Replete Cultures Research Articles

Evidence for a robust photosystem II in the photosynthetic amoeba Paulinella.

Paulinella represents the only known case of an independent primary plastid endosymbiosis, outside Archaeplastida, that occurred c. 120 (million years ago) Ma. These photoautotrophs grow very slowly in replete culture medium with a doubling time of 6-7 d at optimal low light, and are highly sensitive to photodamage under moderate light levels. We used genomic and biophysical methods to investigate the extreme slow growth rate and light sensitivity of Paulinella, which are key to photosymbiont integration. All photosystem II (PSII) genes except psb28-2 and all cytochrome b6 f complex genes except petM and petL are present in Paulinella micropora KR01 (hereafter, KR01). Biophysical measurements of the water oxidation complex, variable chlorophyll fluorescence, and photosynthesis-irradiance curves show no obvious evidence of PSII impairment. Analysis of photoacclimation under high-light suggests that although KR01 can perform charge separation, it lacks photoprotection mechanisms present in cyanobacteria. We hypothesize that Paulinella species are restricted to low light environments because they are deficient in mitigating the formation of reactive oxygen species formed within the photosystems under peak solar intensities. The finding that many photoprotection genes have been lost or transferred to the host-genome during endosymbiont genome reduction, and may lack light-regulation, is consistent with this hypothesis.

Proteomics and expression studies on lipids and fatty acids metabolic genes in Isochrysis galbana under the combined influence of nitrogen starvation and sodium acetate supplementation

The marine alga Isochrysis galbana was grown under the integrated influence of nitrogen starvation and sodium acetate, specifically to understand the associated remodelling of the fatty acid profiles. This is the first comparative proteomics study under four different set of conditions namely nutrient sufficient, with/without sodium acetate and N-deficient with/without sodium acetate. A total set of 546 unique peptides were detected. Due to the addition of sodium acetate, 56 proteins were down-regulated and 7 proteins showed up-regulation respectively in the nutrient complete and the nitrogen starvation media. Both cell growth and docosahexaenoic acid (DHA) accumulation in the nutrient replete cultures were favoured by the addition of reduced carbon source in the form of sodium acetate. The 14 days old sodium acetate supplemented nitrogen starved cells showed highest amounts of total fatty acids and an over accumulation of the oleic acid. Of the seven key genes of the lipid and fatty acid metabolic pathway that were studied, the IgTeCe, IgFad12, and IgFad4 showed over expressions under nitrogen starvation. The gene expressions of lipases and desaturases involved in the fatty acids metabolism were not found to be correlated with the changes observed in the fatty acid profiles under the experimental conditions. This suggested the presence of an alternative metabolic route and/or possibly the variants of fatty acid desaturase genes in I. galbana which was beyond the scope of this study. A translational or post-translational control can not either be ignored.

Photoautotrophic hydrogen production by nitrogen-deprived Chlamydomonas reinhardtii cultures

In this study we have demonstrated the possibility of phototrophic hydrogen production in C. reinhardtii under N-deprived conditions. When tested under air + CO2, and Ar + CO2 N-deprived C. reinhardtii demonstrated decrease in PSII activity mainly due to over reduction of PQ, in addition no ascorbate accumulation was observed in cells. Under air + CO2 atmosphere cells accumulated excessive amounts of starch. When incubated under Ar + CO2 atmosphere cells accumulated starch as nitrogen replete cultures and no hydrogen production was observed. Hydrogen production (86 ml H2 per one l of culture) occurred under Ar + CO2 atmosphere when particular two-step illumination protocol was implicated. In oxygen producing and early oxygen consuming stage cells were illuminated under light intensity 169 μE m−2 s−1. When light was switched to 30 μE m−2 s−1, cultures quickly respired all oxygen and transient to anaerobic conditions with subsequent hydrogen production 2 h later. Actual quantum yield of C. reinhardtii cultures was measured in photobioreactor and maximal quantum efficiency of PSII of dark adapted cells together with JIP test were studied.

