Use of high-performance liquid chromatography to investigate the production of paralytic shellfish toxins by Protogonyaulax spp. in culture

Procedures have been developed for the extraction and high-performance liquid chromatography (HPLC) analysis of paralytic shellfish poisoning (PSP) toxins from Protogonyaulax spp. grown in batch culture. Using these procedures, the toxin content of two isolates of P. tamarensis (NEPCC 183 and 255) and one isolate of P. catenella (NEPCC 355) were examined. Total toxin and individual toxin concentrations were measured for each isolate during the exponential and stationary phases of growth in batch culture. The total toxicity of each isolate as measured by HPLC analysis was found to agree with toxicity as determined by the standard mouse bioassay. Two of the isolates (255 and 355) were found to be toxic and the third (183) was non-toxic. The toxic isolates (255 and 355) both showed higher average total PSP toxin content during the exponential phase (35 and 23 fmol toxin cell^-1, respectively) than during the stationary phase (21 and 8 fmol toxin cell^-1, respectively). These cultures differed dramatically in their toxin composition. P. tamarensis (255) contained a large proportion of the N(21) sulfo toxins (B₁, B₂, C₁, C₂) while P. catenella (355) contained primarily Gonyautoxins 1 through 4. The percent composition of individual toxins was found to be constant throughout the growth cycle for both toxic isolates, even though the total toxin concentration varied. Our results suggest that PSP toxin profiles might be useful as chemotaxonomic indicators.     

Dynamics and physiology of saxitoxin production by the dinoflagellatesAlexandrium spp.

Toxin content (fmol cell^–1) and a suite of elemental and macromolecular variables were measured in batch cultures of the dinoflagellatesAlexandrium fundyense, A. tamarense andAlexandrium sp. from the southern New England region, USA. A different perspective was provided by semicontinuous cultures which revealed sustained, steady-state physiological adaptations by cells to N and P limitation. Two types of variability were investigated. In batch culture, changes in nutrient availability with time caused growth stage variability in toxin content, which often peaked in mid-exponential growth. A second type of variability that could be superimposed on growth stage differences is best exemplified by the high toxin content of cells grown at suboptimal temperatures. Calculations of the net rate of toxin production (R _tox ; fmol cell^–1 d^–1) for these different culture treatments and modes made it possible to separate the dynamics of toxin production from cell division. Over a wide range of growth rates, cells produced toxin at rates approximating those needed to replace losses to daughter cells during division. The exception to this direct proportionality was with P limitation, which was associated with a dramatic increase in the rate of toxin production as cells stopped dividing due to nutrient limitation in batch culture. Growth stage variability in batch culture thus reflects small imbalances (generally within a factor of two) between the specific rates of toxin production and cell division. N limitation and CO₂ depletion both affect pathways involved in toxin synthesis before those needed for cell division; P limitation does the opposite. The patterns of toxin accumulation were the same as for major cellular metabolites or elemental pools. The highest rates of toxin production appear to result from an increased availability of arginine (Arg) within the cell, due to either a lack of competition for this amino acid from pathways involved in cell division or to increased de novo synthesis. There were no significant changes in toxin content with either acclimated growth at elevated salinity, or with short term increases or decreases of salinity. These results demonstrate that toxin production is a complex process which, under some conditions, is closely coupled to growth rate; under other conditions, these processes are completely uncoupled. Explanations for the observed variability probably relate to pool sizes of important metabolites and to the differential response of key biochemical reactions to these pool sizes and to environmental conditions.     

