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Growth, biochemical properties, and chlorophyll fluorescence of symbiotic and free-living dinoflagellates in response to ammonium enrichment
Journal of Experimental Marine Biology and Ecology 438 (2012) 1–6
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Growth, biochemical properties, and chlorophyll fluorescence of symbiotic and free-living dinoflagellates in response to ammonium enrichment Yumi Fuchinoue a, Tomoyo Katayama a, Mitsuko Obata a, Ai Murata a, Robert Kinzie III b, Satoru Taguchi a,⁎ a b
Soka University, Faculty of Engineering, Laboratory of Biological Oceanography, 1–236 Tangi-Cho, Hachiouji, Tokyo 192-8577, Japan Department of Zoology and Hawaii Institute of Marine Biology, University of Hawaii at Manoa, Honolulu, HI 96822, USA
a r t i c l e
i n f o
Article history: Received 27 May 2012 Received in revised form 1 October 2012 Accepted 3 October 2012 Available online 25 October 2012 Keywords: Chlorophyll a Efficiency of red light absorption Maximum quantum efficiency Operating efficiency Package effect
a b s t r a c t The growth, biochemical properties, and chlorophyll fluorescence of cultures of the symbiotic Symbiodinium species from clade A, B, and F and the free-living dinoflagellate Prorocentrum minimum in response to ammonium enrichment were examined following transfer from ammonium-limited to ammonium-enriched conditions. Cultures were grown under a light:dark (L:D) cycle of 300 μmol photons m−2 s−1. Cell growth was initiated on day 1, reached saturation on approximately day 5, and decreased due to ammonium on day 10 (ammonium limitation experiments). Cells were removed from the ammonium limitation series on days 1, 5, and 10 and were enriched to 50 μM ammonium followed by incubation for 4 days (ammonium enrichment experiments). The cell density, cellular carbon, nitrogen, chlorophyll a contents, and chlorophyll fluorescence were measured for 14 days in the ammonium limitation experiments and for 4 days in the ammonium enrichment experiments. The results showed that ammonium disappeared rapidly once the cells were enriched, and the removal rates of ammonium were related to the growth rate under the ammonium enriched conditions. No apparent enhancement of the photosynthetic efficiency (Fv/Fm) in the ammonium-limited conditions was observed for the symbiotic dinoflagellates, whereas a recovery of Fv/Fm from the ammonium-limited conditions was observed within two days for the free-living dinoflagellate. The significant but different linear relationships between the initial slope (α) and the maximum electron transport rate (ETRmax) of ETR vs photon flux density (PFD) curve, which is equivalent to the adaptation index (Ek), observed for Symbiodinium and Prorocentrum may suggest that these species require a different range of PFD for photoacclimation and exhibit a different physiological tolerance to ammonium toxicity. Observations of acclimation and adaptation using variable fluorescence would be useful for understanding the photophysiological strategies of symbiotic and free-living dinoflagellates. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Free-living dinoflagellates may migrate vertically within their photic environment (Wyatt and Horwood, 1973), experiencing a light regime that can change greatly within hours or even minutes. Dinoflagellates belonging to the genus Symbiodinum are associated with a wide range of marine invertebrates, including reef corals, anemones, jellyfish and giant clams. While some invertebrates that host Symbiodinium are planktonic, most are benthic and most of these species, such as corals, clams and anemones are sessile. Therefore, these algae may be exposed to a more predictable light environment with a narrower range of intensities. Depending on their host, Symbiodinium must adapt to the photobehavior of host animals. In associations with planktonic hosts, such as jellyfish, zooxanthellae may experience variable photon fluxes in natural environments (Iglesias-Prieto and Trench, 1994). Algae in symbioses with hosts living in shallow water may experience photodamage (Warner et al., ⁎ Corresponding author. Tel./fax: +81 42 691 8002. E-mail address: [email protected] (S. Taguchi). 0022-0981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2012.10.001
1999). Loss of these damaged Symbiodinium is termed coral bleaching (Glynn, 1996). However, under certain conditions, Symbiodinium cells may exist outside of a host and, thus, experience light conditions more like those of free living planktonic algae. Many invertebrates that host Symbiodinium as adults are not provided with algal symbionts early in their live history as spawned eggs or early-stage larvae. Hosts with this life history pattern (horizontal transition) must acquire algal symbionts if they are to survive (Baird et al., 2009; Weis et al., 2001). Where such free-living Symbiodinium live, and how long they can exist outside of their hosts are questions that are not well understood, but they must exist to form symbiosis with early stages of hosts with this life history. While photophysiological studies have not been performed on populations of naturally occurring free-living Symbiodium cells, experiments with newly infected larvae of the coral Acropora have shown damaging effects at light levels typical of the upper part of the water column (Yakovleua et al., 2009). The source of this pool of Symbiodinium in the environment is not well understood, but it is known that hosts release algal cells into the
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ambient water (Hoegh-Guldberg et al., 1987; Steele, 1977; Stimpson and Kinzie, 1991) and that these cells are viable (Bhagooli and Hidaka, 2004; Ralph et al., 2001). Santos et al. (2009) have demonstrated one possible pathway for these Symbiodinium cells to be produced and released by a host. Thus, while dinoflagellates of the genus Symbiodinium may generally be held within cells of benthic hosts, at least some will experience a photic environment more typical of free-living planktonic forms. Another potential difference between the biology of free-living dinoflagellates and symbiotic forms is related to the ambient nutrient levels these species experience. Free-living phytoplankton can be exposed to a range of nutrient concentrations, but those in tropical oceans generally occur in low-nutrient waters except for polluted coastal waters. Nitrogen levels can often be as low as 0.2 μM for nitrate and undetectable for ammonium (Sharp, 1983). In contrast, algae living in host animal cells may experience very high levels of ammonium. Symbiodinium are known to utilize nutrients such as ammonium released by their host and produce organic matter through photosynthesis, following which the host animals assimilate the organic matter (Hoegh-Guldberg and Williamson, 1999). The concentration of ammonia in the cells of host animals can be as high as 50 μM, and this ammonia can be utilized by Symbiodinium cells (Taguchi and Kinzie, 2001; Yellowlees et al., 1994). However, such concentrations may be toxic for free-living dinoflagellates (Change and McClean, 1997; Leong et al., 2009). Even within Symbiodinium, some variability in acclimation among clades can be expected, as indicated by Robinson and Warner (2006). All cellular chemical constituents are subject to experiencing changes in their cellular contents due the nitrogen availability. Such acclimation may occur in terms of the organisms’ light absorption ability and the distribution of light energy among the photosynthetic machinery in the Photosystem II (Laurion and Roy, 2009). In this study, three clades of Symbiodinium and one free-living dinoflagellate were employed to study acclimation to ammonium enrichment in terms of the maximum quantum efficiency and operating efficiency, as determined by pulse-amplitude-modulated fluorometry (Genty et al., 1989; Schreiber et al., 1994; Shelly et al., 2010). Batch cultures were set up to follow chlorophyll fluorescence together with cell growth under ammonium-limited and ammonium-enriched conditions. The ammonium limitation experiments were designed to produce ammonium-exhausted cells close to growth saturation. Cells were collected from the ammonium limitation experiment on days 1, 5 and 10 and used to initiate the ammonium enrichment experiments. These days corresponded to initial (day 1), log phase (day 5), and fully nitrogen depleted (day 10) cells. The object of this study was to elucidate differences in the responses of the four dinoflagellates (symbiotic Symbiodinium species from clades A, B, and F and the free-living Prorocentrum minimum) to changes in nutrient levels and how these changes were reflected in the photobiology of these cells. The maximum photosynthetic efficiency (Fv/Fm) and photosynthetic characteristics, such as the initial slope (α) and ETRmax estimated from the electron transport rate, were employed to study the maximum photochemical efficiency of open reaction center II (RCIIs) in the dark and the effective photochemical efficiency of RCIIs under light, respectively. We hypothesize that photophysiological difference present between symbiotic and free-living dinoflagellates might be caused by adaptation and acclimation to light regime. 2. Materials and methods 2.1. Culture Strains of zooxanthellae were isolated from three different host animals: clade A from Cassiopea sp.; clade B, from Aiptasia pulchella; and clade F from Montiopora verrucosa (Santos et al., 2004). All strains were obtained from the culture collection at the Hawaii Institute of Marine
