to the seed fiber plate in 1 cm length), PSII photosynthetic parameters, and pigment content, as well as tissue carbon (C) and nitrogen (N) content, were determined at the end of the experiment. PSII Photosynthetic Parameters and Pigmentation. Photosynthetic parameters were measured using the pulse−

■

RESULTS Short-Term Experiment (STE). Seawater chemistry contrasted strongly among the three PCO2 treatments. During the 3 day period of each medium replacement in the STE of 32 days, the continuous variation of was pH was shown in Figure S2, Supporting Information. The average value of seawater chemistry parameters was noted in Table S1, Supporting Information. The pH was noted to increase significantly under C


Article


Figure 1. (a) Germination rate of meiospores, (b) sex ratio of Saccharina japonica under different combinations of PCO2 and light after a 7 day incubation period in the short term experiment; (c) female and (d) male gametophyte sizes of Saccharina japonica under different combination treatments of PCO2 and light after 14 days of incubation in the short term experiment (STE); (e) factorial ANOVA table. Abbreviations: LL = low light; ML = medium light; HL = high light (10, 20, and 30 μmol photons m−2 s−1, respectively). Vertical lines represent standard deviations of five repeat incubations. Values (means ± SD) in bars that have the same letter are not significantly different (p > 0.05).

level had a dramatic effect on gametophyte differentiation and sex ratio (p = 0.000). At PCO2 of 39 Pa, exposure to both LL and HL conditions resulted in a steep decrease in the sex ratio toward more female ones (p = 0.004). At PCO2 of 100 and 200 Pa, although HL increased the sex ratio, the process of gametophyte differentiation was delayed in LL. The male and female gametophytes could not be distinguished from each other. The status of sexual ambiguity in the dimorphic gametophytes was maintained until the end of the STE. A two-way ANOVA indicated that the combined effects of PCO2 and light on female gametophyte size were significant (p =

PCO2 conditions of 39 Pa (p = 0.027), but a slight variation was noted when PCO2’s reached levels of 100 Pa (p = 0.322) and 200 Pa (p = 0.142). Light conditions had an additive influence on seawater chemistry. As light increased from 10 to 20 to 30 μmol photons m−2 s−1, the pH increased significantly at PCO2 levels of 39 Pa (p = 0.001) and 100 Pa (p = 0.019). In the STE, it was noted that increasing PCO2 from 39 to 200 Pa reduced the meiospore germination rate significantly (Figure 1a,e) (p = 0.000). Significant variation in the sex ratio was observed under different combinations of PCO2 and light (Figure 1b,e) (p = 0.024). The average sex ratio in the control group (39 Pa PCO2 + ML) was 0.48 ± 0.12. There was no consistent effect of PCO2 on the sex ratio. In contrast, the light

0.002) (Figure 1e). The high PCO2’s of 100 and 200 Pa combined with HL generally resulted in larger female (p = D


Article


Figure 2. Combined effects of PCO2 and light on the reproductive process of Saccharina japonica, following gametogenesis: (a) combined effects on cell numbers of female gametophytes on the 26th day after gametogenesis; (b) combined effects on fecundity expressed as the ratio of fertile female gametophytes/total females on the 26th day after gametogenesis; (c) combined effects on reproductive success expressed as the ratio of sporophytes to the total number of female gametophytes; (d) numbers of oogonia present on one female gametophyte on the 26th day after gametogenesis cultured under different combinations of PCO2 and light; (e) factorial ANOVA table. Abbreviations: LL = low light; ML = medium light; HL = high light (10, 20, and 30 μmol photons m−2 s−1, respectively). Vertical lines represent standard deviations of five repeat incubations. Values (means ± SD) in bars that have the same letter are not significantly different (p > 0.05).

0.000) and male gametophytes (p = 0.000), compared with control groups (Figure 1c,d). Interestingly, there was an opposite effect of LL on cell size under a PCO2 of 39 Pa, with females being smaller (p = 0.128) and males being larger (p = 0.199) than the controls. Morphologically, the elevated PCO2 changed the cell shape, especially for those under a PCO2 of 100 Pa and HL, where female gametophytes divided into multicellular individuals (shown in Figure S3, Supporting Information). Gametogenesis occurred after 14 days or more, whereupon increasing numbers of fertile female gametophyte began to release eggs. The reproductive process, including the number of female gametophytes, fecundity, and reproductive success, is shown in Figure 2. Results showed that the effects of PCO2 and light on the number of female gametophytes was not consistent, although the combined effect was significant (Figure