Paralytic shellfish poison in the “hiogi” scallop Chlamys nobilis

Attempts were made to analyze the toxin composition of the toxic hiogi scallop Chlamys nobilis. The toxins were partially purified from the digestive glands by column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H⁺ form), resulting in separation into protogonyautoxin (PX), gonyautoxin (GTX) and saxitoxin (STX) fractions. Their total potencies were scored to be <100 mouse units (MU), 3200 MU and 3700 MU, respectively. A 5-min hydrolysis with 0.1 N HCl enhanced the potencies of PX and GTX fractions to 450 MU and 12000 MU respectively, whereas no enhancement occurred in the STX fraction at all. Electrophoretic, thin-layer chromatographic and high performance liquid chromatographic analyses demonstrated that the PX fraction consisted mainly of GTX₈ and its epimer, the GTX fraction of GTX₅, GTX₆, along with two unidentified toxins, and the STX fraction exclusively of two unidentified toxins. This rather unique composition suggested a complex metabolism of PSP in this species.     

Light-shade adaptation by the oceanic dinoflagellates Pyrocystis noctiluca and P. fusiformis

Species-specific rates of photosynthetic carbon uptake (P), chlorophyll a content and P versus irradiance (P-I), have been measured for cells of Pyrocystis noctiluca and P. fusiformis isolated from natural populations collected in the euphotic zone within and below the surface mixed layer in the Sargasso Sea. These same measurements and the assay for ribulose bis-phosphate carboxylase (RuBP-Case), have been made for cultures of P. noctiluca in a 12 h L: 12 h D photoperiod at 9 different constant or at changing light intensities. In nature chl a cell^-1 was constant throughout the euphotic zone. The photosynthetic capacity (P_max), of cells captured below the surface mixed layer was lower by a factor of 10 compared with cells collected from the surface mixed layer. The P_max for P. noctiluca collected and incubated within the surface mixed layer was the same as for cell cultures grown under high light, non nutrient-limiting conditions, suggesting that photosynthesis in the natural system was not nutrient limited. In laboratory cultures under constant low light intensities, chl a cell^-1 increased by a factor of 5 while both P_max and RuBPCase activity decreased by a factor of ca 4 compared with high light intensities. In changing light intensities both P_max and RuBPCase activities were decreased by factors of 4 during low light intervals while chl a cell^-1 approached a constant intermediate value. The change in chl a cell^-1 in response to prolonged exposure to constant low light intensities was first order with a rate constant of 0.33 d^-1. For all irradiance conditions in culture, the P-I dependence could be described by the simple Michaelis-Menten formula. The ratio of P_max to K_I, (the light intensity where P=P_max/2) was a constant with a Coefficient of Variation of 12%: The constancy of this ratio, the parallel changes in RuBPCase activity with P_max and the constant chl a cell^-1 in the Sargasso Sea imply that for P. noctiluca and presumably P. fusiformis in nature, a dark enzymatic step rather than changes in photosynthetic pigment concentrations may regulate the photosynthetic capacity in the changing photic environment.Contribution no. 1141 from McCollum-Pratt Institute and Department of Biology, The Johns Hopkins University. Supported by DOE contract no. EY 76S20 3278, NSF no. OCE 76-02571 and ONR no. N300014-81-C-0062     

Phosphorus- and nitrogen-limited photosynthesis and growth of Gracilaria tikvahiae (Rhodophyceae) in the Florida Keys: an experimental field study

The relative effects of NH ₄ ⁺ (N) and PO ₄ ^3- (P) on growth rate, photosynthetic capacity (P_max), and levels of chemical constituents of the red macroalga Gracilaria tikvahiae McLachlan were assayed during winter and summer, 1983 in inshore waters of the Florida Keys by using in-situ cage cultures. During winter, both N and P enrichment enhanced growth over that of ambient seawater; however, P rather than N accounted for more (60%) of the increased winter growth. During summer, P, but not N, enhanced growth over ambient seawater and accounted for 80% of increased growth. Similarly, P_max was enhanced by both P and N during winter (but mostly by P) and only by P during summer. Elevated C:P, C:N and N:P ratios of G. tikvahiae tissue during winter, but only C:P and N:P ratios during summer, support the pattern of winter N and P limitation and summer P-limitation. This seasonal pattern of N vs P limited growth of G. tikvahiae appears to be a response to seasonally variable dissolved inorganic N (twofold greater concentrations of NH ₄ ⁺ and NO ₃ ^- during summer compared to winter) and constantly low to undetectable concentrations of PO ₄ ^3- . Mean C:P and N:P ratios of G. tikvahiae tissue during the study were 1 818 and 124, respectively, values among the highest reported for macroalgae.     

Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima

Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 ± 1 °C, under a photon flux density (PFD) of 90 ± 5 μmol m⁻² s⁻¹ on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by “midnight”. Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell⁻¹, respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH₄ was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO₃, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h). Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase (“morning” of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases (“afternoon”). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events. Received: 3 September 1998 / Accepted: 30 March 1999     

Nutrient starvation effects on the allelochemical potency of Alexandrium tamarense (Dinophyceae)

Batch culture experiments were performed to investigate potential effects of nutrient starvation on the allelochemical potency of the toxic dinoflagellate Alexandrium tamarense. Triplicate cultures with reduced nitrate (−N) or phosphate (−P) seed were compared to nutrient-replete (+N+P) cultures. Total depletion of the dissolved inorganic limiting nutrient, reduced cell quotas, changed mass ratios of C/N/P and reduced cell yield clearly indicate that treatment cultures at stationary phase were starved by either N or P, whereas growth cessation of +N+P cultures was probably due to carbon limitation and/or a direct effect of high pH. Pulsed addition of the limiting nutrient allowed −N and −P cultures to resume growth. Lytic activity of A. tamarense as quantified by a Rhodomonas bioassay was generally high (EC₅₀ around 100 cells mL⁻¹) and was only slightly modulated by growth phase and/or nutrient starvation. Lytic activity per cell increased with time in both +N+P and −P cultures but not −N cultures. P-starved stationary-phase cells were slightly more lytic than +N+P cultures, but this difference may be due to increased cell size and/or accumulation of extracellular compounds. In conclusion, only slight changes but no general and major increase in lytic activity in response to nutrient starvation was observed.     

DMSP-lyase activity in five marine phytoplankton species: its potential importance in DMS production

Activity of DMSP-lyase, which cleaves dissolved DMSP (henceforth DMSP_d-lyase), was examined in five axenically cultured phytoplankton species, including both DMSP-producing and non-DMSP-producing species. High DMSP_d-lyase activity was found in two DMSP producers, Heterocapsa triquetra strain NIES-7 and Scrippsiella trochoidea strain NIES-369 (Dinophyceae). The DMS production rates at 100 nM DMSP_d were 0.5 fmol cell⁻¹ min⁻¹ for H. triquetra and 0.3 fmol cell⁻¹ min⁻¹ for S. trochoidea. In a non-DMSP producer, Heterosigma akashiwo strain NIES-6 (Raphidophyceae), the DMSP_d-lyase activity was not found. Two DMSP-producing Prymnesiophyceae species, Isochrysis galbana strain CCMP-1323 and Gephyrocapsa oceanica strain NIES-353, did not show any obvious activity either, in contrary to other authors' findings on Phaeocystis sp., another DMSP-producing Prymnesiophyceae species. The comparison of the DMSP_d-lyase activity of the two Dinophyceae species with bacterial DMSP consumption and DMS production activity in Tokyo Bay showed that the DMSP_d-lyase activity of H. triquetra and S. trochoidea could be an important mechanism for DMS production during their blooms. Received: 9 April 1999 / Accepted: 10 December 1999     

Comparisons among species ofAlexandrium (Dinophyceae) grown in nitrogen- or phosphorus-limiting batch culture