The B-Vitamin Mutualism Between the Dinoflagellate Lingulodinium polyedrum and the Bacterium Dinoroseobacter shibae

Recent research has shown that in aquatic systems pairs of prokaryote and eukaryote species exercise symbiotic exchanges of metabolites that are essential for the proliferation of either species. Using dinoflagellate Lingulodinium polyedrum cultures and a factorial design, we examined its growth at different concentrations of vitamin B1 (thiamine) and B12 (cobalamin). When both vitamins were at their lowest concentrations tested, 0.033 pM of B1 and 0.053 pM of B12 the growth was limited. When axenic L. polyedrum was co-cultured with the bacterium Dinoroseobacter shibae, a known B1 and B12 producer, then L. polyedrum grew at the same rate as in culture media supplemented with B1 and B12. In the L. polyedrum vitamin - limited culture (V-L), the abundance of attached and free-living D. shibae was higher than in the vitamin - replete (V-R) culture. In the V-R and V-L co-cultures the measured particulate B12 (pB12) concentration of attached and free-living D. shibae were in the range of 4.7x10-19 to 3x10-18 and 8.4x10-21 to1.2x10-19 (mol cell-1) respectively. Without B12 or B7 (biotin) added to the culture medium of a co-culture of L. polyedrum and D. shibae, the measured dissolved B12 (dB12) concentration was more than 60 pM higher than necessary for un-limited growth rates of L. polyedrum. In the same culture we measured B7 in the L. polyedrum particulate fraction (pB7; 4.7x10-19 to 9.4x10-19 mol cell-1). We suggest that in response to the production of B1 and B12 by D. shibae to supply L. polyedrum requeriments, the latter produced B7, which is required by D. shibae, and in our culture was only produced by L. polyedrum when D. shibae was present. We propose that D. shibae can control L. polyedrum through the release of B1 and B12, and L. polyedrum can control D. shibae through the release of B7. D. shibae is also auxotroph for niacin and 4-amino-benzoic acid, not provided by the culture medium. Therefore, L. polyedrum might affect a similar control through the release of these specific compounds and organic substrate necessary for the growth of D. shibae.

Iron and phosphorus deprivation induce sociality in the marine bloom-forming cyanobacterium Trichodesmium.

Trichodesmium spp. are diazotrophic cyanobacteria that exist as single filaments (trichomes) and as macroscopic colonies of varying shapes formed by aggregating trichomes. The causes and dynamics of colony formation and disassociation are not yet elucidated. we demonstrate that limited availability of dissolved phosphorus (P) or iron (Fe) stimulated trichome mobility and induced colony formation in Trichodesmium erythraeum IMS101 cultures. The specific nutrient limitation differentially affected the rate of colony formation and morphology of the colonies. Fe starvation promoted rapid colony formation (10-48 h from depletion) while 5-7 days were required for colonies to form in P-depleted cultures. Video analyses confirmed that the probability of trichomes to cluster increased from 12 to 35% when transferred from nutrient replete to Fe-depleted conditions. Moreover, the probability for Fe-depleted aggregates to remain colonial increased to 50% from only 10% in nutrient replete cultures. These colonies were also characterized by stronger attachment forces between the trichomes. Enrichment of nutrient-depleted cultures with the limited nutrient-stimulated colony dissociation into single trichomes. We postulate that limited P and Fe availability enhance colony formation of Trichodesmium and primarily control the abundance and distribution of its different morphologies in the nutrient-limited surface ocean.

Growth, stoichiometry and cell size; temperature and nutrient responses in haptophytes.