2a,e) (p = 0.000). However, high PCO2 levels dramatically decreased the fecundity and reproductive success by the 26th (Figure 2b) and 32nd (Figure 2c) days, respectively. With increasing PCO2 from 39 to 200 Pa, fecundity declined from 58.82 ± 1.56% to 10.48 ± 1.81% (Figure 2b). In addition, few, if any, successful sporophytes were detected under the acidified conditions (only 1.42 ± 1.29% at a PCO2 of 100 Pa and zero at a PCO2 of 200 Pa) (Figure 2c). Interestingly, it was found that more than one oogonia appeared on one female gametophyte at a PCO2 of 100 Pa under ML and HL conditions (Figure 2d) and most of the sporophytes that were detected at a PCO2 of 100 Pa were malformed (shown in Figure S4, Supporting Information). Additionally, although the elevated light increased the fecundity and reproductive success in the control groups (Figure 2b,c), it aggravated the negative effect of high PCO2 levels on fecundity (Figure 2b). E


Article

Environmental Science & Technology Long-Term Experiment (LTE). In the LTE, the juvenile sporophytes grew slowly during the first 90 days but relatively quickly during the last 15 days (Figure 3a). A two-way ANOVA

Figure 3. Effect of PCO2 on the growth of Saccharina japonica juvenile sporophytes in the long term experiment. (a) The effect of PCO2 on the length of juvenile sporophytes during incubation; (b) the effect of PCO2 on algal abundance at the end of the experiment. Values (means ± SD) in bars that have the same letter are not significantly different (p > 0.05).

indicated that the elevated PCO2 levels increased the sporophyte length significantly during the incubation period (df = 6, F = 13.990, p = 0.000). Nevertheless, when PCO2 levels increased from 39 to 200 Pa, the algal abundance decreased significantly (df = 2, F = 7.280, p = 0.025) (Figure 3b). The lowest abundance noted at 200 Pa was 37.6% of the abundance level measured at 39 Pa. At the end of the LTE, PSII photosynthetic parameters, including Fv:Fm, YII, rETR, and α, were determined under different PCO2’s (Figure 4a). It was shown that the parameters Fv:Fm (df = 2, F = 7.944, p = 0.021), YII (df = 2, F = 8.667, p = 0.017), and rETR (df = 2, F = 7.900, p = 0.021) increased significantly with the increase of PCO2 from 39 to 200 Pa, except for α, which peaked at a PCO2 of 100 Pa (df = 2, F = 8.667, p = 0.023). There was a parabolic response in trend of pigment concentration as indicated by levels of chlorophyll: of Chl a (df = 2, F = 55033.784, p = 0.000) and Chl c (df = 2, F = 146.490, p = 0.000) to increased PCO2 levels, with the highest values occurring at a PCO2 of 100 Pa (Figure 4b). The altered PCO2 changed the concentrations and the ratio of Chl a and Chl c in the experiment. In contrast to concentration levels, the ratio of Chl a/Chl c was the lowest at a PCO2 of 100 Pa (df = 2, F = 6.500, p = 0.031). Levels of N and C showed a similar trend to

Figure 4. Effect of PCO2 on (a) PSII photosynthetic performance, (b) pigmentation, and (c) element composition of Saccharina japonica juvenile sporophytes at the end of the long-term experiment. Values (means ± SD) in bars that have the same letter are not significantly different (p > 0.05).

those of Chl a and Chl c (Figure 4c). Increasing PCO2 also resulted in higher N (p = 0.006) and C (p = 0.001) content, particularly at a PCO2 of 100 Pa, when the maximum value was reached. Given the greater enhancement of particulate C, compared with N at a PCO2 of 100 Pa, the ratio of C/N was higher than that measured in the control groups (p = 0.000). Nevertheless, it declined at the highest PCO2 of 200 Pa, when the increase of N was higher than that of C. To further test the stress resistance of acidified juvenile sporophytes, the latter were further treated in the laboratory under high light, high temperature, and saturated CO2-adjusted seawater conditions. Results showed that both samples precultured under a PCO2 of 39 and 100 Pa were sensitive to stress induction, in that Fv:Fm and YII had declined steeply by the end of the experiments (shown in Figure S5, Supporting F


