Transcriptional response of Emiliania huxleyi under changing nutrient environments in the North Pacific Subtropical Gyre.

The widespread coccolithophore Emiliania huxleyi is an abundant oceanic phytoplankton, impacting the global cycling of carbon through both photosynthesis and calcification. Here, we examined the transcriptional responses of populations of E. huxleyi in the North Pacific Subtropical Gyre to shifts in the nutrient environment. Using a metatranscriptomic approach, nutrient-amended microcosm studies were used to track the global metabolism of E. huxleyi. The addition of nitrate led to significant changes in transcript abundance for gene pathways involved in nitrogen and phosphorus metabolism, with a decrease in the abundance of genes involved in the acquisition of nitrogen (e.g. N-transporters) and an increase in the abundance of genes associated with phosphate acquisition (e.g. phosphatases). Simultaneously, after the addition of nitrate, genes associated with calcification and genes unique to the diploid life stages of E. huxleyi significantly increased. These results suggest that nitrogen is a major driver of the physiological ecology of E. huxleyi in this system and further suggest that the addition of nitrate drives shifts in the dominant life-stage of the population. Together, these results underscore the importance of phenotypic plasticity to the success of E. huxleyi, a characteristic that likely underpins its ability to thrive across a variety of marine environments.

Transcriptional patterns identify resource controls on the diazotroph Trichodesmium in the Atlantic and Pacific oceans.

The N2-fixing cyanobacterium Trichodesmium is intensely studied because of the control this organism exerts over the cycling of carbon and nitrogen in the low nutrient ocean gyres. Although iron (Fe) and phosphorus (P) bioavailability are thought to be major drivers of Trichodesmium distributions and activities, identifying resource controls on Trichodesmium is challenging, as Fe and P are often organically complexed and their bioavailability to a single species in a mixed community is difficult to constrain. Further, Fe and P geochemistries are linked through the activities of metalloenzymes, such as the alkaline phosphatases (APs) PhoX and PhoA, which are used by microbes to access dissolved organic P (DOP). Here we identified significant correlations between Trichodesmium-specific transcriptional patterns in the North Atlantic (NASG) and North Pacific Subtropical Gyres (NPSG) and patterns in Fe and P biogeochemistry, with the relative enrichment of Fe stress markers in the NPSG, and P stress markers in the NASG. We also observed the differential enrichment of Fe-requiring PhoX transcripts in the NASG and Fe-insensitive PhoA transcripts in the NPSG, suggesting that metalloenzyme switching may be used to mitigate Fe limitation of DOP metabolism in Trichodesmium. This trait may underpin Trichodesmium success across disparate ecosystems.

Epibionts dominate metabolic functional potential of Trichodesmium colonies from the oligotrophic ocean.

Trichodesmium is a genus of marine diazotrophic colonial cyanobacteria that exerts a profound influence on global biogeochemistry, by injecting 'new' nitrogen into the low nutrient systems where it occurs. Colonies of Trichodesmium ubiquitously contain a diverse assemblage of epibiotic microorganisms, constituting a microbiome on the Trichodesmium host. Metagenome sequences from Trichodesmium colonies were analyzed along a resource gradient in the western North Atlantic to examine microbiome community structure, functional diversity and metabolic contributions to the holobiont. Here we demonstrate the presence of a core Trichodesmium microbiome that is modulated to suit different ocean regions, and contributes over 10 times the metabolic potential of Trichodesmium to the holobiont. Given the ubiquitous nature of epibionts on colonies, the substantial functional diversity within the microbiome is likely an integral facet of Trichodesmium physiological ecology across the oligotrophic oceans where this biogeochemically significant diazotroph thrives.

Variable depth distribution of Trichodesmium clades in the North Pacific Ocean.

