http://www.nature.com/nclimate/journal/vaop/ncurrent/full/nclimate1661.html

Similar to findings of Dr. James Cervino about ten years ago, yet we are still debating the human impacts that trigger climate change instead of implementing policies to address them. DV

Jörg Wiedenmann,
Cecilia D’Angelo,
Edward G. Smith,
Alan N. Hunt,
François-Eric Legiret,
Anthony D. Postle
& Eric P. Achterberg

Nature Climate Change (2012)

Received 20 April 2012
Accepted 11 July 2012
Published online 19 August 2012

Mass coral bleaching, resulting from the breakdown of coral–algal symbiosis has been identified as the most severe threat to coral reef survival on a global scale1. Regionally, nutrient enrichment of reef waters is often associated with a significant loss of coral cover and diversity2. Recently, increased dissolved inorganic nitrogen concentrations have been linked to a reduction of the temperature threshold of coral bleaching3, a phenomenon for which no mechanistic explanation is available. Here we show that increased levels of dissolved inorganic nitrogen in combination with limited phosphate concentrations result in an increased susceptibility of corals to temperature- and light-induced bleaching. Mass spectrometric analyses of the algal lipidome revealed a marked accumulation of sulpholipids under these conditions. Together with increased phosphatase activities, this change indicates that the imbalanced supply of dissolved inorganic nitrogen results in phosphate starvation of the symbiotic algae. Based on these findings we introduce a conceptual model that links unfavourable ratios of dissolved inorganic nutrients in the water column with established mechanisms of coral bleaching. Notably, this model improves the understanding of the detrimental effects of coastal nutrient enrichment on coral reefs, which is urgently required to support knowledge-based management strategies to mitigate the effects of climate change.

Main

Shallow-water coral reefs owe their success to the symbiosis of the cnidarian host with dinoflagellates of the genus Symbiodinium (zooxanthellae)4. Recent reports predict that most coral reefs will be lost in the near future as a result of an average surface ocean temperature rise of 1–2 °C and an increased frequency of strong short-term temperature anomalies1. Thermal stress is considered to induce a malfunctioning of the photosynthetic apparatus of the algal symbiont and contribute to a breakdown of the symbiosis manifested by the loss of zooxanthellae and the often fatal bleaching of the corals5. In addition to other factors, anthropogenic eutrophication of coastal waters has been linked to coral reef degradation1. Recently, a connection between terrestrially sourced dissolved inorganic nitrogen (DIN) loading and the upper thermal bleaching thresholds of inshore reefs on the Great Barrier Reef was established3. However, the view that nutrient enrichment is responsible for coral reef decline has been challenged as corals can thrive in high-nutrient water and several experimental studies using increased nutrient levels did not find obvious negative impacts on the physiology of corals6, 7, 8. The lack of consensus can potentially lead to confusion over policy development, government inaction and continued environmental degradation2. Therefore, understanding of the nutrient-dependent processes needs to be urgently improved to promote coral reef resilience by knowledge-based management efforts.

Anthropogenic nutrification often results not only in an increase of dissolved inorganic nutrients such as ammonium, nitrate and phosphate but it also usually modifies the ratio of their concentrations9. In different phytoplankton species, growth becomes chemically unbalanced when the availability of a specific type of nutrient decreases relative to the cellular demand10. This condition is defined as nutrient starvation and results in detrimental effects such as reduced photosynthetic efficiency, measurable as a decrease in fluorescence-based maximum quantum yield of photosystem II photochemistry (Fv/Fm; ref. 10).

Among other factors, phosphate limitation plays a potentially important role in the control of zooxanthellae numbers in the host tissue11. However, several studies have reported an increase in algal cell densities in response to increased concentrations of DIN in the water2, 12, indicating a strong influence of the external nitrogen levels on the proliferation rates of zooxanthellae.

Despite the nutrient limitation that zooxanthellae experience in hospite, the steady-state population of algal cells remains functional because of their full acclimation to this condition. In fact, the limited access to nutrients allows the zooxanthellae to transfer a substantial amount of their photosynthetically fixed carbon to the host cells4 and is therefore of high importance for the functioning of the symbiosis. Recycling of nutrients derived from feeding of the host supports the maintenance of the standing crop of zooxanthellae, but is not sufficient to account for their growth under reef conditions where the supply with food tends to be low4, 13.