Article


(Table S1, Supporting Information). Various studies have indicated that the carbonate chemistry in cultures is determined by the particular experimental setup and the metabolic activity of the algae.21 Photosynthesis and respiration may, respectively, enhance and decrease pH levels. The results of work carried out by Delille et al. also indicated that an elevation of primary production in Macrocystis kelp beds was closely linked to light availability and seawater chemistry, both of which were strongly influenced by biological activity.38 The relatively high pH, obtained under HL, may be attributed to the high level of photosynthesis. S. japonica Survived Projected OA in LTE. Some researchers have pointed out that immediate exposure to OA in the short term might neglect the possibility of phenotypic plasticity of organisms, which could result in variation in the ability of species to acclimate to changing environmental conditions.39 Most algae have some ability to acclimate to changes in light and temperature, but relatively little is known about the extent of, or mechanisms involved in, responding to OA.40 Morphological and physiological acclimation in algae has also been shown to occur during intermediate-term OA studies. In a red tide alga of Phaeocystis globosa, in the short term, a reduction in pH (7.70) decreased the alga’s photosynthesis and light use efficiency; however, algal growth and photosynthetic activity could recover after long-term acclimation under low pH, by increasing its contents of chlorophyll and carotenoids for energy capture and decreasing its nonphotochemcial energy loss.41 In the present study, S. japonica also displayed acclimation to the elevated PCO2 of 100 and 200 Pa. Although nonsuccessful sporophytes were formed after immediate exposure to OA, they were able to survive after long-term acclimation of 105 days (Figure 3a). Moreover, the high PCO2 levels resulted in larger sporophytes (Figure 3a), higher levels of PSII photosynthesis (Figure 4a), higher Chl a and Chl c content (Figure 4b), and higher C and N content (Figure 4c). Nevertheless, plasticity often comes at a cost.42 The biomass abundance of S. japonica sporophytes was also significantly reduced, reaching levels of only 55% and 38% of that of the control at PCO2 of 100 and 200 Pa, respectively (Figure 3b). In terms of photosynthesis, two ways of C utilization, including a C-concentrating mechanism (CCM) that utilizes HCO3− and passive diffusion of CO2, have been adapted to different extents by various seaweeds.43 An OA induced increase in concentration of HCO3− and of CO2 might benefit algae, whose photosynthesis was carbon limited at the present PCO2.11,13,44 Although the CCM has also been detected in some kelp species, they are carbon limited in typical nearshore environments.13,45 It has been found that predicted OA could increase the rETR(max) and net photosynthesis of young vegetative sporophytes of Laminaria hyperborean (Phaeophyceae).27 With the increase in PCO2, it may be expected that the CCM of S. japonica in the current study would be downregulated, allowing for the allocation of more energy into somatic processes.32 As is the case for other noncalcifying autotrophic organisms, it can be expected that S. japonica would benefit from an enhanced supply of CO2, which would stimulate photosynthesis similar to other reported alga.14,27 Such a situation would also result in faster growth, leading to earlier self-thinning, which would be reflected as lower abundance, while the size of the thalli increased.

Information). Specifically, although the acidified samples had a lower resistance to high light and high temperature conditions, they displayed a higher level of acclimation to CO2-saturated seawater compared with the control groups.