Biology, University of Hawaii at Manoa. A culture of the free-living dinoflagellate, Prorocentrum minimum was obtained from the Culture Collection Center at the National Institute of Environmental Science, Japan. Initial cultures were maintained in semi-continuous batch mode in f/2 medium with 50 μM ammonium as the nitrogen source, instead of nitrate (Guillard and Ryther, 1962), at 25 °C, which is the optimum growth temperature as indicated by Suwa et al. (2008), in a light-controlled incubator (Eyela, FLI-301N, Japan) for more than three dilutions in semi-continuous mode. Each dilution lasted for one week. At the end of each dilution, the ammonium concentration was less than 10 μM. The experimental medium was prepared with surface seawater collected in coastal waters off Manazuru, Japan. The mixture of seawater had been aged for longer than one year in the dark at room temperature. The nitrogen concentrations in the aged seawater were below detection limits prior to its use in the experiments. Phosphate, trace metals, and vitamins were added as per the f/2 formulation (Guillard and Ryther, 1962). Photosynthetically active radiation (PAR) was provided using cool-white fluorescence tubes (National, FL40SW, Japan) in 12 h light and 12 h dark cycles at 300 μmol photons m−2 s−1, as determined by a 4π sensor submerged in the culture medium in the culture vessel (Biospherical Instruments, QSL100, USA). 2.2. Incubation experiments and sampling Ammonium-limited experiments were initiated by the addition of 25 mL of the stock cultures to 1 L of the nitrogen depleted f/2 medium. This resulted in an initial cell density of ~ 600 cells mL −1. This procedure did not raise the ammonium concentration appreciably in the experimental bottles (b0.2 μM). The goal of this phase of the study was to maintain ammonium-limited cells for the early part of incubation and produce completely ammonium-depleted cells by day 10 in batch culture mode (ammonium limitation experiments). The subsequent enrichment experiments were performed using cells from the batch culture on days 1, 5, and 10. These cells, in various stages on nitrogen depletion, were used to initiate cultures with a final concentration of 50 μM (ammonium enrichment experiments). For the ammonium limitation experiments, samples were collected daily for 14 days, except for on day 3, 9, and 12. For the ammonium enrichment experiments, cells sampled at the beginning (day 1) and the end of the log phase (day 5) and the stationary phases (day 10) of growth were used to determine the response of the cells to ammonium enrichment for the subsequent four days. 2.3. Cell counts On each sampling day, subsamples were collected to estimate cell density by counting cells using an inverted microscope (Olympus, IMT-2, Japan) (Hasle, 1978). A calibrated hematocytometer (Erma, 35103000, Japan) was employed to count the cells (Guillard, 1978). A minimum of 400 cells per field were counted (Lund et al., 1958). The growth rate was calculated according to the method described by Guillard (1978). Cell size was measured in the ammonium limitation experiments. 2.4. Ammonium analysis Subsamples were collected for analysis of ammonium. Seawater samples were filtered through a 0.45 μm membrane filter (Millipore, Mylex HA, Ireland), and the concentration of ammonium in the filtrates was determined using methodology described in Parsons et al. (1984). The rate of ammonium removal from the medium (Da) was calculated using the following equation: Da ¼ ðlnNH0 –lnNHt Þ=t
ð1Þ
where NH0 and NHt are the ammonium concentrations at time 0 and t, respectively.
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2.5. Particulate organic carbon and nitrogen
2.8. Statistical analysis
Subsamples for analysis of particulate organic carbon and nitrogen were filtered onto glass fiber filters (Whatman, GF/F, England), which had been pre-combusted at 500 °C for 2 h. The amounts of organic carbon and nitrogen on the filters were analyzed using a CHN elemental analyzer (Thermo Finnigan, Flush EA1112 series, Germany). The concentrations of organic carbon and nitrogen were calibrated against acetanilide as a standard (Nagao et al., 2001).
The cellular contents of particulate organic carbon and nitrogen and Chl a were employed to calculate the chemical ratios. The mean with one standard deviation of triplicate values was always calculated and is reported throughout the present paper. Student's t-test and analysis of variance were implemented using Sigma-Plot (System software, version 11.2, San Jose, USA). 3. Results
2.6. Pigment analysis 3.1. Nutrient response Subsamples were collected for analysis of pigments using high-performance liquid chromatography (Beckman, System Gold 168 Diode Array Detector, Fullerton, USA) with a C18 reverse-phase ultrasphere 3 μm column as described by Head and Horn (1993). The obtained peaks were quantified using chlorophyll a (Chl a) standards obtained from the International 14C agency (Denmark). 2.7. Variable fluorescence To measure variable fluorescence, subsamples of the cell suspensions were collected and 3 mL of the sample was transferred to the bottom of a quartz cell in a pulse amplitude modulated fluorometer (PAM, Walz, Water-Pam, Germany) immediately after acclimation to the subsamples for 30 min at 25 °C in a dark laboratory, as described by Obata et al. (2009). Once the maximum fluorescence yield in the dark-adapted state (F0) had become stable, a saturating pulse of 1200 μmol photons at 655 nm m−2 s−1 was supplied to determine the maximum fluorescence yield (Fm) after dark acclimation. The maximum quantum efficiency of PSII (Fv/Fm) for the dark-adapted state was calculated using the method of Schreiber et al. (1994): F v =F m ¼ ðF m –F 0 Þ=F m
ð2Þ
The ammonium concentrations were below detection limits throughout the ammonium limitation experiments. When the cells were exposed to 50 μM ammonium enrichment, the ammonium concentration declined immediately (Fig. 1). The ammonium concentration decreased to approximately 15–25 μM at the end of the ammonium enrichment experiments conducted on day 1 and day 5 and approximately 25–40 μM at the end of the ammonium enrichment experiments on day 10. Therefore, the removal rate decreased with the length of the ammonium limitation experiments and ranged from 42% for the free-living dinoflagellate to 54% for symbiotic Symbiodinium cells from clade A. 3.2. Growth rate The growth rates for symbiotic dinoflagellates ranged from 0.40 ± 0.07 day −1for clade B to 0.55 ± 0.06 d −1 for clade F. The growth rate of Prorocentrum was lower, averaging 0.29 ± 0.05 day −1. When ammonium was introduced to the ammonium-limited cultures, the growth rates of symbiotic Symbiodinium cells from clades A and B obtained from the ammonium limitation experiment initiated on 60
0 0 0 0 F q =F m ¼ F m –F =F m
ð3Þ
The electron transport rate (ETR) was obtained using the following equation (Genty et al., 1989): 0 0 ETR ¼ 0:5f F q =F m PFD
A 40
Ammonium concentration (μM)
The maximum photosynthetic efficiency (Fv/Fm) was obtained under actinic light. For this purpose, light-response curves were generated in triplicate for each culture. Aliquots of 3 mL were taken directly from the experimental bottle, then dark-acclimated for 30 min and the variable fluorescence was determined to adjust the zero level of actinic light. After zero adjustment, the samples were illuminated by the red light-emitting diodes of the actinic light source. The minimum and maximum fluorescence yields in the light-adapted state (F and F’m) were measured with a saturation pulse with a duration of 1 s at a photosynthetic photon flux density of 1835 μmol photons m−2 s−1, following at least 30 s of exposure to actinic light. The maximum fluorescence yields were determined at 12 light levels of actinic light. The PSII operating efficiency (F′q/F′m) was calculated using the equation of Genty et al. (1989):
20
0 60
B 40
ð4Þ 20
where 0.5 is a factor to account for equal number of photons reaching on PSI and PSII, f is the proportional factor (s m 2 μmol photons−1); and PFD is the intensity of actinic light (μmol photons m −2 s −1). The initial slope (α) and the maximum ETRmax were estimated by fitting the following equation (Webb et al., 1974): ETR ¼ ETR
B
max ð1−exp½−PDF=Ek Þ
ð5Þ
where Ek is the adaptation index, which defines the ratio of ETRmax to α.
0
0
2
4
6
8
10
12
14
Days Fig. 1. Changes in the ammonium concentration in the ammonium enrichment experiments initiated on days 1, and 5, and 10 of the ammonium limitation experiments for symbiotic Symbiodinium cells from clades A (A) and Prorocentrum (B).