In analogy to the findings in phytoplankton10, we hypothesize that an increased concentration of DIN in the water can be expected to accelerate proliferation of zooxanthellae2, resulting in phosphate starvation when phosphate availability is low. This scenario might apply particularly to symbiotic algae in corals from coastal regions where the naturally low phosphate and DIN concentrations are altered by anthropogenic inputs14. Here, we tested whether increased DIN levels in combination with limiting phosphate concentrations increases the susceptibility of corals to temperature- and light-induced bleaching.

Using our multicompartment mesocosm15, we cultured seven species of scleractinian corals under a photonflux of ~90 μmol m−2 s−1 in artificial sea water with low nutrient (low DIN/low phosphate; LN/LP); nutrient-replete (high DIN/high phosphate; HN/HP) and imbalanced nutrient (high DIN/ambient phosphate; HN/AP) levels as detailed in the Methods. Over a period of 12 weeks, the low nutrient conditions resulted in pronounced paling of the corals caused by a strong decrease in the density of algal cells (Fig. 1a,b and Supplementary Fig. S1). However, Fv/Fm determined as a measure of the photosynthetic efficiency of zooxanthellae16 was high (>0.5) in nutrient-replete and low nutrient conditions (Fig. 1b and Supplementary Fig. S1). These findings are in close agreement with studies on phytoplankton that demonstrated that Fv/Fm is high and insensitive to nutrient limitation as long as the cells are fully acclimated to this condition10. Montipora foliosa cultured for >five weeks under imbalanced nutrient levels showed higher zooxanthellae densities compared with corals from the nutrient-limited conditions (Fig. 1a,b). In the red- and a purple–green-colour morphs of this species and four other species exposed to imbalanced nutrient levels, however, Fv/Fm dropped below values of zooxanthellae from low nutrient and nutrient-replete conditions, in most cases below the healthy values >0.5 (Fig. 1b,h and Supplementary Fig. S1), indicating a common response among corals from a broad taxonomic range. When corals incubated at imbalanced nutrient levels were exposed to light levels >180 μmol m−2 s−1 they showed strong signs of bleaching, particularly in the light-exposed parts of the colony (Fig. 1c–g). This resembled bleaching patterns often observed during natural bleaching events17. The bleached colonies died partially or completely (Fig. 1g), whereas specimens exposed to lower light levels survived (Fig. 1a and Supplementary Fig. S1). We incubated replicate samples of Euphyllia paradivisia under a photonflux of ~80 μmol m−2 s−1 in replete and imbalanced nutrient conditions to further investigate the role of light in nutrient-mediated bleaching. After four weeks, individuals from both treatments exhibited healthy Fv/Fm values (>0.5) and no visible signs of bleaching. At this point, the light intensity was doubled, reaching a level known to saturate photosynthesis in other shade-acclimated corals18. After 14 days, the samples experiencing imbalanced nutrient levels showed a steep drop in Fv/Fm, suggesting severe photo-inhibition of the zooxanthellae (Fig. 1h). In contrast, photosynthetic competence of algae in the control samples remained essentially unaltered. After three weeks, the corals from imbalanced nutrient conditions lost ~ 50% of their zooxanthellae and also the chlorophyll a content of the algal cells was reduced by >50% (Fig. 1i,j). Increased light levels can cause a reduction in Fv/Fm associated with photodamage of zooxanthellae, stimulate the production of reactive oxygen species and contribute to coral bleaching, particularly if the photosynthetic apparatus was impaired by other factors including temperature stress18, 19. In our experiments, corals in the different treatments were exposed to the same quantity and quality of light, hence the reduction in Fv/Fm and the loss of algal cells suggest that the imbalanced nutrient levels in the water rendered the zooxanthellae more sensitive to light stress.
Figure 1: Bleaching patterns of corals at different nutrient concentrations.
Bleaching patterns of corals at different nutrient concentrations.