Temperature and nutrients are key factors affecting the growth, cell size, and physiology of marine phytoplankton. In the ocean, temperature and nutrient availability often co-vary because temperature drives vertical stratification, which further controls nutrient upwelling. This makes it difficult to disentangle the effects of temperature and nutrients on phytoplankton purely from observational studies. In this study, we carried out a factorial experiment crossing two temperatures (13°and 19°C) with two growth regimes (P-limited, semi-continuous batch cultures [“−P”] and nutrient replete batch cultures in turbidostat mode [“+P”]) for three species of common marine haptophytes (Emiliania huxleyi, Chrysochromulina rotalis and Prymnesium polylepis) to address the effects of temperature and nutrient limitation on elemental content and stoichiometry (C:N:P), total RNA, cell size, and growth rate. We found that the main gradient in elemental content and RNA largely was related to nutrient regime and the resulting differences in growth rate and degree of P-limitation, and observed reduced cell volume-specific content of P and RNA (but also N and C in most cases) and higher N:P and C:P in the slow growing −P cultures compared to the fast growing +P cultures. P-limited cells also tended to be larger than nutrient replete cells. Contrary to other recent studies, we found lower N:P and C:P ratios at high temperature. Overall, elemental content and RNA increased with temperature, especially in the nutrient replete cultures. Notably, however, temperature had a weaker–and in some cases a negative–effect on elemental content and RNA under P-limitation. This interaction indicates that the effect of temperature on cellular composition may differ between nutrient replete and nutrient limited conditions, where cellular uptake and storage of excess nutrients may overshadow changes in resource allocation among the non-storage fractions of biomass (e.g. P-rich ribosomes and N-rich proteins). Cell size decreased at high temperature, which is in accordance with general observations.

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors.

ABSTRACT Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m−2 d−1) and EPA (about 0.35 g m−2 d−1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m−2 d−1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Growth, Chl a content, photosynthesis, and elemental composition in polar and temperate microalgae

AbstractPolar microalgae live under extreme environmental conditions: permanently low temperatures (−1.7°C to +5°C) and extreme variations in irradiance and day length. These conditions may have led to various specific adaptations allowing Arctic phytoplankton to become specialists under these conditions. The goal of this study is to derive, for polar microalgae, empirical relationships between key physiological parameters (growth rate, photosynthesis–irradiance curve parameters, Chl a : C, and N : C ratios) and growth temperature and irradiance in nutrient replete cultures. Ecophysiological characteristics of polar and temperate microalgae were also compared in order to highlight some strategies that are specific to the polar environment. Most of the polar species are psychrophilic. Polar microalgae have low light‐saturated growth rates (μm) and very low light saturation parameters for growth (KE) but similar initial slopes of their growth–irradiance curve (αµ = μm/KE). Low temperatures probably account for low μm and KE in polar species. The C : Chl a ratios (θ) of polar species are similar to those of temperate species although they have much lower growth rates, which implies major differences in energy allocation. Polar microalgae also exhibit very unique photosynthetic properties [low light saturation parameters for photosynthesis (EK), low maximum photosynthetic rates ( ), decreasing and decreasing initial slope of the photosynthesis vs. irradiance curve (α*) with increasing irradiance] and have lower C : N ratios than their temperate cousins, which may be related to a much higher protein content. Some of the implications of these findings in terms of adaptation/acclimation to the environment in which polar species evolve are discussed.

Identifying Aspects of the Post-Transcriptional Program Governing the Proteome of the Green Alga Micromonas pusilla.

Micromonas is a unicellular motile alga within the Prasinophyceae, a green algal group that is related to land plants. This picoeukaryote (<2 μm diameter) is widespread in the marine environment but is not well understood at the cellular level. Here, we examine shifts in mRNA and protein expression over the course of the day-night cycle using triplicated mid-exponential, nutrient replete cultures of Micromonas pusilla CCMP1545. Samples were collected at key transition points during the diel cycle for evaluation using high-throughput LC-MS proteomics. In conjunction, matched mRNA samples from the same time points were sequenced using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as we observed in the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels from both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including codon usage as well as 3’ UTR length and structure. Collectively, our studies provide insights into the regulation of the proteome over a diel cycle as well as the relationships between transcriptional and translational programs in the widespread marine green alga Micromonas.