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Relative growth rate
2.0
1.5
1.0
0.5
0.0 0
1
5
10
Days Fig. 2. Relative growth rates of clade A (open bar), clade B (left hatched bar), clade F (right hatched bar), and Prorocentrum (crosshatched bar) in the ammonium enrichment experiments initiated on days 1, 5, and 10. All values are standardized against the growth rate under the ammonium-limited conditions as indicated on day 0.
day 1 increased. The relative growth rates of clade F changed little until a decrease in the cultures was initiated on day 10. Prorocentrum showed lower growth rates in all three enrichment experiments (Fig. 2). The reduction in the relative growth rates of the three Symbiodinium clades on day 10 compared to day 1 was approximately 58 ± 7%. The growth rate of the free-living dinoflagellate was already suppressed by 30% on day 1 and by 40% on days 5 and 10.
the observed trend was similar within the symbiotic dinoflagellates. The cellular carbon and nitrogen contents of all Symbiodinium clades and the free-living dinoflagellate at the beginning of the ammonium enrichment experiments were approximately 1 ngC cell −1 and 0.1 ngN cell −1, respectively. Regardless of the ammonium enrichment experiment starting on days 1, 5, or 10 of the ammonium limitation experiments, the cellular carbon and nitrogen contents in the free-living dinoflagellate at the end of the ammonium enrichment experiments were significantly higher than those for all Symbiodium clades (p b 0.05). The cellular contents had decreased by more than 50% at the end of the ammonium enrichment experiment initiated on day 1. The cellular contents at the end of the ammonium enrichment experiment initiated on day 10 were higher, reaching more than twice those in the experiments initiated on day 1 in all Symbiodinium clades, whereas the contents in Prorocentrum increased by only 130%. The cellular Chl a contents on day 0 of all ammonium enrichment experiments initiated on days 1, 5, and 10 of the ammonium limitation experiments were similar among the species at approximately 1.6 pg Chl a cell −1and decreased to less than 0.2 pg cell −1 in the ammonium enrichment experiment initiated on day 10 (Fig. 3). The cellular Chl a contents of all species at the end of the ammonium enrichment experiment initiated on day 1 decreased significantly from those on day 0 due to cell division (p b 0.05) and recovered to values three times higher than those on day 0 of the ammonium enrichment experiment initiated on day 10. The highest recovery on day 10 was observed for the free-living dinoflagellate.
3.3. Cellular carbon, nitrogen, and Chl a contents 3.4. Maximum photosynthetic efficiency (Fv/Fm) Representative results are only shown for symbiotic Symbiodinium cells from clade A and the free-living Prorocentrum in Fig. 3 because
0.10
The maximum photosynthetic efficiency observed in Prorocentrum under the ammonium-limited conditions was approximately two-thirds of that observed in Symbiodinium. The significantly different Fv/Fm values indicates a species specific photosynthetic capacity. A continuous decrease in Fv/Fm was observed for Symbiodinium, whereas a sudden decrease in Fv/Fm around day 4 was observed for Prorocentrum. When the cells were transferred to the ammonium-enriched conditions, Fv/Fm showed three significant trends. First, Fv/Fm did not respond to the ammonium enrichment and remained at similar levels during the ammonium enrichment experiment, except in Prorocentrum. Second, Fv/Fm generally decreased with the duration of the ammonium limitation experiments from day 1 to day 10 for Symbiodinum. The ratio of the mean Fv/Fm (with one standard deviation) observed in the ammonium enrichment experiment initiated on day 10 to that on day 1 was 0.91±0.02, indicating a decrease in photosynthetic capability. Third, the maximum Fv/Fm value observed for Prorocentrum was less than 0.4, possibly due to ammonium toxicity, whereas that for Symbiodinium was higher than 0.45.
0.05
3.5. Electron transport rate-photon flux density curve
(ngC cell-1)
Cellular Carbon
1.5
A
B
C
D
1.0
0.5
0.0
0.15
(ngN cell-1)
Cellular Nitrogen
0.20
0.00 2.0
2.0
F
(pg cell-1)
Cellular Chl a
E 1.5
1.5
1.0
1.0
0.5
0.5
0.0
1
5
Days
10
0.0
1
5
10
Days
Fig. 3. Cellular carbon (A and B), nitrogen contents (C and D), and Chl a contents (E and F) at the beginning (dark) and the end of the ammonium enrichment experiment (stippled) in symbiotic Symbiodinium cells from clade A (A, C, and E) and Prorocentrum (B, D, and F). Vertical bars indicate one standard deviation.
When cells were transferred to the ammonium-enriched condition, both α and ETRmax either decreased with time or remained at a level similar to the initial value during the enrichment experiment for both Symbiodinium and Prorocentrum regardless of the duration of the ammonium enrichment experiments. The mean α value for Prorocentrum was the lowest among all of the species investigated in the present study (p b 0.05), whereas the mean ETRmax of Prorocentrum was not different from any of the clades of Symbiodinium (Table 1). The mean α and ETRmax were also not significantly different among the three clades of Symbiodinium. The initial slope (α) and ETRmax ranged from 0.23 to 0.38 e (μmol photons m−2 s−1) −1 and from 30 to 88 e for Symbiodinium, respectively, whereas they ranged from 0.13 to 0.22 e (μmol photons m −2 s−1)−1 and from 26 to 40 e for Prorocentrum, respectively. A significant linear relationship between α and ETRmax was obtained for both Symbiodinium and Prorocentrum (p b 0.01, Fig. 4). The slopes with 95% confidence limits, indicative of the
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4.2. PSII photochemistry Fv/Fm
Table 1 Mean with one standard deviation of α, ETRmax, and EK of the ETR PFD curve for clades A, B, and F and Prorocentrum minimum. Species
α
ETRmax
EK
A B F Prorocentrum minimum
0.267 ± 0.0164 0.328 ± 0.0965 0.304 ± 0.0407 0.170 ± 0.0223
43.8 ± 8.2 57.5 ± 9.0 56.9 ± 15.1 35.1 ± 7.8
165 ± 31 190 ± 20 188 ± 36 210 ± 52
5
Fluorescence responses to ammonium enrichment can vary considerably with the degree of ammonium limitation during the growth of cells, as suggested by Young and Beardall (2003). In the present study, the maximum quantum efficiency (Fv/Fm) indicates a distinctively different pattern of photoacclimation in the transition from ammonium-limited to ammonium-enriched conditions between symbiotic and free-living dinoflagellates. The initial Fv/Fm values are comparable with those previously reported for symbiotic and free-living dinoflagellates under similar conditions (Laurion and Roy, 2009; Suwa et al., 2008), indicating a high potential photosynthetic performance for Photosystem II in actively growing cells. The continuous decrease in Fv/Fm detected in the first half of the ammonium-limited conditions, particularly for Symbiodinium is similar to what was observed by Parkhill et al. (2001) and Young and Beardall (2003). The slope of the temporal decrease in Fv/Fm could be similar under the ammonium- limited conditions for Symbiodinium. After a sudden decrease around day 4, the slight decrease in Fv/Fm observed in the second half of the ammonium-limited conditions for Prorocentrum suggests the possible occurrence of a physiological change in nitrogen metabolism, which will be discussed in the next section.
photoadaptation index, were 262± 32 μmol photons m −2 s −1 for Symbiodinium and approximately 2 times higher than 138 ± 24 μmol photons m−2 s−1for Prorocentrum.
4. Discussion 4.1. Physiological characteristics Autotrophic microalgae acclimate to various environmental conditions by altering their cellular Chl a content (Behrenfeld et al., 2004). Our batch culture experiment involving ammonium limitation showed that the cellular Chl a contents were reduced through increased cell divisions to harvest more photons per Chl a molecule by reducing self-shading within a cell. The growth rates of symbiotic dinoflagellates obtained in the early part (days 1 and 5) of the ammonium limitation experiments were within the range reported for algae within host animals (Chang et al., 1983). By day 10, the cells are in a state of ammonium starvation, and cultures show adverse physiological effects (e.g., Berman-Frank and Dubinsky, 1999). The present cellular Chl a content estimates obtained in the stationary phase (day 10) of growth, ranging from 0.19 to 0.099 pg Chl a cell−1, are several time higher than those obtained at the beginning of the growth phase for both the symbiotic and free-living dinoflagellates (Chan, 1978; Chang et al., 1983; Meson and Sweeney, 1982; Taguchi and Kinzie, 2001). The negative relationship between the cellular contents of Chl a and the growth rate observed on days 1 and 5 is interpreted as the cellular Chl a content being regulated by cell growth as observed by Prezelin (1976). The free-living dinoflagellates are so sensitive to excess ammonium (Goldman and Glibert, 1983) that their growth rate is depressed under the ammonium-enriched conditions, resulting in large cells. The present study suggests that phylogenetically different responses to ammonium concentrations and toxicity are intrinsic differences between symbiotic and free-living dinoflagellates.
