EFFECT OF ZINC AVAILABILITY ON GROWTH,MORPHOLOGY, AND NUTRIENT INCORPORATION IN A COASTAL AND AN OCEANIC DIATOM1

We investigated the effect of Zn availability on growth rate (μ), cell morphology, and elemental stoichiometry and incorporation rate in two marine diatoms. For the coastal diatom Skeletonema costatum (Grev.) Cleve, the half‐saturation constant (K_S) for growth was 4.1 pM Zn²⁺, and growth ceased at ≤ 2.6 pM Zn²⁺, whereas for the oceanic diatom Thalassiosira oceanica Hasle, K_S was 0.5 pM Zn²⁺, and μ remained at ～40%μ_max even at 0.3 pM Zn²⁺. Under Zn‐limiting (Zn‐L) conditions, S. costatum decreased cell size significantly, leading to an 80% increase in surface area to volume ratio (SA/V) at Zn²⁺ of 3.5 pM compared to Zn‐replete (Zn‐R) conditions (at Zn²⁺ of 13.2 pM), whereas T. oceanica’s morphology did not change appreciably. Cell quotas of C, N, P, Si, and chl a significantly decreased under Zn limitation in S. costatum (at Zn²⁺ of 3.5 pM), whereas Zn limitation in T. oceanica (at Zn²⁺ of 0.3 pM) had little effect on quotas. Elemental stoichiometry was ～85C:10N:9Si:1P and 81C:9N:5Si:1P for S. costatum, and 66C:5N:2Si:1P and 52C:6N:2Si:1P for T. oceanica, under Zn‐R and Zn‐L conditions, respectively. Incorporation rates of all elements were significantly reduced under Zn limitation for both diatoms, but particularly for Si in S. costatum, and for C in T. oceanica, despite its apparent tolerance of low Zn conditions. With [Zn²⁺] in some parts of the ocean being of the same order (～0.2 to 2 pM) as our low Zn conditions for T. oceanica, our results support the hypothesis that in situ growth and C acquisition may be limited by Zn in some oceanic species.     

Copepod Population-Specific Response to a Toxic Diatom Diet

Diatoms are key phytoplankton organisms and one of the main primary producers in aquatic ecosystems. However, many diatom species produce a series of secondary metabolites, collectively termed oxylipins, that disrupt development in the offspring of grazers, such as copepods, that feed on these unicellular algae. We hypothesized that different populations of copepods may deal differently with the same oxylipin-producing diatom diet. Here we provide comparative studies of expression level analyses of selected genes of interest for three Calanus helgolandicus populations (North Sea, Atlantic Ocean and Mediterranean Sea) exposed to the same strain of the oxylipin-producing diatom Skeletonema marinoi using as control algae the flagellate Rhodomonas baltica. Expression levels of detoxification enzymes and stress proteins (e.g. glutathione S-transferase, glutathione synthase, superoxide dismutase, catalase, aldehyde dehydrogenases and heat shock proteins) and proteins involved in apoptosis regulation and cell cycle progression were analyzed in copepods after both 24 and 48 hours of feeding on the diatom or on a control diet. Strong differences occurred among copepod populations, with the Mediterranean population of C. helgolandicus being more susceptible to the toxic diet compared to the others. This study opens new perspectives for understanding copepod population-specific responses to diatom toxins and may help in underpinning the cellular mechanisms underlying copepod toxicity during diatom blooms.     

Growth- and nutrient-dependent gene expression in the toxigenic marine dinoflagellate Alexandrium minutum