Understanding marine dissolved organic matter production: Compositional insights from axenic cultures of Thalassiosira pseudonana

Marine dissolved organic matter (DOM) is a key source of carbon and nutrients to microbial life in the oceans, but rapid biological utilization of labile DOM confounds its compositional characterization. In order to characterize potentially bioavailable DOM produced by phytoplankton, DOM from axenic cultures of Thalassiosira pseudonana cultivated in phosphorus (P) replete and low P conditions was extracted using high-recovery electrodialysis (ED) techniques, which resulted in an average dissolved organic carbon (DOC) recovery of 76% ± 7% from all cultures. Low P concentrations resulted in greater cell-normalized production of DOC relative to P replete culture controls at the same growth phase. Despite the different nutrient conditions, DOC composition and DOM molar ratios of carbon to nitrogen (C : N) were similar in all cultures. In contrast, low P concentrations influenced DOM molar carbon to phosphorus (C : P) ratios and dissolved organic phosphorus (DOP) composition. Under P replete and low P conditions, DOM C : P ratios were 130 (± 22) and 2446 (± 519), respectively. 31P Nuclear Magnetic Resonance (NMR) spectroscopy identified P esters (> 90% of DOP) as the dominant P species in DOM produced under P replete conditions, with small or negligible contributions from phosphonates or glycerol P and polyphosphates. However, based on direct fluorometric analysis, DOP from low P cultures was greater than 8 times enriched in dissolved polyphosphate compared to DOP from replete cultures, which is consistent with the growing evidence that polyphosphate is a dynamic component of total P in low P ocean regions.

Effect of phosphorus limitation on coccolith morphology and element ratios in Mediterranean strains of the coccolithophore Emiliania huxleyi

Six Emiliania huxleyi strains isolated in the Mediterranean Sea were grown under phosphorus limitation and replete (control) conditions in batch culture. We tested the hypothesis that increasing phosphorus limitation, resulting from ocean warming, will affect coccolithophores and change their contribution to the PIC:POC ratios. Four strains were isolated in the oligotrophic Algerian basin (western Mediterranean), and two strains in the extremely oligotrophic and often phosphorus depleted Levantine basin (eastern Mediterranean). Samples for particulate inorganic carbon, PIC; particulate organic carbon, POC; particulate organic phosphorus, POP; and total particulate nitrogen, TPN, were taken at equal cell densities in phosphorus limited and control culture conditions (harvesting in exponential and stationary phases respectively). Different morphological features of coccoliths were analyzed. Phosphorus limitation was inferred from: 1. a decrease in growth rate between 22% and 34% compared to phosphorus replete cultures; 2. a reduction in final cell density from ca. 2×106cells/ml to ca. 8×104cells/ml, and 3. markedly lower POP quotas of the limited cells. Differences in the phosphate uptake machinery are suggested based on the fact that three western Mediterranean strains decreased the phosphate concentration in the culture media until the detection limit in the first 3 experimental days while 4–5days were necessary for the other strains. Cellular carbon quotas increased in all strains under phosphorus limitation in comparison to their respective control cultures. The increase in POC quotas ranged between 179% and 260% and in PIC quotas between 43% and 201%. TPN quotas increased under phosphorus limitation between 31% and 86% in five out of six strains. POC:TPN increased in all strains, which suggests a change in the molecular composition of the cells when exposed to phosphorus limitation. Strain specific responses were also observed in the PIC:POC ratio, which decreased in five strains and did not change in another. Phosphorus limitation did not lead to malformations. However, morphological changes of coccoliths occurred, also in a strain specific way, namely the percentage of over-calcified coccoliths was altered by P limitation, but we observed all signs in the responses among different strains. Nevertheless, this morphological feature does not reflect the changes in calcite production (PICp nor PIC:POC). Strain specific responses were not related to the isolation location, suggesting that the isolated western and eastern strains do not represent distinct eco-genotypes featuring different phosphorus usage strategies.