Three species of the dinoflagellate genusAlexandrium (Halim)-two strains of toxic.A. minutum, one each of nontoxicA. tamarense andA. affini-were grown in batch culture in either a low-nitrogen or a low-phosphate medium. Maximum carbon-specific growth rates forA. tamarense were lower (at <0.25 d^-1) than for the other strains, which all exceeded 0.38 d^-1. C-quotas (C content per cell) during exponential growth were similar for all strains (2.5 ng C cell^-1), with cells becoming smaller during the N-limiting stationary phase, but enlarging during prolonged P-deprivation. Values of ¹³C during the exponential phase were low (-25to-30), with most cells during the light phase swimming at the surface when nutrient-replete and migrating to the bottom of the flasks when nutrient-deplete with ¹³C rising to around-15. Biomass could not be estimated reliably from pigmentation, but could be estimated from biovolume (r>0.95), although this was complicated in cultures ofA. minutum by the presence of particles comprized of thecal plates of a similar size to intact cells. Alkaline phosphatase activity was not a reliable indicator a P-status. The most toxic strain tested (A. minutum AL1V) contained the highest concentrations of free amino acids, of arginine (a precursor of paralytic shellfish toxins) and of proline, and also had the lowest C:N mass ratio (at 4.3).A. affini contained the lowest concentrations of arginine, andA. tamarense the highest exponential phase C:N (7.8). For all strains, the mole ratio of intracellular glutamine: glutamate (Gln: Glu, which was abnormally high compared to other algae) could only be used to indicate the presence or absence of N-stress rather than the degree of stress. Additions of ammonium and phosphate resulted in increases in Gln: Glu within 20 min in N-stressed cells and also enhanced toxin content inA. minutum (mainly gonyautoxin) 4 over a 24 h period.     

Different patterns of growth and nitrogen uptake in two clones of marineSynechococcus spp.

We grew marineSynechococcus Clones WH7803 and WH8018 at 150µE m^–2 s^–1 in dilute batch cultures with NH ₄ ⁺ as the limiting nutrient. The maximal uptake capacities for NH ₄ ⁺ and NO ₃ ^- were measured in frequent experiments during log and stationary phases of growth. Clone WH7803, originally isolated from oceanic waters, had a specific uptake rate of NH ₄ ⁺ that approximated the maximum (log phase) specific growth rate (ca ~ 0.025 h^–1). NO ₃ ^- uptake was observed only after nitrogen in the media was depleted; the NO ₃ ^– uptake capacity was ca 12% the capacity for NH ₄ ⁺ uptake throughout the nitrogen depleted period. Growth was arrested upon nitrogen depletion, but resumed soon after reinoculation into fresh media, even after 5 d of starvation. Clone WH8018, originally isolated from coastal waters, revealed a five-fold enhancement in the NH ₄ ⁺ uptake rate relative to growth rate at the time of nitrogen depletion. As nitrogen starvation proceeded, this enhancement was reduced. This clone, too, was able to take up NO ₃ ^- once nitrogen in the media was depleted, but only after ca 20 h. Growth continued for a limited period during nitrogen depletion, but nitrogen-starved cells were slow to recover upon reinoculation into fresh media. We speculate that clonal differences may reflect differences in the molecular regulation of nitrogen assimilation.     

Relative growth of different species of marine algae in wasterwater-seawater mixtures

In recent studies, we developed a combined nutrient removal-marine aquaculture process for the tertiary treatment of wastewater and the production of commercially important shellfish. Part of this process consists of an outdoor mass cultivation system for marine algae. During our previous experiments we observed that marine diatoms almost exclusively are the dominant algal species in our outdoor cultures. To better understand this phenomenon of diatom dominance we grew 16 species of marine algae in continuous monoculture under laboratory conditions simulating to some degree the conditions prevailing in our outdoor experiments. Species such as Skeletonema costatum, Monochrysis lutheri and Tetraselmis sp., which were never dominant in our outdoor cultures, grew as well in monoculture, as Phaeodactylum tricornutum, frequently, the prevalent species outdoors. However, when monocultures of Dunaliella tertiolecta and Thalassiosira pseudonana (3H) were purposely contaminated with P. tricornutum, the latter species quickly became dominant. It is suggested that a complex interaction of environmental factors is usually responsible for the dominance of a particular species; one such factor may be the nitrogen source in the growth media. Under conditions of virtually, complete nitrogen assimilation, the carbon: nitrogen ratio in the algae was high (7 to 8) when NO ₃ ^- –N was the source of nitrogen, and low (4 to 6) when NH ₄ ⁺ –N was the prime form of nitrogen. When algal growth was low, resulting in a large inorganic nitrogen residue, the carbon:nitrogen ratio was low regardless of whether NO ₃ ^- –N or NH ₄ ⁺ –N was the main nitrogen source.Contribution No. 3297 from the Woods Hole Oceanographic Institution.     