■

DISCUSSION Combined Effects of OA at Different Light Levels on the Microscopic Development of S. japonica in STE. In kelp Laminaria hyperborean, different developmental stages were found to respond differently to elevated PCO2.27 Although oogonium formation and vegetative growth of young sporophytes were enhanced with rising PCO2 levels, germination rates of zoospores, weight increase in adult thalli during sorus formation, and the induction of sporogenesis were not influenced by variations in PCO2. In the present study, altered seawater chemistry had dramatic negative effects on S. japonica during early development, shown as a significant reduction of meiospore germination (Figure 1a), female cell numbers (Figure 2a), fecundity (Figure 2b), and reproductive success (Figure 2c). The sex ratio of kelp gametophytes could be altered under various stressors. It has been reported that increasing temperature in culture led to a higher proportion of males, but more females have been observed under other conditions of stress.33 Similar to M. pyrifera, the increased PCO2 also caused a no-significant decline in the sex ratio (Figure 1b) and a larger size of female and male gametophytes (Figure 1c,d). Previous studies have indicated that kelp gametophytes are capable of utilizing bicarbonate and larger gametophytes of Saccharina latissima were observed during high PCO2 conditions. 26,34 This may have been due to higher CO 2 concentrations, which may have resulted in a decrease in the energy levels, attributed to the carbon concentrating mechanism (CCM), which would have benefitted biomass production.35 It has been noted that OA effects are often light dependent.15,17,36 High PCO2 levels (70 Pa) enhanced the growth of Gracilaria lemaneiformis under intermediate light (160 μmol photons m−2 s−1) but no response occurred under low light (30 μmol photons m−2 s−1).37 In contrast, the OA effect on the coccolithophore Emiliania huxleyi was pronounced under low light and generally modulated by energy availability.35 Results in the present study show that the PCO2 levels interact with light and, at different magnitudes, have a variety of effects on the early life history on S. japonica. To eliminate confusion, in the present study, we focused on OA effects and their modulation by light. We found that high light (HL) magnified the positive effects on the gametophyte diameter in females (Figure 1c) and males (Figure 1d). Low light (LL) conditions had a more damaging negative effect of OA. With the increased PCO2 under LL, the process of gametophyte differentiation was delayed until the end of the STE. As was also the case for similar previous studies, light modulation on OA effects may be attributed to the cellular energy state.35 Additionally, it was found that HL performed a strong stimulating effect on fecundity at present PCO2 of 39 Pa but disappeared at elevated CO2 (Figure 2b). It was inferred that the female gametophyte that was ready to release an egg may be sensitive to declining pH induced by elevated PCO2. Light also had an additive influence on the seawater chemistry during 3 days of medium replacement in the STE G


Article


(2012AA052103, 2014AA022003), and the National Science & Technology Pillar Program (2013BAD23B01).

Elevated carbon supply generally changes algal stoichiometry toward a higher C/N ratio.12 Enhancement of carbon utilization and declined nitrogen uptake has been observed in red alga Gracilaria lemaneiformis.46 In contrast, a lowered C/N ratio, under elevated CO2, was reported in Fucus vesiculosus.30 It has been assumed that, in algae, more efficient photosynthesis, under high PCO2 conditions, may lead to a reallocation of nitrogen to other metabolic processes, resulting in higher tissue N content.47 Consistent with our results, the tissue C and N content increased significantly at a PCO2 of 100 Pa but decreased at a PCO2 of 200 Pa (Figure 4c). The lower content at a PCO2 of 200 Pa may be due to the relatively lower pH level of culture medium, which may result in reduction of the C and N metabolism. Further stress resistance experiments showed that the acidified samples had lower resistance to high light and high temperature but displayed higher acclimation to CO2-saturated seawater compared with the control groups (shown in Figure S5, Supporting Information). It is likely that marine organisms might naturally experience various changing conditions, such as UV radiation, high or low light levels, nutrient limitation and eutrophication, warmer temperatures, and so on. There is thus a need to consider the combined effects of multistressors to provide a more ecologically realistic understanding of global climate change. Therefore, it might be inappropriate to define the direct effects of OA only. Additionally, more in situ experiment should be conducted to investigate further the natural OA effects on the whole life stage of S. japonica.