Xanthophyll cycle induction by anaerobic conditions under low light in Chlamydomonas reinhardtii

The effect of anaerobiosis on the induction of the xanthophyll cycle was investigated in Chlamydomonas reinhardtii. The results showed that, anaerobiosis obtained by either sulfur starvation or by bubbling nitrogen in the culture grown in complete medium induced the xanthophyll cycle even when cultures were exposed to low light conditions. The zeaxanthin content reached 35 mmol mol−1 Chl a, after 110 h in anaerobic sulfur-starved cultures, and 30 mmol mol−1 Chl a within 24 h in sulfur replete cultures bubbled with nitrogen. Both starved and non-starved cultures grown under aerobic conditions, did not exhibit any sizeable increase in the zeaxanthin content. Chlorophyll fluorescence measurements revealed a decrease in the maximum photochemical quantum yield of PSII (Fv/Fm) by more than 50 %. The chlorophyll fluorescence kinetics (OJIP) analysis showed a strong rise at the J-step indicating a strong reduction of QA. Our findings demonstrated that anaerobiosis in low light exposed cultures induced the xanthophyll cycle through a strong increase of the level of plastoquinone pool reduction, which was associated to the formation of a trans-thylakoid membranes proton gradient, while in dark anaerobic cultures, no appreciable induction of xanthophyll cycle could be observed, despite the sizeable increase in non–photochemical quenching.

Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum Under Nitrate Deprivation

One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO3−)-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO3−-deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO3−-deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO3− replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.

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 (NaNO 3) and phosphate (NaH 2PO 4) 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 μmol/L Na 2SiO 3. Under nitrate limited (8.82 μmol/L NaNO 3) conditions, the growth of S. costatum was also dominant when phosphate concentrations were from 3.6 to 108 μmol/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 NaNO 3 concentrations between 88.2 and 882 μmol/L with limited phosphate (0.36 μmol/L NaH 2PO 4) 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.

Characterization of putative iron responsive genes as species-specific indicators of iron stress in thalassiosiroid diatoms.

Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations.

Synergy Between Specific Inflammatory Cytokines, IFNγ and TNFα, and Iron Restriction in Erythroid Blockade: A New Model for Anemia of Chronic Disease.

Abstract 159The anemias of chronic disease (ACD) are a common complication of malignancy, inflammation and kidney disorders. In ACD, there is dysregulation of iron homeostasis, decreased proliferation of erythroid progenitors, diminished production of erythropoietin (EPO), and shortened lifespan of RBC. Multiple pathophysiologic mechanisms have been implicated in the development of ACD, including elevated production of hepcidin and inflammatory cytokines, IFNγ, TRAIL, Interleukins-1β, 6, 10, 15, & TNFα. These cytokines are thought to directly inhibit erythroid differentiation through unknown mechanisms. The current study addressed the hypothesis that inhibition of erythropoiesis in ACD may arise through synergistic effects of iron deprivation and specific inflammatory cytokines. To identify relevant cytokines, candidate factors were applied to primary human erythroid progenitors in iron replete and restricted cultures. Peripheral blood human CD34+ progenitors from healthy donors underwent standard prestimulation for 72 hours, followed by culture in unilineage erythroid medium (4.5 U/ml EPO + 25ng/ml SCF) for 4-5 days under iron replete (100% transferrin saturation) or iron restricted (15% transferrin saturation) conditions. Candidate cytokines were screened for effects on viability, proliferation, and differentiation using cell counting and flow cytometric analysis of the erythroid cell surface marker GPA and the megakaryocytic antigen CD41a. Contrary to previous reports, the majority of cytokines (TRAIL & Interleukins-1β, 6, 10, 15) showed no effects on erythroid proliferation or differentiation under iron replete or restricted conditions. By contrast, both IFNγ and TNFα displayed potent inhibitory effects under iron restricted conditions but only weak effects in iron replete cultures. Typically, iron restriction alone reduced the proportion of GPA+ cells by 50%, whereas IFNγ or TNFα combined with iron restriction caused a 90% reduction. While both cytokines cooperated with iron restriction in blocking upregulation of GPA and promoting cell death, each cytokine also had distinctive effects on morphology and differentiation. IFNγ enhanced megakaryocytic development, while TNFα retained cells as immature, CD34+ progenitors. The synergistic inhibition of erythroid differentiation with iron restriction and TNFα was confirmed in vivo using a murine model of dietary iron deprivation coupled with continuous infusion of low-dose TNFα. Regarding the mechanism for this synergy, we have previously shown that erythroid iron deprivation leads to inactivation of the aconitase enzymes, which normally convert citrate to isocitrate, and that provision of exogenous isocitrate abrogates the erythroid inhibition associated with iron deprivation. Accordingly, participation of this pathway was assessed in the more potent erythroid inhibition associated with IFNγ or TNFα plus iron deprivation. Strikingly, isocitrate administration not only abrogated effects due to iron deprivation but also those due to the inflammatory cytokines, leading to complete rescue of erythroid differentiation. To address the underlying basis for erythroid cross-talk of iron and cytokine signaling, we screened pathways implicated in iron metabolism and inflammation. Two relevant pathways identified were Jun kinase (JNK) and calmodulin-associated kinase II (CAMKII), important in TNFα and IFNγ signaling, respectively. In particular, TNFα and iron deprivation synergized in the activation of JNK, and IFNγ and iron deprivation synergized in activating CAMKII. In both cases, isocitrate partially restored the activation to basal levels. As an important negative control, iron deprivation did not affect IFNγ activation of STAT1 phosphorylation, indicating that its effects were not due to upregulation of receptor expression or function. Altogether, these data suggest that among the various cytokines implicated in ACD, only IFNγ and TNFα synergize with iron deprivation in the inhibition of erythropoiesis. These actions occur through cross-talk between intracellular signaling pathways, specifically pathways involving aconitase and cytokine-activated kinases. The connection of aconitase/metabolism with inflammation is novel and has implications for clinical treatment of ACD, as well as for new understanding of erythroid and inflammatory signaling. Disclosures:No relevant conflicts of interest to declare.