4.3. ETR–PDF curve The significant liner relationship between α and ETRmax may suggest that the photosynthetic strategy is similar among Symbiodinium species but different between Symbiodinium and Prorocentrum. The significantly different slopes of the linear relationships and ranges of both values between the two taxonomic groups may be caused by not only by different light ranges being required for photoacclimation but also by different physiological tolerances to ammonium toxicity. Symbiodinium may employ Ek-dependent acclimation (Behrenfeld et al., 2004) to utilize a wide range of irradiances with ample ammonium supplies. Ek-dependent acclimation is also considered as a strategy for Prorocentrum when ammonium is used as the nitrogen source, such as in the present study. When ammonium is enriched, Prorocentrum cells grown in log-phase under the ammonium-limited conditions, can maintain a strategy of Ek-dependent acclimation because they maintain the ability to divide, even at a slow rate due to ammonium toxicity. When ammonium enrichment is applied to ammonium-exhausted Prorocentrum cells, cells with high cellular Chl a contents may adapt an Ek-dependent acclimation strategy to avoid a less efficient photosynthesis phase under low irradiance, while achieving efficient photosynthesis under high irradiance because cell division is extremely depressed due to ammonium toxicity. Ammonium toxicity has been known to occur at concentrations
100
A
B
ETRmax
80
60
40
20
0 0.0
0.1
0.2
0.3
Initial slope ( α )
0.4
0.5
0.0
0.1
0.2
0.3
0.4
0.5
Initial slope ( α )
Fig. 4. Relationship between the initial slope (α) and ETRmax of the ETR PFD curve of Prorocentrum (A) and Symbiodinium (B). Solid circles, open circles, and solid reversed triangles indicate the values on days 1, 5, and 10 for Prorocentrum (A). Solid circles, open circles, and solid reversed triangles indicate values on days 1, 5, and 10 for clade A. Open triangles, solid squares, and open squares indicate values on days 1, 5, and 10 for clade B. Solid diamonds, open diamonds, and solid triangles indicate values on days 1, 5, and 10 for clade F.
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higher than 25 μM for dinoflagellates (Goldman and Glibert, 1983; Leong and Taguchi, 2004). Under the ammonium concentration of 50 μM employed in the present study, low values of μ and α are observed for Prorocentrum caused by ammonium toxicity, but not for Symbiodinium. This leads to a lower range of values and a gentler slope of the linear relationship between α and ETRmax for Prorocentrum than Symbiodinium. Our results are limited to a single experiment involving three clades and one free-living dinoflagellate, and investigation of Fv/Fm, α, and ETRmax as a function of ammonium stress for other clades and free-living dinoflagellates is still required as discussed by Santos et al. (2001). Acknowledgements The present study was partly supported by a grant-in-aid provided by Soka University for ST. All assistance at Scripps Institution of Oceanography to prepare an earlier version of this manuscript is greatly appreciated. [SS] References Baird, A.H., Guest, J.R., Willis, B.L., 2009. Biogeographical and evolutionary patterns in the reproductive biology of scleractinian corals. Ann. Rev. Ecol. Evol. Syst. 40, 551–571. Behrenfeld, M.J., Prasil, O., Babin, M., Bruyant, F., 2004. In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis. J. Phycol. 40, 4–25. Berman-Frank, I., Dubinsky, Z., 1999. Balanced growth in aquatic plants: myth or reality? Bioscience 49, 29–36. Bhagooli, R., Hidaka, M., 2004. Release of zooxanthellae with intact photosynthetic activity by the coral Galaxea fascicularis in response to high temperature stress. Mar. Biol. 145, 329–337. Chan, A.T., 1978. Comparative physiological study of marine diatoms and dinoflagellates in relation to irradiance and cell size. I Growth under continuous light. J. Phycol. 14, 396–402. Chang, S.S., Prezelin, B.B., Trench, R.K., 1983. Mechanisms of photoadpatation in three strains of the symbiotic dinoflagellate Symbiodinium microadriaticum. Mar. Biol. 76, 219–229. Change, F.H., McClean, M., 1997. Growth responses of Alexandrium minimum (Dinophyceae) as a function of three different nitrogen sources and irradiance. N. Z. J. Mar. Freshw. Res. 31, 1–7. Genty, B., Briantais, J., Baker, N., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87–92. Glynn, P.W., 1996. Coral reef bleaching: facts, hypotheses and implications. Glob. Chang. Biol. 2, 495–509. Goldman, J.C., Glibert, P.M., 1983. Kinetics of inorganic nitrogen uptake. In: Carpenter, E.J., Capone, D.G. (Eds.), Nitrogen in the Marine Environment. Academic Press, New York, pp. 233–274. Guillard, R.R.L., 1978. Counting slides. In: Sournia, A. (Ed.), Phytoplankton Manual. UNESCO, Paris, pp. 182–193. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleave) Gran. Can. J. Microbiol. 8, 229–239. Hasle, G.R., 1978. Using the inverted microscope. In: Sournia, A. (Ed.), Phytoplankton Manual. UNESCO, Paris, pp. 191–196. Head, E.J.H., Horn, E.P.W., 1993. Pigment transformation and vertical flux in an area of convergence in the North Atlantic. Deep-Sea Res. II 40, 329–346. Hoegh-Guldberg, O., Williamson, J., 1999. Availability of two forms of dissolved nitrogen to the coral Pocillopa damicornis and its symbiotic zooxanthellae. Mar. Biol. 133, 561–570. Hoegh-Guldberg, O., McCloskey, L.R., Muscatine, L., 1987. Expulsion of zooxanthellae by symbiotic cnidarians from the Red Sea. Coral Reefs 5, 201–204. Iglesias-Prieto, R., Trench, R.K., 1994. Acclimation and adaptation to irradiance in symbiotic dinoflagellates. I. Responses of the photosynthetic unit to changes in photon flux density. Mar. Ecol. Prog. Ser. 113, 163–175. Laurion, I., Roy, S., 2009. Growth and photoprotection in three dinoflagellates (including two strains of Alexandrium tamarense) and one diatom exposed to four weeks of natural and enhanced ultraviolet-B radiation. J. Phycol. 45, 16–33. Leong, S.C.Y., Taguchi, S., 2004. Response of the dinoflagellate Alexandrium tamarense to a range of nitrogen sources and concentrations: growth rate, chemical carbon and nitrogen, and pigments. Hydrobiologia 515, 215–224.
Leong, S.C.Y., Maekawa, M., Taugchi, S., 2009. Carbon and nitrogen acquisition of the toxic dinoflagellate Alexandrium tamarense in response to different nitrogen sources and supply mode. Harmful Algae 9, 48–58. Lund, J.W.G., Kipling, C., LeCren, E.D., 1958. The inverted microscope method of estimating algal numbers and the statistical basis of estimations by counting. Hydrobiologia 11, 143–169. Meson, B.W., Sweeney, B.M., 1982. Adaptation of Ceratium furca and Gonyaulax polyedra (Dinophyceae) to different temperatures and irradiances: growth rates and cell volumes. J. Phycol. 18, 241–245. Nagao, N., Takahashi, K., Hamasaki, K., Toda, T., Taguchi, S., 2001. High ash content in net-plankton samples from shallow coastal water: possible source of error in dry weight measurement of zooplankton biomass. J. Oceanogr. 57, 105–107. Obata, M., Toda, T., Taguchi, S., 2009. Using chlorophyll fluorescence to monitor yields of microbial production. J. Appl. Phycol. 21, 315–319. Parkhill, J.-P., Maillet, G., Cullen, J.J., 2001. Fluorescence-based maximal quantum yield for PSII as a dignostatic of nutrient stress. J. Phycol. 37, 517–529. Parsons, T.R., Maita, Y., Lalli, C.M., 1984. A manual of chemical and biological methods for seawater analysis. Pergamon Press, Oxford . (178 pp.). Prezelin, B.B., 1976. The role of peridinin-chlorphyll a-proteins in the photosynthetic light adaptations of the marine dinoflagellate, Glenodinium sp. Planta 130, 225–233. Ralph, P.J., Gademann, R., Larkum, A.W.D., 2001. Zooxanthellae expelled from bleached corals at 33°C are photosynthetically competent. Mar. Ecol. Prog. Ser. 220, 163–168. Robinson, J.D.R., Warner, M.E., 2006. Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (Pyrrhophyta). J. Phycol. 43, 568–579. Santos, S.R., Taylor, D.J., Coffroth, M.A., 2001. Genetic comparisons of freshly isolated versus cultured symbiotic dinoflagellates: implications for extrapolating to the intact symbiosis. J. Phycol. 37, 900–912. Santos, S.R., Shearer, T.L., Hannes, A.R., Coffroth, M.A., 2004. Fine-scale diversity and specificity in the most prevalent lineage of symbiotic dinoflagellates (Symbiosinium, Dinophyceae) of the Caribbean. Mol. Ecol. 13, 459–469. Santos, S.R., Toyoshima, J., Kinzie III, R.A., 2009. Spatial and temporal dynamics of symbiotic dinoflagellates (Symbiodinium: Dinophyta) in the perforate coral Montipora capitata, Galaxea. J. Coral Reef Stud. 11, 1–9. Schreiber, U., Bilger, W., Neubauer, C., 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. Ecol. Stud. 100, 49–70. Sharp, J.H., 1983. The distributions of inorganic nitrogen and dissolved and particulate organic nitrogen in the sea. In: Carpenter, E.J., Capone, D.G. (Eds.), Nitrogen in the Marine Environment. Academic Press, New York, pp. 1–35. Shelly, K., Holland, D., Beardall, J., 2010. Assessing nutrient status of microalgae using chlorophyll a fluorescence. In: Sugget, D.J., Prail, O., Borowitzka, M.A. (Eds.), Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications. Springer, New York, pp. 223–235. Steele, R.D., 1977. The significance of zooxanthellae containing pellets extruded by seaanemones. Bull. Mar. Sci. 27, 591–594. Stimpson, J., Kinzie III, R.A., 1991. The temporal pattern and rate of release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus) under nitrogenenrichment and control conditions. J. Exp. Mar. Biol. Ecol. 153, 63–74. Suwa, R., Hirose, M., Hidaka, M., 2008. Seasonal fluctuation in zooxanthellar genotype composition