Populations of nitrogen-fixing cyanobacteria in the genus Trichodesmium are critical to ocean ecosystems, yet predicting patterns of Trichodesmium distribution and their role in ocean biogeochemistry is an ongoing challenge. This may, in part, be due to differences in the physiological ecology of Trichodesmium species, which are not typically considered independently in field studies. In this study, the abundance of the two dominant Trichodesmium clades (Clade I and Clade III) was investigated during a survey at Station ALOHA in the North Pacific Subtropical Gyre (NPSG) using a clade-specific qPCR approach. While Clade I dominated the Trichodesmium community, Clade III abundance was >50% in some NPSG samples, in contrast to the western North Atlantic where Clade III abundance was always <10%. Clade I populations were distributed down to depths >80 m, while Clade III populations were only observed in the mixed layer and found to be significantly correlated with depth and temperature. These data suggest active niche partitioning of Trichodesmium species from different clades, as has been observed in other cyanobacteria. Tracking the distribution and physiology of Trichodesmium spp. would contribute to better predictions of the physiological ecology of this biogeochemically important genus in the present and future ocean.

Microbial diversity within the Trichodesmium holobiont.

Nitrogen-fixing cyanobacteria in the genus Trichodesmium play a critical role in the productivity of the tropical and subtropical oligotrophic oceans. The ecological success of these populations is likely associated with the diverse microbial interactions occurring within the Trichodesmium holobiont, especially between Trichodesmium and heterotrophic bacterial epibionts. Yet, the composition of the Trichodesmium holobiont and the processes governing microbial assemblage are not well documented. Here, we used high-resolution 16S rDNA amplicon sequencing to examine the diversity of Trichodesmium and associated epibionts across different ocean regions and colony morphologies (puffs and rafts). Trichodesmium Clade I (i.e., T. thiebautii-like) dominated the colonies in all ocean basins regardless of morphology, although the Trichodesmium community structure significantly varied between morphologies in some regions. On average, Alphaproteobacteria (i.e., Thalassobius), Gammaproteobacteria (i.e., Pseudoalteromonas), Sphingobacteria (i.e., Microscilla and Vibrio) and Flavobacteria dominated the epibiont communities, but community composition and structure significantly differed between regions. Epibionts from the two colony morphologies were taxonomically and functionally distinct in the North Atlantic and North Pacific. These findings suggest that the colony types might define two distinct niches and that epibiont assemblage might be driven in part by selective processes, where epibionts are selected according to their influence on colony metabolism.

Functional group-specific traits drive phytoplankton dynamics in the oligotrophic ocean.

A diverse microbial assemblage in the ocean is responsible for nearly half of global primary production. It has been hypothesized and experimentally demonstrated that nutrient loading can stimulate blooms of large eukaryotic phytoplankton in oligotrophic systems. Although central to balancing biogeochemical models, knowledge of the metabolic traits that govern the dynamics of these bloom-forming phytoplankton is limited. We used eukaryotic metatranscriptomic techniques to identify the metabolic basis of functional group-specific traits that may drive the shift between net heterotrophy and autotrophy in the oligotrophic ocean. Replicated blooms were simulated by deep seawater (DSW) addition to mimic nutrient loading in the North Pacific Subtropical Gyre, and the transcriptional responses of phytoplankton functional groups were assayed. Responses of the diatom, haptophyte, and dinoflagellate functional groups in simulated blooms were unique, with diatoms and haptophytes significantly (95% confidence) shifting their quantitative metabolic fingerprint from the in situ condition, whereas dinoflagellates showed little response. Significantly differentially abundant genes identified the importance of colimitation by nutrients, metals, and vitamins in eukaryotic phytoplankton metabolism and bloom formation in this system. The variable transcript allocation ratio, used to quantify transcript reallocation following DSW amendment, differed for diatoms and haptophytes, reflecting the long-standing paradigm of phytoplankton r- and K-type growth strategies. Although the underlying metabolic potential of the large eukaryotic phytoplankton was consistently present, the lack of a bloom during the study period suggests a crucial dependence on physical and biogeochemical forcing, which are susceptible to alteration with changing climate.

The limit of the genetic adaptation to copper in freshwater phytoplankton.