Effect of food concentration and type of diet on Acartia survival and naupliar development

We have performed life table experiments to investigate the effects of different food types and concentrations on the larval development and survival up to adulthood of Acartia tonsa. The food species offered comprised a wide taxonomic spectrum: the pigmented flagellates Isochrysis galbana, Emiliania huxleyi, Rhodomonas sp., Prorocentrum minimum, the diatom Thalassiosira weissflogii, grown on medium offering enriched macronutrient concentrations and the ciliate Euplotes sp. initially cultured on Rhodomonas. For the ciliate species, also the functional response was studied. In order to avoid limitation by mineral nutrients, food algae have been taken from the exponential growth phase of the nutrient replete cultures. The suitability of Rhodomonas as a food source throughout the entire life cycle was not a surprise. However, in contrast to much of the recent literature about the inadequacy or even toxicity of diatoms, we found that also Thalassiosira could support Acartia-development through the entire life cycle. On the other hand, Acartia could not complete its life cycle when fed with the other food items, Prorocentrum having adverse effects even when mixed with Rhodomonas and Thalassiosira. Isochrysis well supported naupliar survival and development, but was insufficient to support further development until reproduction. With Emiliania and Euplotes, nauplii died off before most of them could reach the first copepodite stages. Acartia-nauplii showed a behavioral preference for Euplotes-feeding over diatom feeding, but nevertheless Euplotes was an insufficient diet to complete development beyond the naupliar stages.

UV-B radiation increases cell permeability and damages nitrogen incorporation mechanisms in Nannochloropsis gaditana

