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Large, predatory, non‐indigenous species such as the cladoceran, Bythotrephes longimanus, can have a profound effect on aquatic food webs, but determining the prey consumed by such species is problematic because they shred prey externally and consume only soft body tissues. We assessed the usefulness of an immunochemical approach, in which putative zooplankton prey were homogenized and polyclonal antibodies against total proteins were raised in rabbits and purified against predator proteins. Using either dot‐blot or enzyme‐linked immunosorbent assay‐plate formats, assays easily detected prey proteins in single B. longimanus. Issues encountered included some degree of cross‐reaction with predator proteins and among prey species for some antisera and questions concerning determination of a correct blank value for assays, however, there are feasible approaches to resolving these issues. Immunochemical assays were readily applied in to a total of 185 individual B. longimanus in the Lake Michigan pelagic ecosystem in summer of 2008 and 2009 and indicated ingestion of cladoceran and copepod species, typically described in the literature, but also dreissenid veliger larvae and the large copepod Limnocalanus macrurus, not usually considered prey for Bythotrephes. Assays results showed some degree of correlation with numerical abundances of zooplankton prey in the water column, suggesting generalist feeding. Protein immunochemical assays offer a quick, sensitive and practical approach to examining planktonic food‐web questions that are compatible with, but have certain advantages over, alternatives such as nucleic acid techniques. [ABSTRACT FROM AUTHOR]

The ability of nutrient-deprived phytoplankton to recover in the short term when nutrients are resupplied has been studied for nitrogen and phosphorus, but the case for silicate ( Si) is poorly understood. Si-limited T halassiosira weissflogii ( Grunow) Fryxell et Hasle (grown in batch culture) was harvested in stationary phase (when cell numbers stopped increasing ~2 d after Si depletion) and senescence (when cell numbers declined ~4 d after Si depletion) and Si was resupplied at different concentrations (from 0 to 100 μM). Cell numbers, proportion of dead cells, variable fluorescence emissions (Fv/Fm), and activities of proteases were measured during Si depletion and for 24 h after Si resupply. As Si was depleted, the specific growth rate declined, dead cells increased from ~2% in log phase, to ~25% in stationary phase to over 35% in senescence, and activities of proteases associated with cell death increased several-fold. Concentration-dependent recovery of growth rate was seen after 24 h for cultures resupplied with Si in stationary phase but not in senescence. However, resupply of Si at 100 μM to stationary phase cultures alone increased protease activity to nearly the levels seen in senescence. Differences in the responses to Si resupply suggest that the ability and time to recover from Si depletion depend not only on the growth phase but also on the concentration resupplied. [ABSTRACT FROM AUTHOR]

When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of programmed cell death with many similarities to apoptosis is induced, including the induction of caspase-like proteases. Many uncertainties about the regulation and mediators that participate in the process remain. To examine the relationship between caspase-like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX-green staining, and the generation of reactive oxygen species (ROS), we used the broad-range caspase inhibitor Boc-D-FMK to block the activity of the whole class of caspase-like proteins simultaneously. In the presence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase-like enzymes in an alga. The fact that caspase-like inhibitors prevent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase-like proteases might have regulatory roles early in cell death, in addition to dismantling functions. [ABSTRACT FROM AUTHOR]

Fucus and Laminaria species, dominant seaweeds in the intertidal and subtidal zones of the temperate North Atlantic, experience tidal cycles that are not synchronized with light:dark (L:D) cycles. To investigate how nutrient assimilation is affected by light cycles, the activity of nitrate reductase (NR) was examined in thalli incubated in outdoor tanks with flowing seawater and natural L:D cycles. NR activity in Laminaria digitata (Huds.) Lamour. showed strong diel patterns with low activities in darkness and peak activities near midday. This diel pattern was controlled by light but not by a circadian rhythm. In contrast, there was no diel variation in NR activity in Fucus serratus L., F. vesiculosus (L.) Lamour., and F. spiralis L. either collected directly from the shore or maintained in the outdoor tanks. In laboratory cultures, transfer to continuous darkness suppressed NR activity in L. digitata, but not in F. vesiculosus; continuous light increased NR activity in L. digitata but decreased activity in F. vesiculosus. Furthermore, 4 d enrichment with ammonium (50 μmol · L−1 pulses), resulted in NR activity declining by >80% in L. digitata, but no significant changes in F. serratus. Seasonal differences in maximum NR activity were present in both genera with activities highest in late winter and lowest in summer. This is the first report of NR activity in any alga that is not strongly regulated by light and ammonium. Because light and tidal emersion do not always coincide, Fucus species may have lost the regulation of NR by light that has been observed in other algae and higher plants. [ABSTRACT FROM AUTHOR]

