Molecular mechanisms by which marine phytoplankton respond to their dynamic chemical environment.

Marine scientists have long been interested in the interactions of marine phytoplankton with their chemical environments. Nutrient availability clearly controls carbon fixation on a global scale, but the interactions between phytoplankton and nutrients are complex and include both short-term responses (seconds to minutes) and longer-term evolutionary adaptations. This review outlines how genomics and functional genomics approaches are providing a better understanding of these complex interactions, especially for cyanobacteria and diatoms, for which the genome sequences of multiple model organisms are available. Transporters and related genes are emerging as the most likely candidates for biomarkers in stress-specific studies, but other genes are also possible candidates. One surprise has been the important role of horizontal gene transfer in mediating chemical-biological interactions.

Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Interactive effects of silver nanoparticles and phosphorus on phytoplankton growth in natural waters.

Increasing amounts of silver nanoparticles (AgNPs) are expected to enter the aquatic ecosystems where their effects on natural phytoplankton communities are poorly understood. We investigated the effects of AgNPs and its interactions with phosphorus (P) supply on the growth kinetics and stoichiometry of natural phytoplankton. Lake water was dosed with AgNPs (carboxy-functionalized capping agent; ∼10-nm particle size; ∼20% Ag w/w) at four different concentrations and five P concentrations and incubated in situ for 3 days. A treatment with ionic silver (AgNO3) was used as a positive control. We found that growth rates, calculated from changes in seston carbon and chlorophyll, responded significantly and interactively (p < 0.0001) to both AgNPs and P. AgNPs reduced the maximum phytoplankton growth rates by 11-85%. In the positive control, no or very little growth was observed. Inhibition of growth rates after exposure to Ag might be related to the reduction in chlorophyll and the inhibition of C and N acquisition rather than P uptake mechanisms. AgNPs, P supply and their interactions also significantly (p < 0.0001) reduced sestonic C:P and N:P ratios and increased C:N, C:Chl and cell-bound Ag stoichiometry. Our results indicate that fate and toxicity of AgNP will vary with phosphorus pollution level in aquatic ecosystems.

Use of agent-based modeling to explore the mechanisms of intracellular phosphorus heterogeneity in cultured phytoplankton.

There can be significant intraspecific individual-level heterogeneity in the intracellular P of phytoplankton, which can affect the population-level growth rate. Several mechanisms can create this heterogeneity, including phenotypic variability in various physiological functions (e.g., nutrient uptake rate). Here, we use modeling to explore the contribution of various mechanisms to the heterogeneity in phytoplankton grown in a laboratory culture. An agent-based model simulates individual cells and their intracellular P. Heterogeneity is introduced by randomizing parameters (e.g., maximum uptake rate) of daughter cells at division. The model was calibrated to observations of the P quota of individual cells of the centric diatom Thalassiosira pseudonana, which were obtained using synchrotron X-ray fluorescence (SXRF). A number of simulations, with individual mechanisms of heterogeneity turned off, then were performed. Comparison of the coefficient of variation (CV) of these and the baseline simulation (i.e., all mechanisms turned on) provides an estimate of the relative contribution of these mechanisms. The results show that the mechanism with the largest contribution to variability is the parameter characterizing the maximum intracellular P, which, when removed, results in a CV of 0.21 compared to a CV of 0.37 with all mechanisms turned on. This suggests that nutrient/element storage capabilities/mechanisms are important determinants of intrapopulation heterogeneity.

Phytoplankton-specific response to enrichment of phosphorus-rich surface waters with ammonium, nitrate, and urea.

Supply of anthropogenic nitrogen (N) to the biosphere has tripled since 1960; however, little is known of how in situ response to N fertilisation differs among phytoplankton, whether species response varies with the chemical form of N, or how interpretation of N effects is influenced by the method of analysis (microscopy, pigment biomarkers). To address these issues, we conducted two 21-day in situ mesocosm (3140 L) experiments to quantify the species- and genus-specific responses of phytoplankton to fertilisation of P-rich lake waters with ammonium (NH(4)(+)), nitrate (NO(3)(-)), and urea ([NH(2)](2)CO). Phytoplankton abundance was estimated using both microscopic enumeration of cell densities and high performance liquid chromatographic (HPLC) analysis of algal pigments. We found that total algal biomass increased 200% and 350% following fertilisation with NO(3)(-) and chemically-reduced N (NH(4)(+), urea), respectively, although 144 individual taxa exhibited distinctive responses to N, including compound-specific stimulation (Planktothrix agardhii and NH(4)(+)), increased biomass with chemically-reduced N alone (Scenedesmus spp., Coelastrum astroideum) and no response (Aphanizomenon flos-aquae, Ceratium hirundinella). Principle components analyses (PCA) captured 53.2-69.9% of variation in experimental assemblages irrespective of the degree of taxonomic resolution of analysis. PCA of species-level data revealed that congeneric taxa exhibited common responses to fertilisation regimes (e.g., Microcystis aeruginosa, M. flos-aquae, M. botrys), whereas genera within the same division had widely divergent responses to added N (e.g., Anabaena, Planktothrix, Microcystis). Least-squares regression analysis demonstrated that changes in phytoplankton biomass determined by microscopy were correlated significantly (p<0.005) with variations in HPLC-derived concentrations of biomarker pigments (r(2) = 0.13-0.64) from all major algal groups, although HPLC tended to underestimate the relative abundance of cyanobacteria. Together, these findings show that while fertilisation of P-rich lakes with N can increase algal biomass, there is substantial variation in responses of genera and divisions to specific chemical forms of added N.