This study shows the response of Nannochloropsis gaditana, a marine nannoplanktonic species, exposed to UV radiation for 7 days. PAR, UV-A and UV-B ratios used were within the range likely to be observed in nature, a photoperiod of 12L:12D was maintained, and light irradiances were modified daily to promote cell acclimation. Growth, pigment content, internal nitrogen and carbon content, and photochemical efficiency using PAM fluorometry were assessed in nutrient replete cultures. Cell size, autofluorescence and cell permeability were analysed by flow cytometry. Results showed a cessation of growth after day 3 and a progressive decrease was observed in Fv/Fm values in cultures exposed to UV-B (plus UV-A and PAR). Flow cytometry analysis also demonstrated an increase in membrane permeability caused by UV-B damage. Cells that showed an increase in membrane permeability also exhibited a proportional decrease in cellular nitrogen content. The results support the conclusion that UV-B radiation can affect N. gaditana nitrogen incorporation mechanisms by direct damage or indirectly by damage to membrane structure and to the photosynthetic apparatus with resulting effects on energy and reductant demand. In contrast, the presence of UV-A radiation was beneficial to cells exposed to PAR plus UV-A when compared to those exposed to only-PAR from day 4. This response resulted in cells with a higher nitrogen content and without changes in membrane permeability.

Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea

Nutritional and environmental conditions were characterized for a batch culture of the marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea for its growth (cells ml −1), cellular toxin content (Qt in fmol cell −1) and toxin composition (mol%). Under a nutrient replete condition, Qt increased with cell growth and peaked at the late stationary phase. Toxin content increased with the nitrate concentration in the culture while it reached a maximum at 5 μM phosphate. When nitrate was replaced with ammonia, Qt decreased by 4.5-fold. Salinity and light intensity were important factors affecting Qt. The latter increased two-fold over the range of salinity from 15 to 30‰, while decreased 38% as light intensity increased from 80 to 220 μE m −2 s −1. Toxin composition varied with growth phase and culture conditions. In nutrient replete cultures, toxin composition varied greatly in the early growth phase (first 3 days) and then C1/C2, C3/C4 and GTX1 remained relatively constant while GTX4 increased from 32 to 46% and GTX5 decreased from 28 to 15%. In general, the composition of GTXs was affected in a much greater extent than C toxins by changes in nutrient conditions, salinity and light intensity. This is especially true with GTX4 and GTX5. These data indicate that the cellular toxin content and toxin composition of A. tamarense HK9301 are not constant, but that they vary with growth phase and culture conditions. Use of toxin composition to identify a toxigenic marine dinoflagellate is not always valid. The data also reveal that high salinity and low light intensity, together with high nitrate and low phosphate concentrations, would favor toxin production by this species.

Elemental composition of marine Prochlorococcus and Synechococcus: Implications for the ecological stoichiometry of the sea

The elemental composition of marine cyanobacteria is an important determinant of the ecological stoichiometry in low‐latitude marine biomes. We analyzed the cellular carbon (C), nitrogen (N), and phosphorus (P) contents of Prochlorococcus (MED4) and Synechococcus (WH8103 and WH8012) under nutrient‐replete and P‐starved conditions. Under nutrient‐replete conditions, C, N, and P quotas (femtogram cell21) of the three strains were 46 ± 4, 9.4 ± 0.9, and 1.0 ± 0.2 for MED4; 92 ± 13, 20 6 3, and 1.8 ± 0.1 for WH8012; and 213 ± 7, 50 ± 2, 3.3 ± 0.5 for WH8103. In P‐limited cultures, they were 61 ± 2, 9.6 ± 0.1, and 0.3 ± 0.1 for MED4; 132 ± 6, 21 ± 2, and 0.5 ± 0.2 for WH8012; and 244 ± 21, 40 ± 4, and 0.8 ± 0.01 for WH8103. P limitation had no effect on the N cell quota of MED4 and WH8012 but reduced the N content of WH8103. The cellular C quota was consistently higher in P‐limited than in nutrient‐replete cultures. All three strains had higher C: P and N: P ratios than the Redfield ratio under both nutrient‐replete and P‐limited conditions. The C:N molar ratios ranged 5–5.7 in replete cultures and 7.1–7.5 in P‐limited cultures; C: P ranged 121–165 in the replete cultures and 464–779 under P limitation; N: P ranged 21–33 in the replete cultures and 59–109 under P limitation. Our results suggest that Prochlorococcus and Synechococcus may have relatively low P requirements in the field, and thus the particulate organic matter they produce would differ from the Redfield ratio (106C : 16N : 1P) often assumed for the production of new particulate organic matter in the sea.