Copper is one of the most frequently used algaecides to control blooms of toxic cyanobacteria in water supply reservoirs. Among the negative impacts derived from the use of this substance is the increasing resistance of cyanobacteria to copper toxicity, as well as changes in the community structure of native phytoplankton. Here, we used the ratchet protocol to investigate the differential evolution and maximum adaptation capacity of selected freshwater phytoplankton species to the exposure of increasing doses of copper. Initially, a dose of 2.5 µM CuSO4·5H2O was able to completely inhibit growth in three strains of the toxic cyanobacterium Microcystis aeruginosa, whereas growth of the chlorophyceans Dictyosphaerium chlorelloides and Desmodesmus intermedius (represented by two different strains) was completely abolished at 12 µM. A significant increase in resistance was achieved in all derived populations during the ratchet experiment. All the chlorophyceans were able to adapt to up to 270 µM of copper sulfate, but 10 µM was the highest concentration that M. aeruginosa strains were able to cope with, although one of the replicates adapted to 30 µM. The recurrent use and increasing doses of copper in water reservoirs could lead to the selection of copper-resistant mutants of both chlorophyceans and cyanobacteria. However, under high concentrations of copper, the composition of phytoplankton community could undergo a drastic change with cyanobacteria being replaced by copper-resistant chlorophyceans. This result stems from a distinct evolutionary potential of these species to adapt to this substance.

The effect of nitrate and phosphate availability on Emiliania huxleyi (NZEH) physiology under different CO2 scenarios.

Growth and calcification of the marine coccolithophorid Emiliania huxleyi is affected by ocean acidification and macronutrients limitation and its response varies between strains. Here we investigated the physiological performance of a highly calcified E. huxleyi strain, NZEH, in a multiparametric experiment. Cells were exposed to different CO2 levels (ranging from 250 to 1314 µatm) under three nutrient conditions [nutrient replete (R), nitrate limited (-N), and phosphate limited (-P)]. We focused on calcite and organic carbon quotas and on nitrate and phosphate utilization by analyzing the activity of nitrate reductase (NRase) and alkaline phosphatase (APase), respectively. Particulate inorganic (PIC) and organic (POC) carbon quotas increased with increasing CO2 under R conditions but a different pattern was observed under nutrient limitation. The PIC:POC ratio decreased with increasing CO2 in nutrient limited cultures. Coccolith length increased with CO2 under all nutrient conditions but the coccosphere volume varied depending on the nutrient treatment. Maximum APase activity was found at 561 µatm of CO2 (pH 7.92) in -P cultures and in R conditions, NRase activity increased linearly with CO2. These results suggest that E. huxleyi's competitive ability for nutrient uptake might be altered in future high-CO2 oceans. The combined dataset will be useful in model parameterizations of the carbon cycle and ocean acidification.

Effects of adaptation, chance, and history on the evolution of the toxic dinoflagellate Alexandrium minutum under selection of increased temperature and acidification.

The roles of adaptation, chance, and history on evolution of the toxic dinoflagellate Alexandrium minutum Halim, under selective conditions simulating global change, have been addressed. Two toxic strains (AL1V and AL2V), previously acclimated for two years at pH 8.0 and 20°C, were transferred to selective conditions: pH 7.5 to simulate acidification and 25°C. Cultures under selective conditions were propagated until growth rate and toxin cell quota achieved an invariant mean value at 720 days (ca. 250 and ca. 180 generations for strains AL1V and AL2V, respectively). Historical contingencies strongly constrained the evolution of growth rate and toxin cell quota, but the forces involved in the evolution were not the same for both traits. Growth rate was 1.5-1.6 times higher than the one measured in ancestral conditions. Genetic adaptation explained two-thirds of total adaptation while one-third was a consequence of physiological adaptation. On the other hand, the evolution of toxin cell quota showed a pattern attributable to neutral mutations because the final variances were significantly higher than those measured at the start of the experiment. It has been hypothesized that harmful algal blooms will increase under the future scenario of global change. Although this study might be considered an oversimplification of the reality, it can be hypothesized that toxic blooms will increase but no predictions can be advanced about toxicity.

Adaptation of microalgae to lindane: a new approach for bioremediation.