Variations in phytoplankton carbon biomass, community assemblages and species succession along Lake Burullus, Northern Egypt.

Phytoplankton assemblages and species succession along Lake Burullus (Southern Mediterranean) is expressed as carbon biomass (mg cm3) using a standard spreadsheet based on the species cell volume cell(-1) carbon relationship. High Chl a levels were measured (maximum 85-126 mg m(-3)) reflecting a dense phytoplankton population (up to 8.3 x 10(3) cell ml(-1) and 5.5 x 10(3) mg cm(-3)) throughout the lake body with maximum concentrations at the western sector of the lake (S1). Adiverse phytoplankton community was determined. Cell count data revealed the dominance of a mixed phytoplankton taxa, however biomass data indicates over-dominance of Bacillariophyceae (up to 98%). Good correlation (r = 0.73, p < 0.05) was found between Chl a and carbon biomass with various cell carbon/Chl a ratio according to variations in community structure. Bacillariophyceae were the most dominant, particularly at the middle (S2) and the western parts (S1) during periods of high nutrient (silicate) and good weather conditions (during spring/summer months). Chlorophyceae were abundant with Scenedesmus sp. mostly dominant, particularly at P-rich sites. Dinoflagellates peaked only during calm and high light summer months (May-July) being at a maximum level at S1. Euglenophyceae were less contributed to total phytoplankton abundance and peaked only; as a transition stage; at S1 during Jannuary and March (winter months). Cyanophyceae were numerous along with maximum peak at S2 affected by the southern drains. Excessive nutrient enrichment into the lake alters the existent structure of phytoplankton community. The water quality index indicated a poor water quality status of the lake.This may led to increase the possibility of toxic algal blooms to invade the lake ecosystem and, in turn, affect the lake fish yield.

Evidence for a three-way trade-off between nitrogen and phosphorus competitive abilities and cell size in phytoplankton.

Trade-offs among functional traits are essential for explaining community structure and species coexistence. While two-way trade-offs have been investigated in many systems, higher-dimensional trade-offs remain largely hypothetical. Here we demonstrate a three-way trade-off between cell size and competitive abilities for nitrogen and phosphorus in marine and freshwater phytoplankton. At a given cell size, competitive abilities for N and P are negatively correlated, but as cell size increases, competitive ability decreases for both nutrients. The relative importance of the two trade-off axes appears to be environment dependent, suggesting different selective pressures: freshwater phytoplankton separate more along the N vs. P competition axis, and marine phytoplankton separate more along the nutrient competition vs. cell size axis. Our results demonstrate the multidimensional nature of key trade-offs among traits and suggest that such trade-offs may drive species interactions and structure ecological communities.

Competition of bloom-forming marine phytoplankton at low nutrient concentrations.

Competition of three bloom-forming marine phytoplankton (diatom Skeletonema costatum, and dinoflagellates Prorocentrum minimum and Alexandrium tamarense) was studied through a series of multispecies cultures with different nitrate (NaNO3) and phosphate (NaH2PO4) levels and excess silicate to interpret red tide algae succession. S. costatum outgrew the other two dinoflagellates in nitrate and phosphate replete cultures with 10 micromol/L Na2SiO3. Under nitrate limited (8.82 micromol/L NaNO3) conditions, the growth of S. costatum was also dominant when phosphate concentrations were from 3.6 to 108 micromol/L. Cell density of the two dinoflagellates only increased slightly, to less than 400 and 600 cells/mL, respectively. Cell density of S. costatum decreased with time before day 12, and then increased to 4000 cells/mL (1.5 mg/L dry biomass) at NaNO3 concentrations between 88.2 and 882 micromol/L with limited phosphate (0.36 micromol/L NaH2PO4) levels. In addition, P. minimum grew well with a maximal cell density of 1690-2100 cells/mL (0.5-0.6 mg/L dry biomass). Although S. costatum initially grew fast, its cell density decreased quickly with time later in the growth phase and the two dinoflagellates were dominant under the nitrate-limited and high nitrate conditions with limited phosphate. These results indicated that the diatom was a poor competitor compared to the two dinoflagellates under limited phosphate; however, it grew well under limited nitrate when growth of the dinoflagellates was near detection limits.

Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom Thalassiosira pseudonana.