and photophysiology in the corals Pavona divaricate and P. decussate. Mar. Ecol. Prog. Ser. 361, 129–137. Taguchi, S., Kinzie III, R.A., 2001. Growth of zooxanthellae in culture with two nitrogen sources. Mar. Biol. 138, 149–155. Warner, M.E., William, K.F., Schmidt, G.W., 1999. Damage to photosystem II in symbiotic dinoflagellates: a determinant of coral bleaching. Proc. Natl. Acad. Sci. U. S. A. 96, 8007–8012. Webb, W.L., Newton, M., Starr, D., 1974. Carbon dioxide exchange of Alnus rubra, a mathematical model. Oecologia 17, 281–291. Weis, V.M., Reynolds, W.S., deBoer, M.D., Krupp, D.A., 2001. Host-symbiont specificity during onset of symbiosis between the dinoflagellates Symbiodinium spp. and planula larvae of the sclerctinian coral Fungia scutaria. Coral Reefs 20, 301–308. Wyatt, T., Horwood, J., 1973. A model which generates red tides. Nature 244, 238–240. Yakovleua, I.M., Baird, A.H., Yamamoto, H.H., Bhagooli, R., Nonaka, M., Hidaka, M., 2009. Algal symbionts increase oxidative damage and death in coral larvae at high temperature. Mar. Ecol. Prog. Ser. 378, 105–112. Yellowlees, D., Rees, T.A., Fitt, W.K., 1994. Effect of ammonium-suplemented seawater on glutamine synthetase and glutamate dehydrogenase activities in host tissue and zooxanthellae of Pocillopora danicornis and on ammonium uptake rates of the zooxanthellae. Pac. Sci. 48, 291–295. Young, E.B., Beardall, J., 2003. Rapid ammonium- and nitrate-induced perturbations to chl a fluorescence in nitrogen-stress Dunaliella tertiolecta (Chlorophyta). J. Phycol. 39, 332–342.
Contents lists available at SciVerse ScienceDirect
Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Growth, biochemical properties, and chlorophyll fluorescence of symbiotic and free-living dinoflagellates in response to ammonium enrichment Yumi Fuchinoue a, Tomoyo Katayama a, Mitsuko Obata a, Ai Murata a, Robert Kinzie III b, Satoru Taguchi a,⁎ a b
Soka University, Faculty of Engineering, Laboratory of Biological Oceanography, 1–236 Tangi-Cho, Hachiouji, Tokyo 192-8577, Japan Department of Zoology and Hawaii Institute of Marine Biology, University of Hawaii at Manoa, Honolulu, HI 96822, USA
a r t i c l e
i n f o
Article history: Received 27 May 2012 Received in revised form 1 October 2012 Accepted 3 October 2012 Available online 25 October 2012 Keywords: Chlorophyll a Efficiency of red light absorption Maximum quantum efficiency Operating efficiency Package effect
a b s t r a c t The growth, biochemical properties, and chlorophyll fluorescence of cultures of the symbiotic Symbiodinium species from clade A, B, and F and the free-living dinoflagellate Prorocentrum minimum in response to ammonium enrichment were examined following transfer from ammonium-limited to ammonium-enriched conditions. Cultures were grown under a light:dark (L:D) cycle of 300 μmol photons m−2 s−1. Cell growth was initiated on day 1, reached saturation on approximately day 5, and decreased due to ammonium on day 10 (ammonium limitation experiments). Cells were removed from the ammonium limitation series on days 1, 5, and 10 and were enriched to 50 μM ammonium followed by incubation for 4 days (ammonium enrichment experiments). The cell density, cellular carbon, nitrogen, chlorophyll a contents, and chlorophyll fluorescence were measured for 14 days in the ammonium limitation experiments and for 4 days in the ammonium enrichment experiments. The results showed that ammonium disappeared rapidly once the cells were enriched, and the removal rates of ammonium were related to the growth rate under the ammonium enriched conditions. No apparent enhancement of the photosynthetic efficiency (Fv/Fm) in the ammonium-limited conditions was observed for the symbiotic dinoflagellates, whereas a recovery of Fv/Fm from the ammonium-limited conditions was observed within two days for the free-living dinoflagellate. The significant but different linear relationships between the initial slope (α) and the maximum electron transport rate (ETRmax) of ETR vs photon flux density (PFD) curve, which is equivalent to the adaptation index (Ek), observed for Symbiodinium and Prorocentrum may suggest that these species require a different range of PFD for photoacclimation and exhibit a different physiological tolerance to ammonium toxicity. Observations of acclimation and adaptation using variable fluorescence would be useful for understanding the photophysiological strategies of symbiotic and free-living dinoflagellates. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Free-living dinoflagellates may migrate vertically within their photic environment (Wyatt and Horwood, 1973), experiencing a light regime that can change greatly within hours or even minutes. Dinoflagellates belonging to the genus Symbiodinum are associated with a wide range of marine invertebrates, including reef corals, anemones, jellyfish and giant clams. While some invertebrates that host Symbiodinium are planktonic, most are benthic and most of these species, such as corals, clams and anemones are sessile. Therefore, these algae may be exposed to a more predictable light environment with a narrower range of intensities. Depending on their host, Symbiodinium must adapt to the photobehavior of host animals. In associations with planktonic hosts, such as jellyfish, zooxanthellae may experience variable photon fluxes in natural environments (Iglesias-Prieto and Trench, 1994). Algae in symbioses with hosts living in shallow water may experience photodamage (Warner et al., ⁎ Corresponding author. Tel./fax: +81 42 691 8002. E-mail address: [email protected] (S. Taguchi). 0022-0981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2012.10.001
1999). Loss of these damaged Symbiodinium is termed coral bleaching (Glynn, 1996). However, under certain conditions, Symbiodinium cells may exist outside of a host and, thus, experience light conditions more like those of free living planktonic algae. Many invertebrates that host Symbiodinium as adults are not provided with algal symbionts early in their live history as spawned eggs or early-stage larvae. Hosts with this life history pattern (horizontal transition) must acquire algal symbionts if they are to survive (Baird et al., 2009; Weis et al., 2001). Where such free-living Symbiodinium live, and how long they can exist outside of their hosts are questions that are not well understood, but they must exist to form symbiosis with early stages of hosts with this life history. While photophysiological studies have not been performed on populations of naturally occurring free-living Symbiodium cells, experiments with newly infected larvae of the coral Acropora have shown damaging effects at light levels typical of the upper part of the water column (Yakovleua et al., 2009). The source of this pool of Symbiodinium in the environment is not well understood, but it is known that hosts release algal cells into the
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ambient water (Hoegh-Guldberg et al., 1987; Steele, 1977; Stimpson and Kinzie, 1991) and that these cells are viable (Bhagooli and Hidaka, 2004; Ralph et al., 2001). Santos et al. (2009) have demonstrated one possible pathway for these Symbiodinium cells to be produced and released by a host. Thus, while dinoflagellates of the genus Symbiodinium may generally be held within cells of benthic hosts, at least some will experience a photic environment more typical of free-living planktonic forms. Another potential difference between the biology of free-living dinoflagellates and symbiotic forms is related to the ambient nutrient levels these species experience. Free-living phytoplankton can be exposed to a range of nutrient concentrations, but those in tropical oceans generally occur in low-nutrient waters except for polluted coastal waters. Nitrogen levels can often be as low as 0.2 μM for nitrate and undetectable for ammonium (Sharp, 1983). In contrast, algae living in host animal cells may experience very high levels of ammonium. Symbiodinium are known to utilize nutrients such as ammonium released by their host and produce organic matter through photosynthesis, following which the host animals assimilate the organic matter (Hoegh-Guldberg and Williamson, 1999). The concentration of ammonia in the cells of host animals can be as high as 50 μM, and this ammonia can be utilized by Symbiodinium cells (Taguchi and Kinzie, 2001; Yellowlees et al., 1994). However, such concentrations may be toxic for free-living dinoflagellates (Change and McClean, 1997; Leong et al., 2009). Even within Symbiodinium, some variability in acclimation among clades can be expected, as indicated by Robinson and Warner (2006). All cellular chemical constituents are subject to experiencing changes in their cellular contents due the nitrogen availability. Such acclimation may occur in terms of the organisms’ light absorption ability and the distribution of light energy among the photosynthetic machinery in the Photosystem II (Laurion and Roy, 2009). In this study, three clades of Symbiodinium and one free-living dinoflagellate were employed to study acclimation to ammonium enrichment in terms of the maximum quantum efficiency and operating efficiency, as determined by pulse-amplitude-modulated fluorometry (Genty et al., 1989; Schreiber et al., 1994; Shelly et al., 2010). Batch cultures were set up to follow chlorophyll fluorescence together with cell growth under ammonium-limited and ammonium-enriched conditions. The ammonium limitation experiments were designed to produce ammonium-exhausted cells close to growth saturation. Cells were collected from the ammonium limitation experiment on days 1, 5 and 10 and used to initiate the ammonium enrichment experiments. These days corresponded to initial (day 1), log phase (day 5), and fully nitrogen depleted (day 10) cells. The object of this study was to elucidate differences in the responses of the four dinoflagellates (symbiotic Symbiodinium species from clades A, B, and F and the free-living Prorocentrum minimum) to changes in nutrient levels and how these changes were reflected in the photobiology of these cells. The maximum photosynthetic efficiency (Fv/Fm) and photosynthetic characteristics, such as the initial slope (α) and ETRmax estimated from the electron transport rate, were employed to study the maximum photochemical efficiency of open reaction center II (RCIIs) in the dark and the effective photochemical efficiency of RCIIs under light, respectively. We hypothesize that photophysiological difference present between symbiotic and free-living dinoflagellates might be caused by adaptation and acclimation to light regime. 2. Materials and methods 2.1. Culture Strains of zooxanthellae were isolated from three different host animals: clade A from Cassiopea sp.; clade B, from Aiptasia pulchella; and clade F from Montiopora verrucosa (Santos et al., 2004). All strains were obtained from the culture collection at the Hawaii Institute of Marine