Lindane is especially worrisome because its persistence in aquatic ecosystems, tendency to bioaccumulation and toxicity. We studied the adaptation of freshwater cyanobacteria and microalgae to resist lindane using an experimental model to distinguish if lindane-resistant cells had their origin in random spontaneous pre-selective mutations (which occur prior to the lindane exposure), or if lindane-resistant cells arose by a mechanism of physiological acclimation during the exposure to the selective agent. Although further research is needed to determine the different mechanisms contributing to the bio-elimination of lindane, this study, however, provides an approach to the bioremediation abilities of the lindane-resistant cells. Wild type strains of the experimental organisms were exposed to increasing lindane levels to estimate lethal concentrations. Growth of wild-type cells was completely inhibited at 5mg/L concentration of lindane. However, after further incubation in lindane for several weeks, occasionally the growth of rare lindane-resistant cells was found. A fluctuation analysis demonstrated that lindane-resistant cells arise only by rare spontaneous mutations that occur randomly prior to exposure to lindane (lindane-resistance did not occur as a result of physiological mechanisms). The rate of mutation from lindane sensitivity to resistance was between 1.48 × 10(-5) and 2.35 × 10(-7) mutations per cell per generation. Lindane-resistant mutants exhibited a diminished fitness in the absence of lindane, but only these variants were able to grow at lindane concentrations higher than 5mg/L (until concentrations as high as 40 mg/L). Lindane-resistant mutants may be maintained in uncontaminated waters as the result of a balance between new resistant mutants arising from spontaneous mutation and resistant cells eliminated by natural selection waters via clone selection. The lindane-resistant cells were also used to test the potential of microalgae to remove lindane. Three concentrations (4, 15 and 40 mg/L) were chosen as a model. In these exposures the lindane-resistant cells showed a great capacity to remove lindane (until 99% lindane was eliminated). Apparently, bioremediation based on lindane-resistant cells could be a great opportunity for cleaning up of lindane- and other chlorinated organics-polluted habitats.

Warming will affect phytoplankton differently: evidence through a mechanistic approach.

Although the consequences of global warming in aquatic ecosystems are only beginning to be revealed, a key to forecasting the impact on aquatic communities is an understanding of individual species' vulnerability to increased temperature. Despite their microscopic size, phytoplankton support about half of the global primary production, drive essential biogeochemical cycles and represent the basis of the aquatic food web. At present, it is known that phytoplankton are important targets and, consequently, harbingers of climate change in aquatic systems. Therefore, investigating the capacity of phytoplankton to adapt to the predicted warming has become a relevant issue. However, considering the polyphyletic complexity of the phytoplankton community, different responses to increased temperature are expected. We experimentally tested the effects of warming on 12 species of phytoplankton isolated from a variety of environments by using a mechanistic approach able to assess evolutionary adaptation (the so-called ratchet technique). We found different degrees of tolerance to temperature rises and an interspecific capacity for genetic adaptation. The thermal resistance level reached by each species is discussed in relation to their respective original habitats. Our study additionally provides evidence on the most resistant phytoplankton groups in a future warming scenario.

Evolutionary changes in growth rate and toxin production in the cyanobacterium Microcystis aeruginosa under a scenario of eutrophication and temperature increase.

Toxic blooms of the cyanobacterium Microcystis aeruginosa affect humans and animals in inland water systems worldwide, and it has been hypothesized that the development of these blooms will increase under the future scenario of global change, considering eutrophication and temperature increase as two important consequences. The importance of genetic adaptation, chance and history on evolution of growth rate, and toxin production of M. aeruginosa was studied under these new conditions. The experiment followed the idea of "replaying life's tape" by means of the simultaneous propagation of 15 independent isolates of three M. aeruginosa strains, which were grown under doubled nutrient concentration and temperature during c. 87 generations. Adaptation by new mutations that resulted in the enhancement of growth rate arose during propagation of derived cultures under the new environmental conditions was the main component of evolution; however, chance also contributed in a lesser extension to evolution of growth rate. Mutations were selected, displacing the wild-type ancestral genotypes. In contrast, the effect of selection on mutations affecting microcystin production was neutral. Chance and history were the pacemakers in evolution of toxin production. Although this study might be considered an oversimplification of the reality, it suggest that a future scenario of global change might lead to an increase in M. aeruginosa bloom frequency, but no predictions about the frequency of toxicity can be made.

GENETIC ADAPTATION AND ACCLIMATION OF PHYTOPLANKTON ALONG A STRESS GRADIENT IN THE EXTREME WATERS OF THE AGRIO RIVER-CAVIAHUE LAKE (ARGENTINA)(1).

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal-rich water to moderate stressful conditions) of the Agrio River-Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria-Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal-rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10(-7) resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

Adaptation of green microalgae to the herbicides simazine and diquat as result of pre-selective mutations.