a, Representative replicate colonies of M. foliosa cultured under photonfluxes of ~90 μmol m−2 s−1 and HN/HP, LN/LP or HN/AP conditions. b, Fv/Fm and zooxanthellae densities of colonies exposed to different nutrient concentrations. c–g, Bleaching of corals from HN/AP conditions under photonfluxes >180 μmol m−2 s−1. c–e, Pronounced bleaching of light-exposed areas in A. microphthalma (c) and M. foliosa (d). Arrows indicate the direction of the incident light the corals experienced during the treatment. e, Replicate colonies of E. paradivisia exposed to ~90 μmol m−2 s−1 (left colony) and ~180 μmol m−2 s−1 (right colony). f, Acropora valida. g, Bleaching of Porites lobata colonies resulted in complete or partial mortality. h–j, Effects of increased light intensity on E. paradivisia incubated under HN/HP or HN/AP conditions. h, Fv/Fm is strongly reduced in corals from HN/AP after the doubling of the light intensity. i,j, Bleaching of these samples is caused by loss of zooxanthellae and reduced chlorophyll a content per algal cell. The horizontal dashed line in b and h signifies the level above which Fv/Fm values are considered to be in a healthy range. Colour scales are provided in a and g to facilitate the comparison of coral colours. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01) as detailed in the Supplementary Information. We evaluated whether this reduced photosynthetic efficiency could be caused by phosphate starvation of a proliferating symbiont population. First, we allowed M. foliosa colonies to adjust for six weeks in low-nutrient sea water to photonfluxes of ~30 μmol m−2 s−1. This acclimation was necessary to prevent a light-stress-driven loss of zooxanthellae after the transfer to sea water with imbalanced nutrient levels. Within 14 days after this transfer, the symbiont densities doubled, reporting an accordingly increased nutrient demand (Supplementary Fig. S2). Accordingly, zooxanthellae from corals kept under imbalanced nutrient conditions and a photonflux of ~90 μmol m−2 s−1 showed increased acidic and alkaline phosphatase activity indicating an increased demand for phosphorus20 (Fig. 2a). a, Increased activity of acidic and alkaline phosphatases in zooxanthellae from HN/AP treatments. b, Mass spectrometric analysis of the algal lipid content revealed a strong increase in SQDG in phosphate-starved zooxanthellae as determined by a precursor scan of the characteristic fragment of a mass of 225 under positive ionization (225+) c, Under phosphate starvation, the ratios of the zooxanthellae lipids, SQDG, PG and PC, are disturbed by the strong increase of SQDGs. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01) as detailed in the Supplementary Information. Photosynthetic organisms respond to LP stress by substituting phospholipids such as phosphatidylglycerol (PG) with sulpholipids, in particular with sulphoquinovosyldiacylglycerol (SQDG), to maintain the functionality of photosynthetic membranes and the embedded photosystems21. We observed a marked increase of SQDGs by one order of magnitude in zooxanthellae of M. foliosa kept under imbalanced nutrient conditions (Fig. 2b,c). These findings demonstrate that the stress symptoms observed under increased light intensities resulted indeed from phosphate starvation of the algal cells in hospite. The changes in the algal lipid complement offer potential explanations for the detrimental effects of phosphate starvation. The increase in SQDGs results in a shift in lipid ratios that will presumably alter the normal ionic character of photosynthetic membranes required for maintaining the proper assembly and functioning of the photosynthetic apparatus21. Most interestingly, malfunctioning photosystems and increased oxidative stress associated with photoinhibition of zooxanthellae have been shown to promote coral bleaching5, 22. Moreover, Tchernov and colleagues found that the specific lipid composition of different zooxanthellae strains is correlated with their susceptibility to thermal bleaching23. Taken together, our results allow to link the effects of phosphate starvation conveniently with established downstream processes of coral bleaching5, 19. As temperature stress is the main cause of mass coral bleaching on the global scale, we tested the impact of increased temperatures and the influence of additional light stress on phosphate-starved corals. After incubation (>five weeks) at ambient temperatures under a photonflux of ~90 μmol m−2 s−1 and imbalanced nutrient levels, Acropora polystoma showed a reduction in photosynthetic efficiency (Fig. 3a). The Fv/Fm values, though, were still in the healthy range of >0.5. Photosynthetic efficiency diminished slightly with increasing temperature. A subsequent increment in light intensity resulted in a remarkable decrease in Fv/Fm, which dropped earlier below