■

■

(1) Koch, M.; Bowes, G.; Ross, C.; Zhang, X. H. Climate change and ocean acidification effects on seagrasses and marine macroalgae. Global Change Biol. 2013, 19 (1), 103−132. (2) Feely, R. A.; Sabine, C. L.; Lee, K.; Berelson, W.; Kleypas, J.; Fabry, V. J.; Millero, F. J. Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 2004, 305 (5682), 362−366. (3) Doney, S. C. The growing human footprint on coastal and openocean biogeochemistry. Science 2010, 328 (5985), 1512−1516. (4) Caldeira, K.; Wickett, M. E. Anthropogenic carbon and ocean pH. Nature 2003, 425 (6956), 365. (5) Orr, J. C.; Fabry, V. J.; Aumont, O.; Bopp, L.; Doney, S. C.; Feely, R. A.; Gnanadesikan, A.; Gruber, N.; Ishida, A.; Joos, F.; Key, R. M.; Lindsay, K.; Maier-Reimer, E.; Matear, R.; Monfray, P.; Mouchet, A.; Najjar, R. G.; Plattner, G. K.; Rodgers, K. B.; Sabine, C. L.; Sarmiento, J. L.; Schlitzer, R.; Slater, R. D.; Totterdell, I. J.; Weirig, M. F.; Yamanaka, Y.; Yool, A. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 2005, 437 (7059), 681−686. (6) Lotze, H. K.; Lenihan, H. S.; Bourque, B. J.; Bradbury, R. H.; Cooke, R. G.; Kay, M. C.; Kidwell, S. M.; Kirby, M. X.; Peterson, C. H.; Jackson, J. B. C. Depletion, degradation, and recovery potential of estuaries and coastal seas. Science 2006, 312 (5781), 1806−1809. (7) Brewer, P. G.; Peltzer, W. T. Limits to marine life. Science 2009, 324 (5925), 347−348. (8) Hofmann, G. E.; Barry, J. P.; Edmunds, P. J.; Gates, R. D.; Hutchins, D. A.; Klinger, T.; Sewell, M. A. The effect of ocean acidification on calcifying organisms in marine ecosystems: An organism-to-ecosystem perspective. Annu. Rev. Ecol. Evol. Syst. 2010, 41, 127−147. (9) Kroeker, K. J.; Gambi, M. C.; Micheli, F. Community dynamics and ecosystem simplification in a high-CO2 ocean. Proc. Natl. Acad. Sci. 2013, 110 (31), 12721−12726. (10) Kroeker, K. J.; Kordas, R. L.; Crim, R.; Hendriks, I. E.; Ramajo, L.; Singh, G. S.; Duarte, C. M.; Gattuso, J. P. Impacts of ocean acidification on marine organisms: Quantifying sensitivities and interaction with warming. Global Change Biol. 2013, 19 (6), 1884− 1896. (11) Zou, D. Effects of elevated atmospheric CO2 on growth, photosynthesis and nitrogen metabolism in the economic brown seaweed, Hizikia f usiforme (Sargassaceae, Phaeophyta). Aquaculture 2005, 250 (3), 726−735. (12) van de Waal, D. B.; Verschoor, A. M.; Verspagen, J. M. H.; van Donk, E.; Huisman, J. Climate-driven changes in the ecological stoichiometry of aquatic ecosystems. Front. Ecol. Environ. 2010, 8 (3), 145−152. (13) Swanson, A. K.; Fox, C. H. Altered kelp (Laminariales) phlorotannins and growth under elevated carbon dioxide and ultraviolet-B treatments can influence associated intertidal food webs. Global Change Biol. 2007, 13 (8), 1696−1709. (14) Olischläger, M.; Wiencke, C. Ocean acidification alleviates lowtemperature effects on growth and photosynthesis of the red alga Neosiphonia harveyi (Rhodophyta). J. Exp. Bot. 2013, 64 (18), 5587− 5597. (15) Harvey, B. P.; Gwynn-Jones, D.; Moore, P. J. Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidifcation and warming. Ecol. Evol. 2013, 3 (4), 1016−1030. (16) Gao, K. S.; Xu, J. T.; Gao, G.; Li, Y.; Hutchins, D. A.; Huang, B. Q.; Wang, L.; Zheng, Y.; Jin, P.; Cai, X. N.; Häder, D.; Li, W.; Xu, K.; Liu, N.; Riebesell, U. Rising CO2 and increased light exposure synergistically reduce marine primary productivity. Nat. Clim. Change 2012, 2 (7), 519−523. (17) García-Gómez, C.; Gordillo, F. J. L.; Palma, A.; Lorenzoa, M. R.; Segovia, M. Elevated CO2 alleviates high PAR and UV stress in the unicellular chlorophyte Dunaliella tertiolecta. Photochem. Photobiol. Sci. 2014, 13 (9), 1347−1358.

ASSOCIATED CONTENT

* Supporting Information S

Table S1, Variation in seawater chemistry parameters during each medium replacement period in STE; Figure S1, PSII photosynthetic performance of (a,c,e,g) female and (b,d,f,h) male gametophytes of Saccharina japonica under different combination treatments of PCO2 and light after 14 days incubation in the short term experiment; Figure S2, The continuous variation of pH during each medium replacement in STE; Figure S3, Photographs of (a) female and (b) male gametophytes cultured under different combination treatments of PCO2 and light after 14 days incubation in STE; Figure S4, (a) photographs of oogonia present on female gametophyte on the 26th day after gametogenesis cultured under different combinations of PCO2 and light; (b) photographs of malformed sporophytes recorded on the 32nd day after gametogenesis cultured under pH 7.8 combined with ML and HL; Figure S5, Comparable variation in PSII photosynthetic parameters of (a) Fv:Fm and (b) YII during stress treatments in RE. This material is available free of charge via the Internet at http://pubs.acs. org/.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 532 85830360; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (41176153, 31200187, 41306179), HiTech Research and Development Program (863) of China H