Biology, University of Hawaii at Manoa. A culture of the free-living dinoflagellate, Prorocentrum minimum was obtained from the Culture Collection Center at the National Institute of Environmental Science, Japan. Initial cultures were maintained in semi-continuous batch mode in f/2 medium with 50 μM ammonium as the nitrogen source, instead of nitrate (Guillard and Ryther, 1962), at 25 °C, which is the optimum growth temperature as indicated by Suwa et al. (2008), in a light-controlled incubator (Eyela, FLI-301N, Japan) for more than three dilutions in semi-continuous mode. Each dilution lasted for one week. At the end of each dilution, the ammonium concentration was less than 10 μM. The experimental medium was prepared with surface seawater collected in coastal waters off Manazuru, Japan. The mixture of seawater had been aged for longer than one year in the dark at room temperature. The nitrogen concentrations in the aged seawater were below detection limits prior to its use in the experiments. Phosphate, trace metals, and vitamins were added as per the f/2 formulation (Guillard and Ryther, 1962). Photosynthetically active radiation (PAR) was provided using cool-white fluorescence tubes (National, FL40SW, Japan) in 12 h light and 12 h dark cycles at 300 μmol photons m−2 s−1, as determined by a 4π sensor submerged in the culture medium in the culture vessel (Biospherical Instruments, QSL100, USA). 2.2. Incubation experiments and sampling Ammonium-limited experiments were initiated by the addition of 25 mL of the stock cultures to 1 L of the nitrogen depleted f/2 medium. This resulted in an initial cell density of ~ 600 cells mL −1. This procedure did not raise the ammonium concentration appreciably in the experimental bottles (b0.2 μM). The goal of this phase of the study was to maintain ammonium-limited cells for the early part of incubation and produce completely ammonium-depleted cells by day 10 in batch culture mode (ammonium limitation experiments). The subsequent enrichment experiments were performed using cells from the batch culture on days 1, 5, and 10. These cells, in various stages on nitrogen depletion, were used to initiate cultures with a final concentration of 50 μM (ammonium enrichment experiments). For the ammonium limitation experiments, samples were collected daily for 14 days, except for on day 3, 9, and 12. For the ammonium enrichment experiments, cells sampled at the beginning (day 1) and the end of the log phase (day 5) and the stationary phases (day 10) of growth were used to determine the response of the cells to ammonium enrichment for the subsequent four days. 2.3. Cell counts On each sampling day, subsamples were collected to estimate cell density by counting cells using an inverted microscope (Olympus, IMT-2, Japan) (Hasle, 1978). A calibrated hematocytometer (Erma, 35103000, Japan) was employed to count the cells (Guillard, 1978). A minimum of 400 cells per field were counted (Lund et al., 1958). The growth rate was calculated according to the method described by Guillard (1978). Cell size was measured in the ammonium limitation experiments. 2.4. Ammonium analysis Subsamples were collected for analysis of ammonium. Seawater samples were filtered through a 0.45 μm membrane filter (Millipore, Mylex HA, Ireland), and the concentration of ammonium in the filtrates was determined using methodology described in Parsons et al. (1984). The rate of ammonium removal from the medium (Da) was calculated using the following equation: Da ¼ ðlnNH0 –lnNHt Þ=t
ð1Þ
where NH0 and NHt are the ammonium concentrations at time 0 and t, respectively.
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2.5. Particulate organic carbon and nitrogen
2.8. Statistical analysis
Subsamples for analysis of particulate organic carbon and nitrogen were filtered onto glass fiber filters (Whatman, GF/F, England), which had been pre-combusted at 500 °C for 2 h. The amounts of organic carbon and nitrogen on the filters were analyzed using a CHN elemental analyzer (Thermo Finnigan, Flush EA1112 series, Germany). The concentrations of organic carbon and nitrogen were calibrated against acetanilide as a standard (Nagao et al., 2001).
The cellular contents of particulate organic carbon and nitrogen and Chl a were employed to calculate the chemical ratios. The mean with one standard deviation of triplicate values was always calculated and is reported throughout the present paper. Student's t-test and analysis of variance were implemented using Sigma-Plot (System software, version 11.2, San Jose, USA). 3. Results
2.6. Pigment analysis 3.1. Nutrient response Subsamples were collected for analysis of pigments using high-performance liquid chromatography (Beckman, System Gold 168 Diode Array Detector, Fullerton, USA) with a C18 reverse-phase ultrasphere 3 μm column as described by Head and Horn (1993). The obtained peaks were quantified using chlorophyll a (Chl a) standards obtained from the International 14C agency (Denmark). 2.7. Variable fluorescence To measure variable fluorescence, subsamples of the cell suspensions were collected and 3 mL of the sample was transferred to the bottom of a quartz cell in a pulse amplitude modulated fluorometer (PAM, Walz, Water-Pam, Germany) immediately after acclimation to the subsamples for 30 min at 25 °C in a dark laboratory, as described by Obata et al. (2009). Once the maximum fluorescence yield in the dark-adapted state (F0) had become stable, a saturating pulse of 1200 μmol photons at 655 nm m−2 s−1 was supplied to determine the maximum fluorescence yield (Fm) after dark acclimation. The maximum quantum efficiency of PSII (Fv/Fm) for the dark-adapted state was calculated using the method of Schreiber et al. (1994): F v =F m ¼ ðF m –F 0 Þ=F m
ð2Þ
The ammonium concentrations were below detection limits throughout the ammonium limitation experiments. When the cells were exposed to 50 μM ammonium enrichment, the ammonium concentration declined immediately (Fig. 1). The ammonium concentration decreased to approximately 15–25 μM at the end of the ammonium enrichment experiments conducted on day 1 and day 5 and approximately 25–40 μM at the end of the ammonium enrichment experiments on day 10. Therefore, the removal rate decreased with the length of the ammonium limitation experiments and ranged from 42% for the free-living dinoflagellate to 54% for symbiotic Symbiodinium cells from clade A. 3.2. Growth rate The growth rates for symbiotic dinoflagellates ranged from 0.40 ± 0.07 day −1for clade B to 0.55 ± 0.06 d −1 for clade F. The growth rate of Prorocentrum was lower, averaging 0.29 ± 0.05 day −1. When ammonium was introduced to the ammonium-limited cultures, the growth rates of symbiotic Symbiodinium cells from clades A and B obtained from the ammonium limitation experiment initiated on 60
0 0 0 0 F q =F m ¼ F m –F =F m
ð3Þ
The electron transport rate (ETR) was obtained using the following equation (Genty et al., 1989): 0 0 ETR ¼ 0:5f F q =F m PFD
A 40
Ammonium concentration (μM)
The maximum photosynthetic efficiency (Fv/Fm) was obtained under actinic light. For this purpose, light-response curves were generated in triplicate for each culture. Aliquots of 3 mL were taken directly from the experimental bottle, then dark-acclimated for 30 min and the variable fluorescence was determined to adjust the zero level of actinic light. After zero adjustment, the samples were illuminated by the red light-emitting diodes of the actinic light source. The minimum and maximum fluorescence yields in the light-adapted state (F and F’m) were measured with a saturation pulse with a duration of 1 s at a photosynthetic photon flux density of 1835 μmol photons m−2 s−1, following at least 30 s of exposure to actinic light. The maximum fluorescence yields were determined at 12 light levels of actinic light. The PSII operating efficiency (F′q/F′m) was calculated using the equation of Genty et al. (1989):
20
0 60
B 40
ð4Þ 20
where 0.5 is a factor to account for equal number of photons reaching on PSI and PSII, f is the proportional factor (s m 2 μmol photons−1); and PFD is the intensity of actinic light (μmol photons m −2 s −1). The initial slope (α) and the maximum ETRmax were estimated by fitting the following equation (Webb et al., 1974): ETR ¼ ETR
B
max ð1−exp½−PDF=Ek Þ
ð5Þ
where Ek is the adaptation index, which defines the ratio of ETRmax to α.