critical levels (<0.5) in phosphate-starved individuals compared with nutrient-replete controls (Fig. 3a). A similarly pronounced decrease of Fv/Fm was observed when higher light levels were followed by an increase in temperatures, suggesting that light and temperature act together to promote bleaching in phosphate-starved corals (Fig. 3c). These results are in agreement with reports that bleaching of the fire coral Millepora alcicornis from a high-light habitat occurred one week earlier compared with a low-light habitat during the period of heat stress in 1998 (ref. 24). Over the full duration of our experiments, Fv/Fm of phosphate-starved zooxanthellae was always lower compared with the controls and at the end their density reached only ~ 40% of the nutrient-replete counterparts (Fig. 3b,d), resulting in a bleached appearance of the corals from imbalanced nutrient levels (Supplementary Fig. S3). The changes in light and temperature conditions over time are indicated by the labels of the abscissas. a,c, Time courses of combined stress experiments indicate that phosphate starvation, temperature and light stress result in reduced Fv/Fm values in zooxanthellae of A. polystoma. The horizontal dashed line in a and c indicates the threshold above which Fv/Fm values are considered to be in a healthy range. b,d, The lower photosynthetic efficiency is associated with reduced zooxanthellae numbers determined at the end of the respective experiment. e, Survival rates of replicate colonies of A. microphthalma from HN/HP or HN/AP sea water exposed to light and temperature stress. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01) as detailed in the Supplementary Information. In a combined light- and temperature-stress experiment we tested whether phosphate starvation also affects survival rates of corals. After a three-week treatment period, 100% mortality was observed among phosphate-starved Acropora microphthalma (Fig. 3e). In contrast, all control samples were visibly bleached, but survived the treatment. In phosphate-starved M. foliosa, the cover of the skeleton with living tissue was reduced by ~ 70% (Supplementary Fig. S4). Our findings indicate that phosphate starvation lowers the threshold of corals to suffer from light- and temperature-driven bleaching. We propose a new conceptual model of nutrient effects on coral bleaching (Fig. 4). This model assumes that a transition of zooxanthellae from a nutrient-limited to a nutrient-starved (here: phosphate) state leads to changes in the lipid composition of the algal membranes. Under thermal stress and in combination with light exposure, the altered photosynthetic membranes and embedded photosystems would show the previously described malfunctions of photosynthesis23, 25 that ultimately result in the breakdown of the coral–algal symbiosis and loss of zooxanthellae. Stress from low nutrient concentrations can arise owing to increased cellular demand during chemically unbalanced growth, but also may occur in waters in which specific nutrients become exhausted over time. Our findings suggest that the most severe impact on coral health might actually not arise from the over-enrichment with one group of nutrients (for example, DIN) but from the resulting relative depletion of other types (for example, phosphate) that is caused by the increased demand of proliferating zooxanthellae populations. This view is substantiated by the finding that the photosynthetic efficiency of zooxanthellae is reduced under a combination of limited iron availability and high temperatures26. Figure 4: Conceptual model of nutrient-starvation stress in zooxanthellae using the example of phosphate starvation. Conceptual model of nutrient-starvation stress in zooxanthellae using the example of phosphate starvation. a, Nutrient limitation in a steady-state population where the growth rate is determined by the rate of nutrient supply. Cells are fully acclimated and show no signs of stress. b, Phosphate starvation of zooxanthellae is induced by the transition from a nutrient-limited (a), to nutrient-starved state (b), owing to an increased cellular P demand caused by growth rates being accelerated through an increased DIN supply. The prioritized distribution of phosphate resources by the algae results in an altered composition of (thylakoid) membranes and a reduced threshold for light- and heat-induced bleaching. Our results have strong implications for coastal management. They suggest that reef resilience could benefit from considering local nutrient profiles and adjusting agricultural and tertiary wastewater-treatment practices in the proximity of coral reefs to reach favourable nutrient ratios in reef waters while working towards overall lower nutrient loadings. Finally, our findings support the view that local management of nutrient enrichment could