In low-phosphorus (P) marine systems, phytoplankton replace membrane phospholipids with non-phosphorus lipids, but it is not known how rapidly this substitution occurs. Here, when cells of the model diatom Thalassiosira pseudonana were transferred from P-replete medium to P-free medium, the phospholipid content of the cells rapidly declined within 48 h from 45±0.9 to 21±4.5% of the total membrane lipids; the difference was made up by non-phosphorus lipids. Conversely, when P-limited T. pseudonana were resupplied with P, cells reduced the percentage of their total membrane lipids contributed by a non-phosphorus lipid from 43±1.5 to 7.3±0.9% within 24 h, whereas the contribution by phospholipids rose from 2.2±0.1 to 44±3%. This dynamic phospholipid reservoir contained sufficient P to synthesize multiple haploid genomes, suggesting that phospholipid turnover could be an important P source for cells. Field observations of phytoplankton lipid content may thus reflect short-term changes in P supply and cellular physiology, rather than simply long-term adjustment to the environment.

[Dynamic studies on the effect of nutrients on the growth of Microcystis aeruginosa].

The effects of nitrogen and phosphorus on the growth of Microcystis aeruginosa were investigated by using the Monod equation. The semi-saturation constants of Microcystis aeruginosa to TP (KsP) and TN (KsN) were calculated. The results show that KsN is higher than KsP. This indicates that the effect of TP on the growth of Microcystis aeruginos is further more significant than that of TN. Further analysis finds that extant quantity (X) and special growth rate (micro) of Microcystis aeruginosa increase with TP or TN, and there exists a point of inflection. When the concentrations of TP and TN ranged from 0.005mg/L to 0.2mg/L and from 0.01mg/L to 2mg/L respectively with TP or TN as the single limiting substrate, the growth rate of Microcystis aeruginosa increased rapidly. Because Microcystis aeruginosa exhibits different affinity with TP and TN (according to the semi-saturation constants), the effect of N/P ratio on the growth of Microcystis aeruginosa does not demonstrate at a constant value. No constant ratio can be used to determine the limiting nutrient elements on the growth of Microcystis aeruginosa in any aquatic environment. The effect of N/P ratio on the growth of Microcystis aeruginosa depends on both the N and P concentrations and the N/P ratio.

The impact of surface-adsorbed phosphorus on phytoplankton Redfield stoichiometry.

The Redfield ratio of 106 carbon:16 nitrogen:1 phosphorus in marine phytoplankton is one of the foundations of ocean biogeochemistry, with applications in algal physiology, palaeoclimatology and global climate change. However, this ratio varies substantially in response to changes in algal nutrient status and taxonomic affiliation. Here we report that Redfield ratios are also strongly affected by partitioning into surface-adsorbed and intracellular phosphorus pools. The C:N:surface-adsorbed P (80-105 C:15-18 N:1 P) and total (71-80 C:13-14 N:1 P) ratios in natural populations and cultures of Trichodesmium were close to Redfield values and not significantly different from each other. In contrast, intracellular ratios consistently exceeded the Redfield ratio (316-434 C:59-83 N:1 intracellular P). These high intracellular ratios were associated with reduced N2 fixation rates, suggestive of phosphorus deficiency. Other algal species also have substantial surface-adsorbed phosphorus pools, suggesting that our Trichodesmium results are generally applicable to all phytoplankton. Measurements of the distinct phytoplankton phosphorus pools may be required to assess nutrient limitation accurately from elemental composition. Deviations from Redfield stoichiometry may be attributable to surface adsorption of phosphorus rather than to biological processes, and this scavenging could affect the interpretation of marine nutrient inventories and ecosystem models.

Studies of the effect of Psi-APONIN from Nannochloris sp. on the Florida red tide organism Karenia brevis.

Studies were conducted on the conditions under which the red tide organism, Karenia brevis (a.k.a., Gymnodinium breve), was treated with Nannochloris sp. The latter organism is known to produce cytolytic agents called Apparent Oceanic Naturally Occurring Cytolin (APONINs). Conventional wisdom might suggest that brevetoxins would be released upon destruction of the single-celled dinoflagellate K. brevis and that efforts to treat red tide outbreaks would lead to release of brevetoxins and enhanced toxicity toward marine species. Earlier studies described conditions by which K. brevis cells were converted to a non-motile form when cultures of K. brevis were treated with an isolate (Psi-APONIN) produced by Nannochloris sp. but when centrifuged only a small amount of the toxin was released. The present study confirms that the toxin is not released when the K. brevis is undisturbed, however, when the culture is stressed (stirred with a magnetic stirring bar for 24, 48, and 72h) toxin was released, and the toxicity could be measured using a Microtox analyzer. In the study, it was found that at as few as eighty cells of K. brevis produced a toxic effect of 20% as measured by the effect on Vibrio fischeri. Nannochloris sp. had no effect on the bacteria used in the Microtox analyzer, nor did interaction of Nannochloris sp. with K. brevis in the short term. This effect is presumed to be due to the production of Psi-APONIN and conversion of K. brevis to a non-motile or resting form.