Aquatic ecosystems located close to agricultural areas are increasingly polluted by herbicides. We evaluated the capacity for adaptation of green microalgae to lethal concentrations of the herbicide simazine in one strain of Dictyosphaerium chlorelloides and two strains of Scenedesmus intermedius, as well as adaptation to the herbicide diquat in one of the strains of S. intermedius. A Luria-Delbrück fluctuation analysis was carried out in order to distinguish between resistant cells arising from physiological adaptation (acclimatization) or post-adaptive mutation (both events occurring after the exposure to the herbicides), and adaptation due to mutations before the exposure to the herbicides. Simazine-resistant cells arose by rare spontaneous mutations before the exposure to simazine, with a rate of 3.0 x 10(-6) mutants per cell per generation in both strains of S. intermedius, and of 9.2 x 10(-6) mutants per cell per generation in D. chlorelloides. Diquat-resistant cells in S. intermedius arose by pre-selective mutations with a rate of 17.9 x 10(-6) per cell per generation. Rare, pre-selective mutations may allow the survival of green microalgae in simazine- or diquat-polluted waters, via herbicide-resistant selection. Therefore, human-synthesized pollutants, such as the herbicides simazine and diquat, could cause the emergence of evolutionary novelties in aquatic environments.

Toxicity and adaptation of Dictyosphaerium chlorelloides to extreme chromium contamination.

Metals are often spilled by industries into inland water environments, with adverse consequences. Numerous papers have reported that heavy metals produce massive destruction of algae. Nevertheless, algal populations seem to become tolerant when they have had previous exposures to heavy metals. Because the mechanisms allowing heavy metal tolerance of algae are not yet known, the present study analyzed the effect of hexavalent chromium on growth and photosynthetic performance of Dictyosphaerium chlorelloides, stressing on the adaptation mechanisms to chromium contamination. Growth and photosynthetic performance of algal cells were inhibited by Cr(VI) at 10 mg/L, and the 72-h median inhibition concentration was established as 1.64 and 1.54 mg/L, respectively. However, after further incubation for a three month period in an environment with 25 mg/L of chromium, some rare, chromium-resistant cells occasionally were found. A Luria-Delbrück fluctuation analysis was performed to distinguish between resistant algae arising from rare, spontaneous mutations and resistant algae arising from physiological adaptation and other adaptive mechanisms. Resistant cells arose only by spontaneous mutations before the addition of chromium, with a rate of 1.77 x 10(-6) mutants per cell division. From a practical point of view, the use of both chromium-sensitive and chromium-resistant genotypes could make possible a specific algal biosensor for chromium.

Adaptation of the chlorophycean Dictyosphaerium chlorelloides to stressful acidic, mine metal-rich waters as result of pre-selective mutations.

Several species of microalgae, closely related to mesophilic lineages, inhabit the extreme environment (pH 2.5, high levels of metals) of the Spain's Aguas Agrias Stream water (AASW). Consequently, AASW constitutes an interesting natural laboratory for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective or post-selective origin of adaptation processes allowing the existence of microalgae in AASW, a Luria-Delbrück fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In the analysis, AASW was used as selective factor. Preselective, resistant D. chlorelloides cells appeared with a frequency of 1.1 x 10(-6) per cell per generation. AASW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new AASW-resistant cells arising by mutation and AASW-resistant mutants eliminated by natural selection (equilibrium at c. 12 AASW-resistants per 10(7) wild-type cells). We propose that the microalgae inhabiting this stressful environment could be the descendents of chance mutants that arrived in the past or are even arriving at the present.

Adaptation of phytoplankton to novel residual materials of water pollution: an experimental model analysing the evolution of an experimental microalgal population under formaldehyde contamination.

The adaptation mechanisms of microalgae to grow in contaminated waters were analysed using a chlorophyta species under formaldehyde exposure as experimental model. Cultures initially collapsed after exposure to 16 ppm formaldehyde, but occasionally resistant cells were able to grow after further incubation. Resistant cells arose by rare spontaneous mutations that appeared before the exposure to formaldehyde (mutation rate=3.61 x 10(-6)), and not as result of physiological mechanisms. Although mutations may be the mechanisms that should allow the survival of microalgae in polluted waters in a world under rapid global change, mutants have a diminished growth rate.