NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY1

Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4‐fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient‐replete and nitrogen (N)‐limited semi‐continuous cultures. N‐limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two‐fold increase (1%–4% to 5%–9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4‐fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation.     

INFLUENCE OF PHOSPHORUS LIMITATION ON TOXICITY AND PHOTOSYNTHESIS OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) MONITORED BY IN‐LINE DETECTION OF VARIABLE CHLOROPHYLL FLUORESCENCE1

The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexandrium minutum were examined in batch culture experiments. Cell division was greatly impaired in P‐limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equivalent to P‐replete cultures. The cellular concentration of paralytic shellfish toxins was consistent over the growth cycle of control cultures from lag phase into logarithmic growth phase, with toxins repeatedly lost to daughter cells during division. The low level of cell division in P‐limited cultures resulted in a 10‐fold increase of cellular toxin compared with controls, but this dropped upon P spiking due to increased rates of cell division. The history of phosphorus supply had an important effect on toxin concentration, with the P‐limited and the P‐spiked cultures showing values 2‐fold higher than the P‐replete cultures. Toxin profiles of the A. minutum strain used in these experiments were dominated by the N₁‐hydroxy toxins, gonyautoxins (GTX) GTX1 and GTX4, which were approximately 40 times more abundant than their analogues, GTX2 and GTX3, in P‐limited cultures. The dominance of the N₁‐hydroxy toxins increased significantly in control cultures as they advanced through logarithmic growth. In‐line measurements of the variable chl fluorescence of light‐adapted cells indicated consistent photochemical efficiency under P‐replete conditions. P limitation induced a drop in fluorescence‐based photochemical efficiency that was reversible by P spiking. There was an inverse linear relationship between in‐line fluorescence and cell toxin quota (r = ?0.88). Monitoring fluorescence in‐line may be valuable in managing efficient biotechnological production of toxins.     

Inorganic carbon promotes photosynthesis,growth, and maximum biomass of phytoplankton in eutrophic water bodies

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Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

Monitoring cellular C:N ratio in phytoplankton by means of FTIR‐spectroscopy

Statistical growth rate modelling can be applied in a variety of ecological and biotechnological applications. Such models are frequently based on Monod or Droop equations and, especially for the latter, require reliable determination of model input parameters such as C:N quotas. Besides growth rate modelling, a C:N quota quantification can be useful for monitoring and interpretation of physiological acclimation to abiotic and biotic disturbances (e.g., nutrient limitations). However, as high throughput C:N quota determination is difficult to perform, alternatives need to be established. Fourier‐transformed infrared (FTIR) spectroscopy is used to analyze a variety of biochemical, chemical, and physiological parameters in phytoplankton. Hence, a quantification of the C:N quota should also be feasible. Therefore, using FTIR spectroscopy, six phytoplankton species from among different phylogenetic groups have been analyzed to determine the effect of nutrient limitation on C:N quota patterns. The typical species‐specific response to increasing nitrogen limitation was an increase in the C:N quota. Irrespective of this species specificity, we were able to develop a reliable multi‐species C:N quota prediction model based on FTIR spectroscopy using the partial least square regression (PLSR) algorithm. Our data demonstrate that the PLSR approach is more robust in C:N quota quantification (R² = 0.93) than linear correlation of C:N quota versus growth rate (R² ranges from 0.74 to 0.86) or biochemical information based on FTIR spectra (R² ranges from 0.82 to 0.89). This accurate prediction of C:N values may support high throughput measurements in a broad range of future approaches.     