The toxigenic marine dinoflagellate Alexandrium minutum forms toxic blooms causing paralytic shellfish poisoning (PSP), primarily in coastal waters, throughout the world. We examined effects on physiology and gene expression patterns associated with growth and nutrient starvation in a toxic strain of A. minutum. Bloom-relevant factors, including growth rate, intracellular toxin content, allelochemical activity and nutrient status were investigated in A. minutum cultures grown under different environmental regimes. Allelochemical activity of A. minutum cultures, quantified with a cryptomonad Rhodomonas bioassay, increased with age but was independent of nutrient status.The phenotypic data were integrated and compared with gene expression in cell samples taken at selected points along the growth curve. We observed 489 genes consistently differentially expressed between exponentially growing and growth-limited cultures. The expression pattern of stationary-phase cultures was characterized by conspicuous down-regulation of translation-associated genes, up-regulation of sequences involved in intracellular signalling and some indications of increased activity of selfish genetic elements such as transposons. Treatment-specific patterns included five genes regulated in parallel in all nutrient-limited cultures. The conspicuous decrease in photosynthetic performance identified in N-starved cultures was paralleled by down-regulation of chloroplast-associated genes.The particular gene expression patterns we identified as specifically linked with exponential growth, cessation of growth or nutrient limitation may be suitable biomarkers for indicating the beginning of growth limitation in field- or mesocosm studies.     

Conserved Gene Order and Expanded Inverted Repeats Characterize Plastid Genomes of Thalassiosirales

Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Variable plastid genome sizes and extensive genome rearrangements have been observed across the diatom phylogeny, but little is known about plastid genome evolution within order- or family-level clades. The Thalassiosirales is one of the more comprehensively studied orders in terms of both genetics and morphology. Seven complete diatom plastid genomes are reported here including four Thalassiosirales: Thalassiosira weissflogii, Roundia cardiophora, Cyclotella sp. WC03_2, Cyclotella sp. L04_2, and three additional non-Thalassiosirales species Chaetoceros simplex, Cerataulina daemon, and Rhizosolenia imbricata. The sizes of the seven genomes vary from 116,459 to 129,498 bp, and their genomes are compact and lack introns. The larger size of the plastid genomes of Thalassiosirales compared to other diatoms is due primarily to expansion of the inverted repeat. Gene content within Thalassiosirales is more conserved compared to other diatom lineages. Gene order within Thalassiosirales is highly conserved except for the extensive genome rearrangement in Thalassiosira oceanica. Cyclotella nana, Thalassiosira weissflogii and Roundia cardiophora share an identical gene order, which is inferred to be the ancestral order for the Thalassiosirales, differing from that of the other two Cyclotella species by a single inversion. The genes ilvB and ilvH are missing in all six diatom plastid genomes except for Cerataulina daemon, suggesting an independent gain of these genes in this species. The acpP1 gene is missing in all Thalassiosirales, suggesting that its loss may be a synapomorphy for the order and this gene may have been functionally transferred to the nucleus. Three genes involved in photosynthesis, psaE, psaI, psaM, are missing in Rhizosolenia imbricata, which represents the first documented instance of the loss of photosynthetic genes in diatom plastid genomes.     

The use of banquettes of Posidonia oceanica as a source of fiber and minerals in ruminant nutrition. An observational study

In the search for new food sources that contribute to the optimization of livestock production this paper discusses the possibility of using waste (called banquettes) of a marine plant commonly found on the Mediterranean coasts: Posidonia oceanica. The idea stems from the use of a waste that in summertime generates large costs because it is considered bothersome on the beaches. Thus, tons and tons of residues are collected each year from the beach, being destined for incineration. However, alternative uses for these residues are suggested, such as forage that is particularly relevant for the Mediterranean coast, where the weather does not support abundant grass growth. With this purpose, samples of banquettes of P. oceanica from six different points of a touristic place located in the Murcia Region (S.E. of Spain) were collected in April 2012 on the same day directly from the beach above the water line, washed with distilled water and sun-dried for 48 h. Approximately 500 g of each sample of plant material was chopped and two subsamples of 200 g each were placed in airtight plastic containers and sent to the laboratory for mineral and chemical analysis. This report provides data on the nutritional composition of P. oceanica such as mineral contents (As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn) and chemical composition (ash, CP, ether extract (EE), NDF and ADF, respectively) and ADL. Finally, the in sacco rumen disappearance of dry matter (DM), organic matter (OM), CP and NDF were studied. Our results showed that minerals, except for Fe, where within the range of values reported for P. oceanica in other parts of the Mediterranean region. Given the high Fe content further studies assessing the antagonic Fe–Cu interaction and its effect on animal health should be addressed. In relation to chemical composition, it is clear that this seagrass is a poor protein source and has levels of DM degradability at 24 h, similar to those obtained for cereal straw. The information summarized here shows some potential for the use of banquettes of P. oceanica as forage source in ruminant nutrition.     