Article

Environmental Science & Technology (18) Gachon, C. M.; Strittmatter, M.; Müller, D. G.; Kleinteich, J.; Küpper, F. C. Detection of differential host susceptibility to the marine oomycete pathogen Eurychasma dicksonii by real-time PCR: Not all algae are equal. Appl. Environ. Microbiol. 2009, 75 (2), 322−328. (19) Raybaud, V.; Beaugrand, G.; Goberville, E.; Delebecq, G.; Destombe, C.; Valero, M.; Davoult, D.; Morin, P.; Gevaert, F. Decline in kelp in west Europe and climate. PLoS One 2013, 8 (6), No. e66044. (20) Kroeker, K. J.; Kordas, R. L.; Crim, R. N.; Singh, G. G. Metaanalysis reveals negative yet variable effects of ocean acidification on marine organisms. Ecol. Lett. 2010, 13 (11), 1419−1434. (21) Roleda, M. Y.; Morris, J. N.; McGraw, C. M.; Hurd, C. L. Ocean acidification and seaweed reproduction: Increased CO2 ameliorates the negative effect of lowered pH on meiospore germination in the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae). Global Change Biol. 2012, 18 (3), 854−864. (22) Gaitán-Espitia, J. D.; Hancock, J. R.; Padilla-Gamiño, J. L.; Rivestb, E. B.; Blanchettec, C. A.; Reedc, D. C.; Hofmann, G. E. Interactive effects of elevated temperature and pCO2 on early-lifehistory stages of the giant kelp Macrocystis pyrifera. J. Exp. Mar. Biol. Ecol. 2014, 457, 51−58. (23) Broadgate, W.; Riebesell, U.; Armstrong, C.; Brewer, P.; Denman, K.; Feely, R.; Gao, K. S.; Gattuso, J. P.; Isensee, K.; Kleypas, J.; Laffoley, D.; Orr, J.; Pörtner, H. O.; de Rezende, C. E.; Schmidt, D.; Urban, E.; SCOR; Waite, A.; Valdés,L. Ocean Acidification Summary for Policymakers−Third Symposium on the Ocean in a High-CO2 World, International Geosphere-Biosphere Programme, Stockholm, Sweden, 2013. (24) Bartsch, I.; Wiencke, C.; Bischof, K.; Buchholzc, C. M.; Bucka, B. H.; Eggertd, A.; Feuerpfeild, P.; Hanelte, D.; Jacobsenf, S.; Karezg, R.; Karstend, U.; Molisc, M.; Roledac, M. Y.; Schubertd, H.; Schumannd, R.; Valentina, K.; Weinbergerh, F.; Wieseh, J. The genus Laminaria sensulato: Recent insights and developments. Eur. J. Phycol. 2008, 43 (1), 1−86. (25) Zhang, Y. C.; Jiang, P.; Gao, J. T.; Liao, J. M.; Sun, S. J.; Shen, Z. L.; Qin, S. Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta). Sci. China Ser. C 2008, 51 (12), 1116− 1120. (26) Redmond, S. Effects of increasing temperature and ocean acidification on the microstages of two populations of Saccharina latissima in the Northwest Atlantic. Master’s thesis, University of Connecticut, 2013. (27) Olischläger, M.; Bartsch, I.; Gutow, L.; Wiencke, C. Effects of ocean acidification on different life-cycle stages of the kelp Laminaria hyperborea (Phaeophyceae). Bot. Mar. 2012, 55 (5), 511−525. (28) Tseng, C. K. Algal biotechnology industries and research activities in China. J. Appl. Phycol. 2001, 13, 375−380. (29) Guillemin, M. L.; Sepúlveda, R. D.; Correa, J. A.; Destombe, C. Differential ecological responses to environmental stress in the life history phases of the isomorphic red alga Gracilaria chilensis (Rhodophyta). J. Appl. Phycol. 2013, 25, 215−224. (30) Hiriart-Baer, V. P.; Arciszewski, T. J.; Malkin, S. Y.; Guildford, S. J.; Hecky, R. E. Use of pulse-amplitude-modulated fluorescence to assess the physiological status of Cladophora sp. along a water quality gradient. J. Phycol. 2008, 44 (6), 1604−1613. (31) Jeffrey, S. W.; Humphrey, G. F. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 1975, 167, 191−194. (32) Gutow, L.; Rahman, M. M.; Bartl, K.; Saborowski, R.; Bartsch, I.; Wiencke, C. Ocean acidification affects growth but not nutritional quality of the seaweed Fucus vesiculosus (Phaeophyceae, Fucales). J. Exp. Mar. Biol. Ecol. 2014, 453, 84−90. (33) Oppliger, L. V.; Correa, J. A.; Faugeron, S.; Beltrán, J.; Tellier, F.; Valero, M.; Destombe, C. Sex ratio variation in the Lessonia nigrescens complex (Laminariales, Phaeophyceae): Effect of latitude, temperature, and marginality. J. Phycol. 2011, 47 (1), 5−12.