Dark CO2 uptake by the diatom Chaetoceros simplex in response to nitrogen pulsing

In a continuing investigation of dark CO₂ uptake by nitrogen-limited cultures of the marine diatom Chaetoceros simplex (Bbsm), we expanded on several of our earlier conclusions regarding the potential application of this physiological response for measuring the degree and type of nitrogen limitation in phytoplankton populations. First, the duration over which the maximal enhancement of dark ¹⁴CO₂ uptake was sustained after NH ₄ ⁺ enrichment was a function both of the concentration of added NH ₄ ⁺ and the standing crop of phytoplankton nitrogen — in effect, the total N demand. Second, pulsing with NH ₄ ⁺ for a given degree of N-limitation always produced the same level of enhanced dark CO₂ uptake regardless of whether the cultures were preconditioned with oxidized or reduced nitrogen. In contrast, urea pulsing led to reduced dark CO₂ uptake, but the effect was most pronounced in cells grown on NO ₃ ^– . And third, the assay could be used to distinguish readily between no, moderate, and severe N limitation. The degree of severe N limitation was quantitatively correlated with the degree of enhanced dark CO₂ uptake, but this relationship was not so clear in the region of moderate N limitation. The main advantage of the assay is that it is a relatively simple and effective alternative to more complicated techniques for gauging the degree and form of N limitation in phytoplankton. Further evaluation will be required, both in the laboratory and field, before the assay can be calibrated for quantitative use.Contribution No. 5982 from the Woods Hole Oceanographic Institution     

Regulation of the Si and C uptake and of the soluble free-silicon pool in a synchronised culture of<Emphasis Type="Italic"> Cylindrotheca fusiformis</Emphasis> (Bacillariophyceae): effects on the Si/C ratio

Silicon and carbon uptake rates were studied over a 24 h light/dark cycle in a synchronised culture of the marine diatom Cylindrotheca fusiformis (Reimann et Lewin) using ³²Si and ¹⁴C. The silicic acid uptake rate per cell (^cSi) varied between 1.2 and 20.0 fmol Si cell^–1 h^–1 and was closely correlated to the G2+M phase of the cell cycle. A linear and significant relationship was determined between the percentage of cells present in G2+M and ^cSi. Evolution of the soluble free-silicon pool was studied simultaneously. The concentration of the total soluble free pool of silicon (Q^PSi) varied from 1% to 7% of the total silicon content. A significant difference of 1.5 fmol Si cell^–1 between Q^PSi and the labelled free pool (Q^npSi) was measured, indicating the presence of an unlabelled fraction of the pool. The concentration of Q^npSi was around 1.0 fmol Si cell^–1 prior to cell division and did not change as a function of ^cSi, which indicated a feedback mechanism coupling uptake into the free pool and incorporation into the frustule. In parallel, ¹⁴C uptake variation (^cC) was measured during the division of the population. The value of ^cC varied between 0.44 and 0.78 pmol C cell^–1 h^–1 and appeared to be maximal when cells were in the G1 phase. This variation of ^cC marginally affected the total carbon content of the cells (Q^TC) in comparison with the light/dark cycle. The variations in the Si/C ratio, from 0.021 to 0.046, demonstrated the different control mechanisms of Si and C metabolisms during the course of the cell- and photocycle.Communicated by S.A. Poulet, Roscoff     