0
0
2
4
6
8
10
12
14
Days Fig. 1. Changes in the ammonium concentration in the ammonium enrichment experiments initiated on days 1, and 5, and 10 of the ammonium limitation experiments for symbiotic Symbiodinium cells from clades A (A) and Prorocentrum (B).
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Relative growth rate
2.0
1.5
1.0
0.5
0.0 0
1
5
10
Days Fig. 2. Relative growth rates of clade A (open bar), clade B (left hatched bar), clade F (right hatched bar), and Prorocentrum (crosshatched bar) in the ammonium enrichment experiments initiated on days 1, 5, and 10. All values are standardized against the growth rate under the ammonium-limited conditions as indicated on day 0.
day 1 increased. The relative growth rates of clade F changed little until a decrease in the cultures was initiated on day 10. Prorocentrum showed lower growth rates in all three enrichment experiments (Fig. 2). The reduction in the relative growth rates of the three Symbiodinium clades on day 10 compared to day 1 was approximately 58 ± 7%. The growth rate of the free-living dinoflagellate was already suppressed by 30% on day 1 and by 40% on days 5 and 10.
the observed trend was similar within the symbiotic dinoflagellates. The cellular carbon and nitrogen contents of all Symbiodinium clades and the free-living dinoflagellate at the beginning of the ammonium enrichment experiments were approximately 1 ngC cell −1 and 0.1 ngN cell −1, respectively. Regardless of the ammonium enrichment experiment starting on days 1, 5, or 10 of the ammonium limitation experiments, the cellular carbon and nitrogen contents in the free-living dinoflagellate at the end of the ammonium enrichment experiments were significantly higher than those for all Symbiodium clades (p b 0.05). The cellular contents had decreased by more than 50% at the end of the ammonium enrichment experiment initiated on day 1. The cellular contents at the end of the ammonium enrichment experiment initiated on day 10 were higher, reaching more than twice those in the experiments initiated on day 1 in all Symbiodinium clades, whereas the contents in Prorocentrum increased by only 130%. The cellular Chl a contents on day 0 of all ammonium enrichment experiments initiated on days 1, 5, and 10 of the ammonium limitation experiments were similar among the species at approximately 1.6 pg Chl a cell −1and decreased to less than 0.2 pg cell −1 in the ammonium enrichment experiment initiated on day 10 (Fig. 3). The cellular Chl a contents of all species at the end of the ammonium enrichment experiment initiated on day 1 decreased significantly from those on day 0 due to cell division (p b 0.05) and recovered to values three times higher than those on day 0 of the ammonium enrichment experiment initiated on day 10. The highest recovery on day 10 was observed for the free-living dinoflagellate.
3.3. Cellular carbon, nitrogen, and Chl a contents 3.4. Maximum photosynthetic efficiency (Fv/Fm) Representative results are only shown for symbiotic Symbiodinium cells from clade A and the free-living Prorocentrum in Fig. 3 because
0.10
The maximum photosynthetic efficiency observed in Prorocentrum under the ammonium-limited conditions was approximately two-thirds of that observed in Symbiodinium. The significantly different Fv/Fm values indicates a species specific photosynthetic capacity. A continuous decrease in Fv/Fm was observed for Symbiodinium, whereas a sudden decrease in Fv/Fm around day 4 was observed for Prorocentrum. When the cells were transferred to the ammonium-enriched conditions, Fv/Fm showed three significant trends. First, Fv/Fm did not respond to the ammonium enrichment and remained at similar levels during the ammonium enrichment experiment, except in Prorocentrum. Second, Fv/Fm generally decreased with the duration of the ammonium limitation experiments from day 1 to day 10 for Symbiodinum. The ratio of the mean Fv/Fm (with one standard deviation) observed in the ammonium enrichment experiment initiated on day 10 to that on day 1 was 0.91±0.02, indicating a decrease in photosynthetic capability. Third, the maximum Fv/Fm value observed for Prorocentrum was less than 0.4, possibly due to ammonium toxicity, whereas that for Symbiodinium was higher than 0.45.
0.05
3.5. Electron transport rate-photon flux density curve
(ngC cell-1)
Cellular Carbon
1.5
A
B
C
D
1.0
0.5
0.0
0.15
(ngN cell-1)
Cellular Nitrogen
0.20
0.00 2.0
2.0
F
(pg cell-1)
Cellular Chl a
E 1.5
1.5
1.0
1.0
0.5
0.5
0.0
1
5
Days
10
0.0
1
5
10
Days
Fig. 3. Cellular carbon (A and B), nitrogen contents (C and D), and Chl a contents (E and F) at the beginning (dark) and the end of the ammonium enrichment experiment (stippled) in symbiotic Symbiodinium cells from clade A (A, C, and E) and Prorocentrum (B, D, and F). Vertical bars indicate one standard deviation.
When cells were transferred to the ammonium-enriched condition, both α and ETRmax either decreased with time or remained at a level similar to the initial value during the enrichment experiment for both Symbiodinium and Prorocentrum regardless of the duration of the ammonium enrichment experiments. The mean α value for Prorocentrum was the lowest among all of the species investigated in the present study (p b 0.05), whereas the mean ETRmax of Prorocentrum was not different from any of the clades of Symbiodinium (Table 1). The mean α and ETRmax were also not significantly different among the three clades of Symbiodinium. The initial slope (α) and ETRmax ranged from 0.23 to 0.38 e (μmol photons m−2 s−1) −1 and from 30 to 88 e for Symbiodinium, respectively, whereas they ranged from 0.13 to 0.22 e (μmol photons m −2 s−1)−1 and from 26 to 40 e for Prorocentrum, respectively. A significant linear relationship between α and ETRmax was obtained for both Symbiodinium and Prorocentrum (p b 0.01, Fig. 4). The slopes with 95% confidence limits, indicative of the
Y. Fuchinoue et al. / Journal of Experimental Marine Biology and Ecology 438 (2012) 1–6
4.2. PSII photochemistry Fv/Fm
Table 1 Mean with one standard deviation of α, ETRmax, and EK of the ETR PFD curve for clades A, B, and F and Prorocentrum minimum. Species
α
ETRmax
EK
A B F Prorocentrum minimum
0.267 ± 0.0164 0.328 ± 0.0965 0.304 ± 0.0407 0.170 ± 0.0223
43.8 ± 8.2 57.5 ± 9.0 56.9 ± 15.1 35.1 ± 7.8
165 ± 31 190 ± 20 188 ± 36 210 ± 52
5
Fluorescence responses to ammonium enrichment can vary considerably with the degree of ammonium limitation during the growth of cells, as suggested by Young and Beardall (2003). In the present study, the maximum quantum efficiency (Fv/Fm) indicates a distinctively different pattern of photoacclimation in the transition from ammonium-limited to ammonium-enriched conditions between symbiotic and free-living dinoflagellates. The initial Fv/Fm values are comparable with those previously reported for symbiotic and free-living dinoflagellates under similar conditions (Laurion and Roy, 2009; Suwa et al., 2008), indicating a high potential photosynthetic performance for Photosystem II in actively growing cells. The continuous decrease in Fv/Fm detected in the first half of the ammonium-limited conditions, particularly for Symbiodinium is similar to what was observed by Parkhill et al. (2001) and Young and Beardall (2003). The slope of the temporal decrease in Fv/Fm could be similar under the ammonium- limited conditions for Symbiodinium. After a sudden decrease around day 4, the slight decrease in Fv/Fm observed in the second half of the ammonium-limited conditions for Prorocentrum suggests the possible occurrence of a physiological change in nitrogen metabolism, which will be discussed in the next section.
photoadaptation index, were 262± 32 μmol photons m −2 s −1 for Symbiodinium and approximately 2 times higher than 138 ± 24 μmol photons m−2 s−1for Prorocentrum.