(34) Zhang, X.; Hu, H.; Tan, T. Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatif ida (Phaeophyceae). Phycologia 2006, 45 (6), 642−647. (35) Rokitta, S. D.; Rost, B. Effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi. Limnol. Oceanogr. 2012, 57 (2), 607−618. (36) Lefebvre, S. C.; Harris, G.; Webster, R.; Leonardos, N.; Geider, R. J.; Raines, C. A.; Read, B. A.; Garrido, J. L. Characterization and expression analysis of the Lhcf gene family in Emiliania huxleyi (Haptophyta) reveals differential responses to light and CO2. J. Phycol. 2010, 46 (1), 123−134. (37) Zou, D. H.; Gao, K. S. Effects of elevated CO2 on the red seaweed Gracilaria lemaneiformis (Gigartinales, Rhodophyta) grown at different irradiance levels. Phycologia 2009, 48 (6), 510−517. (38) Delille, B.; Borges, A. V.; Delille, D. Influence of giant kelp beds (Macrocystis pyrifera) on diel cycles of pCO2 and DIC in the SubAntarctic coastal area. Estuarine, Coastal Shelf Sci. 2009, 81 (1), 114− 122. (39) Tatters, A. O.; Schnetzer, A.; Fu, F.; Lie, A. Y.A.; Caron, D. A.; Hutchins, D. A. Short-versus long-term responses to changing CO2 in a coastal dinoflagellate bloom: Implications for interspecific competitive interactions and community structure. Evolution 2013, 67 (7), 1879−1891. (40) Pespeni, M. H.; Barney, B. T.; Palumbi, S. R. Differences in the regulation of growth and biomineralization genes revealed through long-term common-garden acclimation and experimental genomics in the purple sea urchin. Evolution 2013, 67 (7), 1901−1914. (41) Chen, S. W.; Beardall, J.; Gao, K. S. A red tide alga grown under ocean acidification up-regulates its tolerance to lower pH by increasing its photophysiological functions. Biogeosci. Discuss. 2014, 11 (5), 6303−6328. (42) Godbold, J. A.; Calosi, P. Ocean acidification and climate change: Advances in ecology and evolution. Philos. Trans. R. Soc., B 2013, 368 (1627), 20120448. (43) Raven, J. A.; Cockell, C. S.; La Rocha, C. L. The evolution of inorganic carbon concentrating mechanisms in photosynthesis. Philos. Trans. R. Soc., B 2008, 363 (1504), 2641−2650. (44) Diaz-Pulido, G. M.; Gouezo, M.; Tilbrook, B.; Dove, S.; Anthony, K. High CO2 enhances the competitive strength of seaweeds over corals. Ecol. Lett. 2011, 14 (2), 156−162. (45) Axelsson, L.; Mercado, J.; Figueroa, F. Utilization of HCO3− at high pH by the brown macroalga Laminaria saccharina. Eur. J. Phycol. 2000, 35 (1), 53−59. (46) Xu, Z.; Zou, D.; Gao, K. Effects of elevated CO2 and phosphorus supply on growth, photosynthesis and nutrient uptake in the marine macroalga Gracilaria lemaneiformis (Rhodophyta). Bot. Mar. 2010, 53 (2), 123−129. (47) Falkenberg, L. J.; Russell, B. D.; Connell, S. D. Contrasting resource limitations of marine primary producers: Implications for competitive interactions under enriched CO2 and nutrient regimes. Oecologia 2013, 172 (2), 575−583.

I


Effects of CO2 and Seawater Acidification on the Early Stages of

Recommend Documents