Predator-induced defence in a dinoflagellate generates benefits without direct costs

Inducible defences in phytoplankton are often assumed to come at a cost to the organism, but trade-offs have proven hard to establish experimentally. A reason for this may be that some trade-off costs only become evident under resource-limiting conditions. To explore the effect of nutrient limitation on trade-offs in toxin-producing dinoflagellates, we induced toxin production in Alexandrium minutum by chemical cues from copepods under different levels of nitrogen limitation. The effects were both nitrogen- and grazer-concentration dependent. Induced cells had higher cellular toxin content and a larger fraction of the cells was rejected by a copepod, demonstrating the clear benefits of toxin production. Induced cells also had a higher carbon and nitrogen content, despite up to 25% reduction in cell size. Unexpectedly, induced cells seemed to grow faster than controls, likely owing to a higher specific nutrient affinity due to reduced size. We thus found no clear trade-offs, rather the opposite. However, indirect ecological costs that do not manifest under laboratory conditions may be important. Inducing appropriate defence traits in response to threat-specific warning signals may also prevent larger cumulative costs from expressing several defensive traits simultaneously.Subject terms: Ecology, Microbial ecology     

Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation

Lipid production by the red yeast Rhodosporidium toruloides was explored under nutrient limitation. To determine the compositional profiles of R. toruloides cells, samples were prepared using a continuous cultivation process under nutrient limitation and analyzed via several methods, including Fourier transform infrared spectroscopy and elemental analysis. Under nitrogen limitation, as the dilution rate increased, the cellular lipid content decreased but the carbohydrate and protein contents increased. Under carbon limitation, the cellular lipid, protein, and carbohydrate contents remained relatively constant at the different dilution rates. Moreover, the cellular elemental composition was essentially identical under nitrogen and carbon limitation at a high dilution rate of 0.20 h⁻¹. We also analyzed the consumed carbon to nitrogen (C/N) under different nutrition conditions. The results indicated that the consumed C/N had a major influence on cell metabolism and product formation, which contributed to our understanding of the physiological characteristics of R. toruloides.

     

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP. LMECYA 31 (CYANOBACTERIA)1

We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per‐cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment.     

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY‐labeled conjugate of brevetoxin congener PbTx‐2 (B‐PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid‐rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20–200 μm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B‐PbTx. Uptake and intracellular fluorescence of B‐PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY‐conjugated K. brevis associated secondary metabolites, brevenal (B‐Bn) and brevisin (B‐Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently‐tagged brevetoxin conjugates in non‐toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B‐PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment.     

UNCOUPLING OF SILICON COMPARED WITH CARBON AND NITROGEN METABOLISMS AND THE ROLE OF THE CELL CYCLE IN CONTINUOUS CULTURES OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) UNDER LIGHT,NITROGEN, AND PHOSPHORUS CONTROL1

The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light‐ (E), nitrogen‐ (N), and phosphorus‐ (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2‐ to 6‐fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions.     

Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

Long‐term nitrogen loading alleviates phosphorus limitation in terrestrial ecosystems

Increased human‐derived nitrogen (N) deposition to terrestrial ecosystems has resulted in widespread phosphorus (P) limitation of net primary productivity. However, it remains unclear if and how N‐induced P limitation varies over time. Soil extracellular phosphatases catalyze the hydrolysis of P from soil organic matter, an important adaptive mechanism for ecosystems to cope with N‐induced P limitation. Here we show, using a meta‐analysis of 140 studies and 668 observations worldwide, that N stimulation of soil phosphatase activity diminishes over time. Whereas short‐term N loading (≤5 years) significantly increased soil phosphatase activity by 28%, long‐term N loading had no significant effect. Nitrogen loading did not affect soil available P and total P content in either short‐ or long‐term studies. Together, these results suggest that N‐induced P limitation in ecosystems is alleviated in the long‐term through the initial stimulation of soil phosphatase activity, thereby securing P supply to support plant growth. Our results suggest that increases in terrestrial carbon uptake due to ongoing anthropogenic N loading may be greater than previously thought.     