4. Discussion 4.1. Physiological characteristics Autotrophic microalgae acclimate to various environmental conditions by altering their cellular Chl a content (Behrenfeld et al., 2004). Our batch culture experiment involving ammonium limitation showed that the cellular Chl a contents were reduced through increased cell divisions to harvest more photons per Chl a molecule by reducing self-shading within a cell. The growth rates of symbiotic dinoflagellates obtained in the early part (days 1 and 5) of the ammonium limitation experiments were within the range reported for algae within host animals (Chang et al., 1983). By day 10, the cells are in a state of ammonium starvation, and cultures show adverse physiological effects (e.g., Berman-Frank and Dubinsky, 1999). The present cellular Chl a content estimates obtained in the stationary phase (day 10) of growth, ranging from 0.19 to 0.099 pg Chl a cell−1, are several time higher than those obtained at the beginning of the growth phase for both the symbiotic and free-living dinoflagellates (Chan, 1978; Chang et al., 1983; Meson and Sweeney, 1982; Taguchi and Kinzie, 2001). The negative relationship between the cellular contents of Chl a and the growth rate observed on days 1 and 5 is interpreted as the cellular Chl a content being regulated by cell growth as observed by Prezelin (1976). The free-living dinoflagellates are so sensitive to excess ammonium (Goldman and Glibert, 1983) that their growth rate is depressed under the ammonium-enriched conditions, resulting in large cells. The present study suggests that phylogenetically different responses to ammonium concentrations and toxicity are intrinsic differences between symbiotic and free-living dinoflagellates.
4.3. ETR–PDF curve The significant liner relationship between α and ETRmax may suggest that the photosynthetic strategy is similar among Symbiodinium species but different between Symbiodinium and Prorocentrum. The significantly different slopes of the linear relationships and ranges of both values between the two taxonomic groups may be caused by not only by different light ranges being required for photoacclimation but also by different physiological tolerances to ammonium toxicity. Symbiodinium may employ Ek-dependent acclimation (Behrenfeld et al., 2004) to utilize a wide range of irradiances with ample ammonium supplies. Ek-dependent acclimation is also considered as a strategy for Prorocentrum when ammonium is used as the nitrogen source, such as in the present study. When ammonium is enriched, Prorocentrum cells grown in log-phase under the ammonium-limited conditions, can maintain a strategy of Ek-dependent acclimation because they maintain the ability to divide, even at a slow rate due to ammonium toxicity. When ammonium enrichment is applied to ammonium-exhausted Prorocentrum cells, cells with high cellular Chl a contents may adapt an Ek-dependent acclimation strategy to avoid a less efficient photosynthesis phase under low irradiance, while achieving efficient photosynthesis under high irradiance because cell division is extremely depressed due to ammonium toxicity. Ammonium toxicity has been known to occur at concentrations
100
A
B
ETRmax
80
60
40
20
0 0.0
0.1
0.2
0.3
Initial slope ( α )
0.4
0.5
0.0
0.1
0.2
0.3
0.4
0.5
Initial slope ( α )
Fig. 4. Relationship between the initial slope (α) and ETRmax of the ETR PFD curve of Prorocentrum (A) and Symbiodinium (B). Solid circles, open circles, and solid reversed triangles indicate the values on days 1, 5, and 10 for Prorocentrum (A). Solid circles, open circles, and solid reversed triangles indicate values on days 1, 5, and 10 for clade A. Open triangles, solid squares, and open squares indicate values on days 1, 5, and 10 for clade B. Solid diamonds, open diamonds, and solid triangles indicate values on days 1, 5, and 10 for clade F.
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higher than 25 μM for dinoflagellates (Goldman and Glibert, 1983; Leong and Taguchi, 2004). Under the ammonium concentration of 50 μM employed in the present study, low values of μ and α are observed for Prorocentrum caused by ammonium toxicity, but not for Symbiodinium. This leads to a lower range of values and a gentler slope of the linear relationship between α and ETRmax for Prorocentrum than Symbiodinium. Our results are limited to a single experiment involving three clades and one free-living dinoflagellate, and investigation of Fv/Fm, α, and ETRmax as a function of ammonium stress for other clades and free-living dinoflagellates is still required as discussed by Santos et al. (2001). Acknowledgements The present study was partly supported by a grant-in-aid provided by Soka University for ST. All assistance at Scripps Institution of Oceanography to prepare an earlier version of this manuscript is greatly appreciated. [SS] References Baird, A.H., Guest, J.R., Willis, B.L., 2009. Biogeographical and evolutionary patterns in the reproductive biology of scleractinian corals. Ann. Rev. Ecol. Evol. Syst. 40, 551–571. Behrenfeld, M.J., Prasil, O., Babin, M., Bruyant, F., 2004. In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis. J. Phycol. 40, 4–25. Berman-Frank, I., Dubinsky, Z., 1999. Balanced growth in aquatic plants: myth or reality? Bioscience 49, 29–36. Bhagooli, R., Hidaka, M., 2004. Release of zooxanthellae with intact photosynthetic activity by the coral Galaxea fascicularis in response to high temperature stress. Mar. Biol. 145, 329–337. Chan, A.T., 1978. Comparative physiological study of marine diatoms and dinoflagellates in relation to irradiance and cell size. I Growth under continuous light. J. Phycol. 14, 396–402. Chang, S.S., Prezelin, B.B., Trench, R.K., 1983. Mechanisms of photoadpatation in three strains of the symbiotic dinoflagellate Symbiodinium microadriaticum. Mar. Biol. 76, 219–229. Change, F.H., McClean, M., 1997. Growth responses of Alexandrium minimum (Dinophyceae) as a function of three different nitrogen sources and irradiance. N. Z. J. Mar. Freshw. Res. 31, 1–7. Genty, B., Briantais, J., Baker, N., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87–92. Glynn, P.W., 1996. Coral reef bleaching: facts, hypotheses and implications. Glob. Chang. Biol. 2, 495–509. Goldman, J.C., Glibert, P.M., 1983. Kinetics of inorganic nitrogen uptake. In: Carpenter, E.J., Capone, D.G. (Eds.), Nitrogen in the Marine Environment. Academic Press, New York, pp. 233–274. Guillard, R.R.L., 1978. Counting slides. In: Sournia, A. (Ed.), Phytoplankton Manual. UNESCO, Paris, pp. 182–193. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleave) Gran. Can. J. Microbiol. 8, 229–239. Hasle, G.R., 1978. Using the inverted microscope. In: Sournia, A. (Ed.), Phytoplankton Manual. UNESCO, Paris, pp. 191–196. Head, E.J.H., Horn, E.P.W., 1993. Pigment transformation and vertical flux in an area of convergence in the North Atlantic. Deep-Sea Res. II 40, 329–346. Hoegh-Guldberg, O., Williamson, J., 1999. Availability of two forms of dissolved nitrogen to the coral Pocillopa damicornis and its symbiotic zooxanthellae. Mar. Biol. 133, 561–570. Hoegh-Guldberg, O., McCloskey, L.R., Muscatine, L., 1987. Expulsion of zooxanthellae by symbiotic cnidarians from the Red Sea. Coral Reefs 5, 201–204. Iglesias-Prieto, R., Trench, R.K., 1994. Acclimation and adaptation to irradiance in symbiotic dinoflagellates. I. Responses of the photosynthetic unit to changes in photon flux density. Mar. Ecol. Prog. Ser. 113, 163–175. Laurion, I., Roy, S., 2009. Growth and photoprotection in three dinoflagellates (including two strains of Alexandrium tamarense) and one diatom exposed to four weeks of natural and enhanced ultraviolet-B radiation. J. Phycol. 45, 16–33. Leong, S.C.Y., Taguchi, S., 2004. Response of the dinoflagellate Alexandrium tamarense to a range of nitrogen sources and concentrations: growth rate, chemical carbon and nitrogen, and pigments. Hydrobiologia 515, 215–224.
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