Nitrate uptake and assimilation in Thalassiosira weissflogii and Phaeodactylum tricornutum: interactions with photosynthesis and with the uptake of other ions

Interactions of the nitrate, phosphate, and ammonium uptake systems and the interactions of these systems with photosynthesis were investigated for Thalassiosira weissflogii and Phaeodactylum tricornutum preconditioned in continuous culture. The cultures were supplied with NO _- ³ and PO ₌ ⁴ in an N:P atomic ratio of 15:1, and residual concentrations of both nutrients in the growth chamber were very low. The rate of NO _- ³ uptake was reduced by the addition of NH ₊ ⁴ or PO ₌ ⁴ . The rate of PO ₌ ⁴ uptake by T. weissflogii was reduced by the addition of NH ₊ ⁴ . The rate of carbon fixation was reduced by NO _- ³ additions and slightly reduced by the addition of PO ₌ ⁴ . There were two components of NO _- ³ uptake, one light-dependent and one light-independent. Uptake inhibition by added PO ₌ ⁴ acted on the light-independent component. The change in the C fixation rate due to added NO _- ³ was equal to the rate of NO _- ³ uptake by the light-dependent component on a molar basis. Nitrate assimilation (reduction) rates were calculated from the time course of extracellular and intracellular NO _- ³ concentrations. The light-dark change in the assimilation rate was similar to the light-dark change in the uptake rate, suggesting close coupling between the light-dependent components of uptake and assimilation. The assimilation rate dropped upon exhaustion of extracellular NO _- ³ , implying that an uptake-coupled component of assimilation is unavailable for the assimilation of intracellular NO _- ³ . The reduction in the C fixation rate due to NO _- ³ was temporally associated with uptake rather than assimilation, but may reflect interaction with either the light-dependent uptake step or the closely coupled assimilation. Phosphate additions reduced the rate of NO _- ³ uptake, while the rate of assimilation was unaffected.     

Effect of water temperature and light intensity on growth rate and toxicity change in Protogonyaulax tamarensis

The effect of water temperature and light intensity on the growth rate and the toxicity of Protogonyaulax tamarensis was examined using a monoclonal culture isolated from Ofunato Bay, Japan in March, 1984. The growth rate decreased with the decrease of either light intensity or temperature. The amount of toxin produced increased concomitantly with the decrease of the growth rate. However, the increase of the toxicity under low growth rate was less remarkable when the growth rate was lowered by the decrease of light intensity. This indicates that photosynthesis plays an important role in the production of toxin in P. tamarensis.     

Development of rapid ammonium uptake during starvation of batch and chemostat cultures of the marine diatom Thalassiosira pseudonana

Cell nitrogen quotas and uptake rates following ammonium additions were measured during ammonium-limited growth transients obtained by starving batch and chemostat cultures of Thalassiosira pseudonana (Clone 3 H). During starvation, cell quotas decreased by more than 50% in batch cultures. In chemostat cultures, the drop in cell quota during starvation decreased with dilution rate, from more than 50% at 1.45 d^-1, to less than 10% at 0.22 d^-1. Minimal levels of 3 to 4×10^-2 pg-at. N cell^-1 were reached after 24 h starvation in both batch and chemostat cultures. Uptake rates over the first minute of perturbation experiments were 3 times the long-term (10 to 30 min) rates. In batch cultures, specific uptake rates increased from 4 d^-1 to 20 d^-1 after 24 h starvation. Uptake rates per cell were independent of starvation time and dilution rate in chemostat cultures, but lower in non-starved batch cultures. The implications of these data for models of phytoplankton growth are discussed: the data support models which predict a depression in average growth rates when diatoms encounter microscale patches in oligotrophic environments.     

Regulation of growth in Laminaria digitata: use of in-vivo nitrate reductase activities as an indicator of nitrogen limitation in field populations of Laminaria spp.