reduce the effects of global climate change on coral reefs2, 3 and should help the design of functioning marine reserves. Methods The experiments were conducted in the coral mesocosm of the coral reef laboratory at the National Oceanography Centre Southampton. A detailed description of the experimental set-up is provided in ref. 15. This aquarium system has been running since 2007 and comprises three identical units. Each unit consists of two reservoir bins containing heating and filtration equipment and several experimental tanks. In the connected mode, a body of 2,250 l of artificial sea water circulates through the system. The artificial sea water was prepared by dissolving PRO-REEF salt mixture (Tropic Marin) in demineralized water. Five per cent of the water is changed on a weekly basis and an iron supplement is added daily. For the present experiment, phosphate levels were kept at low levels (<0.07 μM) by the application of Rowaphos phosphate-removal matrix (Rowa) and the addition of ethanol (2.5 ml per 1,000 l per day; ref. 15). Phosphate and nitrate levels were increased or maintained by continuous low-level dosing of sodium nitrate or disodium hydrogen phosphate solutions using peristaltic pumps. Corals were fed with frozen rotifers (Tropical Marine Centre) at a density of 0.5 g (frozen weight) per 135 l twice a week. If not stated otherwise, the temperature of the system was kept constant at 24 °C. Temperature-stress treatments were conducted in experimental tanks (40 l) equipped with precision heaters (Jaeger). These tanks were supplied with water from the connected unit with a flow rate of 80 l h−1. Current inside the experimental tanks (flow rate: 1,800 l h−1) was generated by Nanostream 6015 (Tunze). Corals were illuminated with metal halide lamps fitted with Aqualine 10000 burners (Aqua Medic) on a 10 hr/14 hr light/dark cycle. Changes in light intensity were achieved by altering the distance of the lamps to the samples. The corals used here were kept in our system for at least 2.5 years. For experimental purposes, the mother colonies were fragmented and the fragments were glued on tiles. They were allowed to regenerate and grow for at least two months before entering an experiment. Diagnostic restriction digests of the polymerase chain reaction-amplified small subunit ribosomal DNA gene of zooxanthellae of the corals under study revealed clade C as the dominant symbiont strain in the brown-, red- and purple–green-colour morphs of Montipora spp. (foliosa) and A. microphthalma27. Using the same approach, we found a prevalence of clade C also in E. paradivisia; Porites lobata and in the encrusting Montipora species. The zooxanthellae complement of A. polystoma consisted of a combination of clade C (~ 40%) with a yet unidentified strain (~ 60%). A detailed description of the methods is provided in the Supplementary Information. Briefly, nitrate, nitrite and phosphate at micromolar concentrations were determined using standard colourimetric techniques with a nutrient autoanalyser (Seal Analytical)28. Nanomolar levels of phosphate were determined with a colourimetric method using a 2 m liquid waveguide capillary cell with a miniaturized detector (Ocean Optics)29. Ammonium measurements were undertaken following a modified version of the method by Holmes using a FP-2020 Fluorescence Detector (Jasco). Ammonium levels found in our mesocosm were very low (<0.7% of total DIN) compared with the combined nitrite (~10%) and nitrate concentrations (~ 90%). Therefore, here DIN was considered to be represented by nitrite+nitrate values. Different units of the tank system were adjusted to low-nutrient (DIN ~0.7 μM/phosphate ~0.006 μM), nutrient-replete (DIN ~6.5 μM/phosphate ~0.3 μM) and imbalanced-nutrient (DIN >3 μM/phosphate ~0.07 μM) conditions15.

Zooxanthellae were counted using a haemocytometer and their pigment content was determined by spectrophotometric analysis of acetone extracts as described previously30. The maximum quantum yield of photosystem II photochemistry of zooxanthellae was measured with a submersible pulse-amplitude modulated fluorometer (Diving-PAM) according to previous recommendations16. The hydrolysis of para-nitrophenyl phosphate was determined colourimetrically as measure of alkaline and acidic phosphatase activity in zooxanthellae as described20. Specific lipids of interest for these studies, SQDG, PG and phosphatidylcholine (PC) molecular species, were analysed in detail using a Waters Micromass Quattro Ultima triple quadrupole mass spectrometer (Micromass) equipped with an electrospray ionization interface. To quantify the loss of tissue, coral fluorescence was documented and analysed for live tissue quantification using ImageJ (http://rsbweb.nih.gov/ij/) and MATLAB (MathWorks).
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