Increased freshwater discharge shifts the trophic balance in the coastal zone of the northern Baltic Sea

Increased precipitation is one projected outcome of climate change that may enhance the discharge of freshwater to the coastal zone. The resulting lower salinity, and associated discharge of both nutrients and dissolved organic carbon, may influence food web functioning. The scope of this study was to determine the net outcome of increased freshwater discharge on the balance between auto‐ and heterotrophic processes in the coastal zone. By using long‐term ecological time series data covering 13 years, we show that increased river discharge suppresses phytoplankton biomass production and shifts the carbon flow towards microbial heterotrophy. A 76% increase in freshwater discharge resulted in a 2.2 times higher ratio of bacterio‐ to phytoplankton production (P_b:P_p). The level of P_b:P_p is a function of riverine total organic carbon supply to the coastal zone. This is mainly due to the negative effect of freshwater and total organic carbon discharge on phytoplankton growth, despite a concomitant increase in discharge of nitrogen and phosphorus. With a time lag of 2 years the bacterial production recovered after an initial decline, further synergistically elevating the microbial heterotrophy. Current climate change projections suggesting increased precipitation may therefore lead to increased microbial heterotrophy, thereby decreasing the transfer efficiency of biomass to higher trophic levels. This prognosis would suggest reduced fish production and lower sedimentation rates of phytoplankton, a factor of detriment to benthic fauna. Our findings show that discharge of freshwater and total organic carbon significantly contributes to the balance of coastal processes at large spatial and temporal scales, and that model's would be greatly augmented by the inclusion of these environmental drivers as regulators of coastal productivity.     

Nitrogen deposition and warming – effects on phytoplankton nutrient limitation in subarctic lakes

The aim of this study was to predict the combined effects of enhanced nitrogen (N) deposition and warming on phytoplankton development in high latitude and mountain lakes. Consequently, we assessed, in a series of enclosure experiments, how lake water nutrient stoichiometry and phytoplankton nutrient limitation varied over the growing season in 11 lakes situated along an altitudinal/climate gradient with low N‐deposition (<1 kg N ha^?1 yr^?1) in northern subarctic Sweden. Short‐term bioassay experiments with N‐ and P‐additions revealed that phytoplankton in high‐alpine lakes were more prone to P‐limitation, and with decreasing altitude became increasingly N‐ and NP‐colimited. Nutrient limitation was additionally most obvious in midsummer. There was also a strong positive correlation between phytoplankton growth and water temperature in the bioassays. Although excess nutrients were available in spring and autumn, on these occasions growth was likely constrained by low water temperatures. These results imply that enhanced N‐deposition over the Swedish mountain areas will, with the exception of high‐alpine lakes, enhance biomass and drive phytoplankton from N‐ to P‐limitation. However, if not accompanied by warming, N‐input from deposition will stimulate limited phytoplankton growth due to low water temperatures during large parts of the growing season. Direct effects of warming, allowing increased metabolic rates and an extension of the growing season, seem equally crucial to synergistically enhance phytoplankton development in these lakes.     

Inter- and intra-annual variability in the phytoplankton community of a high mountain lake: the influence of external (atmospheric) and internal (recycled) sources of phosphorus