Rapid Evolution of a Sexual Reproduction Gene in Centric Diatoms of the Genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene β-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

Copper toxicity response influences mesotrophic Synechococcus community structure

Picocyanobacteria from the genus Synechococcus are ubiquitous in ocean waters. Their phylogenetic and genomic diversity suggests ecological niche differentiation, but the selective forces influencing this are not well defined. Marine picocyanobacteria are sensitive to Cu toxicity, so adaptations to this stress could represent a selective force within, and between, ‘species’, also known as clades. Here, we compared Cu stress responses in cultures and natural populations of marine Synechococcus from two co‐occurring major mesotrophic clades (I and IV). Using custom microarrays and proteomics to characterize expression responses to Cu in the lab and field, we found evidence for a general stress regulon in marine Synechococcus. However, the two clades also exhibited distinct responses to copper. The Clade I representative induced expression of genomic island genes in cultures and Southern California Bight populations, while the Clade IV representative downregulated Fe‐limitation proteins. Copper incubation experiments suggest that Clade IV populations may harbour stress‐tolerant subgroups, and thus fitness tradeoffs may govern Cu‐tolerant strain distributions. This work demonstrates that Synechococcus has distinct adaptive strategies to deal with Cu toxicity at both the clade and subclade level, implying that metal toxicity and stress response adaptations represent an important selective force for influencing diversity within marine Synechococcus populations.     

Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta)

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.     

Effects of UV radiation and nitrate limitation on the production of biogenic sulfur compounds by marine phytoplankton

We tested the effects of UV radiation (UVR) and nitrate limitation on the production of dimethylsulfide (DMS), particulate dimethylsulfoniopropionate (DMSPp), and particulate dimethylsulfoxide (DMSOp) in natural seawater from the Gulf of Mexico and in phytoplankton cultures. DMS/Chl a ratios in PAR-only and PAR + UV-exposed seawater were 0.44–2.0 and 0.46–1.9 nmol DMS μg⁻¹ Chl a, respectively, whereas the ratios in cultures of Amphidinium carterae were 1.0–2.2 nmol μg⁻¹ in PAR-exposed samples and 0.91–2.1 nmol μg⁻¹ in PAR + UV-exposed samples. These results suggested that UVR did not substantially affect DMS/Chl a ratios in seawater and A. carterae culture samples. Similarly, UVR had no significant effect on DMSOp/Chl a in seawater samples (0.83–1.6 nmol DMSO μg⁻¹ Chl a for PAR + UV vs. 0.70–1.5 nmol μg⁻¹ for PAR-exposed seawater samples, respectively) or in A. carterae cultures (0.20–1.3 and 0.19–0.88 nmol DMSO μg⁻¹ Chl a in PAR + UV- and PAR-exposed cultures, respectively). In an experiment with the diatom, Thalassiosira oceanica, the culture was grown in high nitrate (30 μM) or low nitrate (6 μM) media and exposed to PAR-only or PAR + UV. The low nitrate, PAR-only samples showed an increase of intracellular dimethylsulfoniopropionate (DMSP) concentration from 2.1 to 15 mmol L⁻¹ in 60 h, but the increase occurred only after cultures reached the stationary phase. Cultures of T. oceanica grown under UVR had lower growth rates than those under PAR-only (μ′ = 0.17 and 0.32 d⁻¹, respectively) and perhaps did not experience severe nitrate limitation even in the low nitrate treatment. These results suggest that the elevated UVR in low nitrate environments could result in reduction of DMSP in some species, whereas DMSP concentrations would not be affected in eutrophic areas.     