Diatoms are unicellular brown algae that likely arose from the endocytobiosis of a red alga into a single-celled heterotroph and that constitute an algal class of major importance in phytoplankton communities around the globe. The first whole-genome sequence from a diatom species, Thalassiosira  pseudonana Hasle et Heimdal, was recently reported, and features that are central to diatom physiology and ecology, such as silicon and nitrogen metabolism, iron uptake, and carbon concentration mechanisms, were described. Following this initial study, the basic cellular systems controlling cell signaling, gene expression, cytoskeletal structures, and response to stress have been cataloged in an attempt to obtain a global view of the molecular foundations that sustain such an ecologically successful group of organisms. Comparative analysis with several microbial, plant, and metazoan complete genome sequences allowed the identification of putative membrane receptors, signaling proteins, and other components of central interest to diatom ecophysiology and evolution. Thalassiosira  pseudonana likely perceives light through a novel phytochrome and several cryptochrome photoreceptors; it may lack the conserved RHO small-GTPase subfamily of cell-polarity regulators, despite undergoing polarized cell-wall synthesis; and it possesses an unusually large number of heat-shock transcription factors, which may indicate the central importance of transcriptional responses to environmental stress. The availability of the complete gene repertoire will permit a detailed biochemical and genetic analysis of how diatoms prosper in aquatic environments and will contribute to the understanding of eukaryotic evolution. [ABSTRACT FROM AUTHOR]

Measurements of pH drift were used to assess the ability of 38 red algal seaweeds to use bicarbonate and to deplete the dissolved inorganic carbon pool (DIC) from seawater medium. Subtidal algae were typically restricted to the use of DIC in the form of dissolved CO2, reducing the initial DIC by only 9%. Intertidal species used both dissolved CO2 and bicarbonate and reduced initial DIC by as much as 70%. DIC reductions and pH compensation points for the intertidal species tested were strongly correlated with their vertical zonation on the rocky shoreline (analysis of variance). DIC acquisition efficiency increased with tidal height, but species from the upper edge of the intertidal demonstrated a reversal of this trend. This general pattern associated with tidal height was observed not only among intertidal red algae in general, but also among four species of the genusPorphyra(P. tortaV. Krishnamurthy,P. papenfussiiKrishnamurthy,P. perforataJ. Agardh,P. fucicolaKrishnamurthy) and among four populations of the broadly distributed speciesMastocarpus papillatus(C. Agardh). TheMastocarpusobservations suggest either that individuals of this species may be able to express alternate strategies for carbon acquisition or that intertidal height may select for survivorship of genotypes with different carbon acquisition strategies. Taken together, these data suggest that the carbon acquisition strategy of intertidal red algae may be an important physiological set of adaptations that is under active evolutionary selection. These physiological differences were not related to phylogeny, tested as membership in red algal families and orders. [ABSTRACT FROM AUTHOR]

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro- and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species (Apedinella spinifera), growth rate and final yield was no different between the media but in one case (Emiliania huxleyi) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species. [ABSTRACT FROM AUTHOR]

Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. We surveyed caseinolytic and leucine aminapeptidase (LAP) activities in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii (Gru.) Fryxell et Husle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi (Lohm.) Hay et Mohler, and the cyanobacterium Synechococcus sp. (WH 5701). LAP activity was very low at pH <6 and peaked between pH 7.7 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4-5 range and broad maxima above pH 8. Thus, acidic vacuolar proteases apparently represented only a small faction of total protease activity. Attempts to classify proteases using selective inhibitors were inconclusive. Neither the serine/cysteine protease inhibitor leupeptin nor the aspartic protease inhibitor pepstatin A inhibited caseinolytic or LAP acitivty in any species. The metalloprotease inhibitor EDTA was only effective against LAP activity in some species, causing average decreases of 30-50%, whereas the cysteine/serine protease inhibitor phenyl methyl sulfonyl fluoride achieved at best a 30-60% decrease in caseinolytic activity. Caseinolytic activities were remarkably stable. At pH 7.5 and 25°C, extracts of d. tertiolecta, E. hyxleyi, and Synechococcus showed no changes in activity after 24 h, whereas activity declined by less than 50% in the other species. Incubation of cell extracts for 1 h at 25°C in pH 7.5 buffer did not alter patterns of cell proteins, suggesting that endogenous proteases did not effectively degrade endogenous proteins. Casein zymograms were used to identify >2000-and <20-kDa proteases in homogenates of log-phase T. weissflogii: only the smaller protease was found in d. tertiolecta. Antibodies to the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechrococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics. [ABSTRACT FROM AUTHOR]