1. The inter‐ and intra‐annual changes in the biomass, elemental (carbon (C), nitrogen (N) and phosphorus (P)) and taxonomical composition of the phytoplankton in a high mountain lake in Spain were studied during 3 years with different physical (fluctuating hydrological regime) and chemical conditions. The importance of internal and external sources of P to the phytoplankton was estimated as the amount of P supplied via zooplankton recycling (internal) or through ice‐melting and atmospheric deposition (external). 2. Inter‐annual differences in phytoplankton biomass were associated with temperature and total dissolved phosphorus. In 1995, phytoplankton biomass was positively correlated with total dissolved phosphorus. In contrast, the negative relationship between zooplankton and seston biomass (direct predatory effects) and the positive relationship between zooplankton P excretion and phytoplankton biomass in 1997 (indirect P‐recycling effects), reinforces the primary role of zooplankton in regulating the total biomass of phytoplankton but, at the same time, encouraging its growth via P‐recycling. 3. Year‐to‐year variations in seston C : P and N : P ratios exceeded intra‐annual variations. The C : P and N : P ratios were high in 1995, indicating strong P limitation. In contrast, in 1996 and 1997, these ratios were low during ice‐out (C : P < 100 and N : P < 10) and increased markedly as the season progressed. Atmospheric P load to the lake was responsible for the decline in C : P and N : P ratios. 4. Intra‐annual variations in zooplankton stoichiometry were more pronounced than the overall differences between 1995 and 1996. Thus, the zooplankton N : P ratio ranged from 6.9 to 40.1 (mean 21.4) in 1995, and from 10.4 to 42.2 (mean 24.9) in 1996. The zooplankton N : P ratio tended to be low after ice‐out, when the zooplankton community was dominated by copepod nauplii, and high towards mid‐ and late‐season, when these were replaced by copepodites and adults. 5. In 1995, the minimum demands for P of phytoplankton were satisfied by ice‐melting, atmospheric loading and zooplankton recycling over 100%. In order of importance, atmospheric inputs (> 1000%), zooplankton recycling (9–542%), and ice‐melting processes (0.37–5.16%) satisfied the minimum demand for P of phytoplankton during 1996 and 1997. Although the effect of external forces was rather sporadic and unpredictable in comparison with biologically driven recycle processes, both may affect phytoplankton structure and elemental composition. 6. We identified three conceptual models representing the seasonal phosphorus flux among the major compartments of the pelagic zone. While ice‐melting processes dominated the nutrient flow at the thaw, biologically driven processes such as zooplankton recycling became relevant as the season and zooplankton ontogeny progressed. The stochastic nature of P inputs associated with atmospheric events can promote rapid transitional changes between a community limited by internal recycling and one regulated by external load. 7. The elemental composition of the zooplankton explains changes in phytoplankton taxonomic and elemental composition. The elemental negative balance (seston N : P < zooplankton N : P, low N : P recycled) during the thaw, would promote a community dominated by species with a high demand for P (Cryptophyceae). The shift to an elemental positive balance (seston N : P > zooplankton N : P, high N : P recycled) in mid‐season would skew the N : P ratio of the recycled nutrients, favouring dominance by chrysophytes. The return to negative balance, as a consequence of the ontogenetic increase in zooplankton N : P ratio and the external P inputs towards the end of the ice‐free season, could alleviate the limitation of P and account for the appearance of other phytoplankton classes (Chlorophyceae or Dinophyceae).     

Intraspecific trait variation and trade‐offs within and across populations of a toxic dinoflagellate

Intraspecific trait diversity can promote the success of a species, as complementarity of functional traits within populations may enhance its competitive success and facilitates resilience to changing environmental conditions. Here, we experimentally determined the variation and relationships between traits in 15 strains of the toxic dinoflagellate Alexandrium ostenfeldii derived from two populations. Measured traits included growth rate, cell size, elemental composition, nitrogen uptake kinetics, toxin production and allelochemical potency. Our results demonstrate substantial variation in all analysed traits both within and across populations, particularly in nitrogen affinity, which was even comparable to interspecific variation across phytoplankton species. We found distinct trade‐offs between maximum nitrogen uptake rate and affinity, and between defensive and competitive traits. Furthermore, we identified differences in trait variation between the genetically similar populations. The observed high trait variation may facilitate development and resilience of harmful algal blooms under dynamic environmental conditions.     

Unimodal size scaling of phytoplankton growth and the size dependence of nutrient uptake and use

Phytoplankton size structure is key for the ecology and biogeochemistry of pelagic ecosystems, but the relationship between cell size and maximum growth rate (μ_max) is not yet well understood. We used cultures of 22 species of marine phytoplankton from five phyla, ranging from 0.1 to 10⁶ μm³ in cell volume (V_cell), to determine experimentally the size dependence of growth, metabolic rate, elemental stoichiometry and nutrient uptake. We show that both μ_max and carbon‐specific photosynthesis peak at intermediate cell sizes. Maximum nitrogen uptake rate (V_maxN) scales isometrically with V_cell, whereas nitrogen minimum quota scales as V_cell^0.84. Large cells thus possess high ability to take up nitrogen, relative to their requirements, and large storage capacity, but their growth is limited by the conversion of nutrients into biomass. Small species show similar volume‐specific V_maxN compared to their larger counterparts, but have higher nitrogen requirements. We suggest that the unimodal size scaling of phytoplankton growth arises from taxon‐independent, size‐related constraints in nutrient uptake, requirement and assimilation.     