Physiological and Biochemical Response of the Photosynthetic Apparatus of Two Marine Diatoms to Fe Stress

Flavodoxin is a small electron-transfer protein capable of replacing ferredoxin during periods of Fe deficiency. When evaluating the suitability of flavodoxin as a diagnostic indicator for Fe limitation of phytoplankton growth, we examined its expression in two marine diatoms we cultured using trace-metal-buffered medium. Thalassio-sira weissflogii and Phaeodactylum tricornutum were cultured in ethylenediaminetetraacetic acid-buffered Sargasso Sea water containing from 10 to 1000 nM added Fe. Trace-metal-buffered cultures of each diatom maintained high growth rates across the entire range of Fe additions. Similarly, declines in chlorophyll/cell and in the ratio of photosystem II variable-to-maximum fluorescence were negligible (P. tricornutum) to moderate (T. weissflogii; 54% decline in chlorophyll/cell and 22% decrease in variable-to-maximum fluorescence). Moreover, only minor variations in photosynthetic parameters were observed across the range of additions. In contrast, flavodoxin was expressed to high levels in low-Fe cultures. Despite the inverse relationship between flavodoxin expression and Fe content of the medium, its expression was seemingly independent of any of the indicators of cell physiology that were assayed. It appears that flavodoxin is expressed as an early-stage response to Fe stress and that its accumulation need not be intimately connected to limitations imposed by Fe on the growth rate of these diatoms.     

Genetic variability of Posidonia oceanica (L.) Delile in relation to local factors and biogeographic patterns

To evaluate genetic differences of Posidonia oceanica (L.) Delile both at smaller (within a meadow) and larger scale (Mediterranean basin), plants of P. oceanica were analyzed by PCR technique and compared using random amplified polymorphic DNA (RAPD) markers. Results were associated to known differences in phenology. At the small-scale level, P. oceanica shoots collected in the bay of Monterosso al Mare (Liguria, NW Mediterranean Sea) showed genetic differences among sampling stations, with a decrease in genetic diversity along an anthropogenic disturbance gradient. At basin level, genetic differences were detected among 11 P. oceanica shoots coming from different regions of the Mediterranean, and transplanted to the Port-Cros National Park (France) between 1989 and 1991: Izmir, Turkey; Athens, Greece; Taranto, Italy; Ischia Island, Italy; Lavezzi, France; Port-Cros, France; Banyuls, France; Palma de Majorca, Balearic Islands, Spain; Marsa Bay, Algiers. By cluster analysis two major Mediterranean groups were distinguished, the Eastern Mediterranean Group (EMG) and the Western Mediterranean Group (WMG). This suggests that eastern and western populations of P. oceanica have diverged during the colonization of the Mediterranean (after near extinction of the Mediterranean biota in the Messinian period, approximately 5.6 million years ago), and have experienced little gene flow between them. Cluster analysis also indicated that previously described phenological differences among P. oceanica populations in different sectors of the Mediterranean are not mere phenotypic responses to different climatic and hydrological conditions but may well have a genetic basis.     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.     

IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions

The most common strategy to produce recombinant proteins using Escherichia coli as expression vector is fed-batch culture, since high cell density cultures strategies have successfully been applied. Several methodologies to limit the specific growth rate in order to control E. coli metabolism have been defined, demonstrating that cultures can be grown under glucose limitation up to high cell densities without accumulation of acetic acid. However, under induction conditions it has been observed that E. coli metabolism is reorganized again and leads to acetic acid accumulation, causing inhibition of cell growth and decreasing protein expression efficiency.We propose a double limitation strategy (glucose and IPTG) for E. coli fed-batch cultures to avoid the deregulation of the metabolism in the induction phase. Reducing the concentration of IPTG while keeping glucose growth limitation, the accumulation of acetic acid decreased. At an IPTG concentration of 0.03 mmol/g DCW no accumulation of acetic acid was observed during the induction phase, in contraposition to what has normally been observed.Although a slight reduction of protein expression rate was observed when applying this double limitation strategy, the bioprocess volumetric productivity was enhanced due to the capability to prolong the induction phase, reaching higher levels of protein production. Another advantage of this strategy is the reduction of media cost due to the lower level of IPTG used.