Although the spectral quality of light in the ocean varies considerably with depth, the effect of light quality on different physiological processes in marine phytoplankton remains largely unknown. In cases here experiments are performed under full spectral irradiance, the meaning of these experiments in situ is thus unclear. In this study. We determined whether variations in spectral quality affected the sinking rates of marine diatoms. Semicontinuous batch cultures of Thalassiorira weissflogii(Gru.) Fryxell et Hasle and Dityturn brightwellii (t. West) Grunow in Van Huerk were grown under continuous red, white, or blue light. For T. weissflogii, sinking rates (SETCOL method) were twice as high (&ap; 0.2 m-d[SUP-1]) for cells grown under red light as for cells grown under white or blue light (&ap;0.08 m-d[SUP-1]), but there were no significant differences in carbohydrate content (&ap;105 fg-μm[SUP-3]) or silica content (&ap;17 fg-μm[SUP-3] to account for the difference in sinking rates. Thalassiosira weissflogii grown under blue light was significantly smaller (495 μm[SUP3]) than cells grown under red light (661 μm[SUP3]) which could contribute to its reduced sinking rate. However, cells grown under white light were similar in size to those grown under red light but had sinking rates not different from those of cells grown under blue light, indicating the involvement of factors other than size. There were no significant differences in sinking rate (&ap; 0.054 m-d[SUP-1]) or slica content (&ap;20 fg μm[SUP-1] in D. brightwelliigrown under red, white, or blue light, but cells grown under red light were significantly (20%) larger and contamed significantly (20%) more carbohydrate per μm[SUP3] than cells grown under white or blue light Spectral quality had no consistent effect on sinking rate, hichemical composition carbohydrate or silica content). In sinking rate of cells grown under white light compared to those grown under blue light supports the ecological validity of sinking rate studies done under white light. [ABSTRACT FROM AUTHOR]

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called "in vivo") assays, which are more loosely controlled and lead to difficulties in assessing enzyme characteristics such as the half-saturation constant (K[SUBm]). In this paper, we describe an in vitro NR assay developed for F.gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min[SUP-1] g[SUP-1] wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K[SUBm]) for NADH was 0.18 (±0.05) mM and for nitrate, K[SUBm] = 0. 99 (± 0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO[SUB3,SUP-]·g[SUP-1] wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to nearFebruary levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay. optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions. [ABSTRACT FROM AUTHOR]

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the tow rates is not well established under steady-stale conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira Pseudonana (Hustedt) Hasle and Heimdal, Growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 901 μmol quanta·m[SUP-2]·s[SUP-1]), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of Bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostal cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates ( <25% of nutrient-replete growth rates), but chemo stats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates in creased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field. [ABSTRACT FROM AUTHOR]

Light-limited cultures of the marine diatom Thalassiosira pseudonana (Hustedt Hasle and Heimdal (3H clone) were grown over a range of growth rates between 0.06 and 1.64 d[sup-1]. Variations in cell volume, cell quotas of carbon, nitrogen, and protein, and maximal activity of the enzyme nucleoside diphosphate kinase (NDPK) were measured and examined as a function of growth rate. NDPK from T. pseudonana showed K[subm] values of 0.24 and O. 68 mM for thymidine 5′-diphosphate and adenosine 5′-triphosphate (ATP), respectively, which are similar to those found for NDPK from a variety of organisms, from bacteria to mammals. An apparent activation enthalpy of 3.52 kCal·mol[sup-1] was determined from Arrhenius plots. No thermodynamic transition points were noted over a temperature range from l0° to 25°C. NDPK activity was significantly correlated with growth rate but not with cell volume, carbon, nitrogen, or protein, for inter-specific comparisons, normalization of enzyme activity to cell number may be most meaningful. NDPK activity per cell versus growth rate followed a U-shaped relationship, being relatively constant between 0.5 and 1.0 d-t and rising at higher and lower growth rates. Over this range, enzyme activity may be regulated by substrate concentration (ATP or other nucleoside triphosphates) or by adenylate energy charge. At higher growth rates where energy charge and substrate concentrations are probably high, changes in enzyme concentration appear to be required. The reasons for a rise in enzyme activity at low growth rate is unclear. Simultaneous measurement of nucleoside di- and triphosphate levels alongside NDPK measurements may help clarify the relationship, but these preliminary experiments indicate that NDPK is of limited usefulness as an index of in situ growth rate. [ABSTRACT FROM AUTHOR]