Oligotrophication of a large,deep lake alters food quantity and quality constraints at the primary producer–consumer interface

To assess nutritional consequences associated with lake oligotrophication for aquatic consumers, we analyzed the elemental and biochemical composition of natural seston and concomitantly conducted laboratory growth experiments in which the freshwater key herbivore Daphnia was raised on natural seston of the nowadays (2008) oligotrophic Lake Constance throughout an annual cycle. Food quality mediated constraints on Daphnia performance were assessed by comparing somatic growth rates with seston characteristics (multiple regression analysis) and by manipulating the elemental and biochemical composition of natural seston experimentally (nutrient supplementation). Results were compared to similar experiments carried out previously (1997) during a mesotrophic phase of the lake. In the oligotrophic phase, particulate carbon and phosphorus concentrations were lower, fatty acid concentrations were higher, and the taxonomic composition of phytoplankton was less diverse, with a more diatom‐ and cryptophytes‐dominated community, compared to the previous mesotrophic phase. Multiple regression analysis indicated a shift from a simultaneous limitation by food quantity (in terms of carbon) and quality (i.e. α‐linolenic acid) during the mesotrophic phase to a complex multiple nutrient limitation mediated by food quantity, phosphorus, and omega‐3 fatty acids in the following oligotrophic phase. The concomitant supplementation experiments also revealed seasonal changes in multiple resource limitations, i.e. the prevalent limitation by food quantity was accompanied by a simultaneous limitation by either phosphorus or omega‐3 fatty acids, and thus confirmed and complemented the multiple regression approach. Our results indicate that seasonal and annual changes in nutrient availabilities can create complex co‐limitation scenarios consumers have to cope with, which consequently may also affect the efficiency of energy transfer in food webs.     

Molecular response of Anabaena flos-aquae to differing concentrations of phosphorus: A combined Fourier transform infrared and X-ray microanalytical study

The response of Anabaena flos‐aquae to varying levels of P availability was determined using the combined techniques of Fourier transform infrared (FTIR) microspectroscopy and energy‐dispersive X‐ray microanalysis (EDXRMA). Batch cultures of Anabaena were grown at initial P concentrations of 50 μg (low‐P culture), 500 μg (intermediate‐P) and 5000 μg (high‐P) (PO₄‐P L^?1) and were monitored for total biovolume, chlorophyll a, media nutrients (PO₄‐P and NO₃‐N), cellular P quota (EDXRMA) and carbon allocation (FTIR spectroscopy). The high‐P culture showed a sixfold increase in total biovolume over the sampling period. Phosphorus in the media remained at more than ～4000 μg L^?1 and intracellular P concentration (P quota) as determined by EDXRMA showed no decline, remaining at more than 0.20% dry weight (DW). The intermediate‐P culture showed a similar increase in total biovolume, but P concentrations in the media fell to ～20 μg L^?1, and this was reflected in a decline in the cellular P quota from 0.24% to 0.06% DW. Although the high‐ and intermediate‐P treatments differed markedly in both internal and external P, analysis of the FTIR spectra from the two treatments, using both hierarchical cluster analysis (HCA) and principal component analysis (PCA), indicated no difference in carbon allocation. Cells from the low‐P treatment showed strong evidence for P deficiency. P concentrations in the media were undetectable (<5 μg L^?1), total biovolume was much reduced, and P quotas were low, falling from 0.08% to 0.01% DW. HCA and PCA clearly separated FTIR spectra from low‐P cells from those of intermediate‐ and high‐P cultures, with low‐P cells having increased absorbance in the region of the spectra associated with carbohydrate. Both FTIR spectroscopy and EDXRMA have the resolution to study the elemental and macromolecular composition of single species within mixed phytoplankton populations and the combined use of these techniques has considerable potential for the study of cyanobacterial responses to environmental stress.