The seasonal variation in growth rate of a population of Laminaria digitata (Huds.) Lamour growing at Arbroath, Scotland was studied between August 1981 and September 1982, and was found to follow the biphasic annual cycle typical of this genus. Growth rates were maximum (0.3 cm cm^-1 mo^-1) in early June and minimum (0.05 cm cm^-1 mo^-1) between September and January. An analysis of the relationship between the seasonal changes in environmental factors (inorganic nitrogen concentrations, irradiance and temperature) with those of growth rate and the accumulation or mobilisation of cellular reserves of carbohydrates and nitrate, indicated that growth was nitrogen-limited between June and October and light-limited (with a possible co-involvement of temperature) for the remainder of the year. These conclusions were supported by the seasonal changes in the ratio of actual: potential in-vivo nitrate reductase activities in L. digitata, thus confirming the suitability of this technique for monitoring the occurrence of nitrogen limitation in Laminaria spp. The seasonal changes in blade nitrate reductase activities closely followed those of growth rate, with maximum activities [0.3 mol NO ₃ ^- reduced g^-1 (wet wt) h^-1] being present in late May and minimum levels [0.01 mol NO ₃ ^- reduced g^-1 (wet wt) h^-1] occurring between November and March. The correlation observed between nitrate reductase activities and growth rate is consistent with the ability of Laminaria spp. to store excess inorganic nitrogen, available during winter and early spring, as NO ₃ ^- , and with the requirement to conserve enzyme protein during the summer period of nitrogen limitation.     

Inorganic nitrogen metabolism inUlva rigida illuminated with blue light

Inorganic nitrogen metabolism in blue light was studied for the green algaUlva rigida C. Agardh collected in the south of Spain (Punta Carnero, Algeciras) in the winter of 1987. NH₄ ⁺ has been reported to inhibit NO₃ ^- uptake; however,U. rigida showed a net NO₃ ^- uptake even when the NH₄ ⁺ concentration of the external medium was three or four times greater than the concentration of NO₃ ^-. NO₃ ^- uptake rates were similar in both darkness and in blue light of various photon fluence rates (PFR) ranging from 17 to 160 mol m^-2 s^-1. Since NO₃ ^- uptake is an active mechanism involving the consumption of ATP, respiratory metabolism can provide enough ATP to maintain the energetic requirement of NO₃ ^- transport even in darkness. In contrast, NO₃ ^- reduction inU. rigida was highly dependent on the net photosynthetic rate. After 7 h in blue light, intracellular NO₃ ^- concentrations ([NO₃ ^-] _i ) were higher in specimens exposed to intensities below the light compensation point (LCP) than in those incubated at a PFR above the LCP. When PFR is below the light compensation point, NO₃ ^- reduction is low, probably because all the NADH produced by the cells is oxidized in the respiratory chain in order to produce ATP to maintain a steady NO₃ ^- transport rate. The total nitrogen (TN) and carbon (TC) contents decreased from darkness to 33 mol m^-2 s^-1 in blue light. In this range, catabolic processes prevailed over anabolic ones. In contrast, increases in TN and TC contents were observed above the light compensation point. The C : N ratio increased with light intensity, reaching a stable value of 17 at 78 mol m^-2 s^-1 in blue light. Intracellular NO₃ ^- concentration and NO₃ ^- reduction appear to be directly controlled by light intensity. This external control of [NO₃ ^-]_i and the small capacity ofU. rigida to retain incorporated NO₃ ^-, NO₂ ^- and NH₄ ⁺ ions may explain its nitrophilic character.     

Growth and competition of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. I. Nutrient effects

Two marine diatoms, Phaeodactylum tricornutum (Bohlin) and Thalassiosira pseudonana (Hasle and Heimdal), were grown both separately and together in batch cultures on a mixture of waste water and seawater enriched with different components of f medium. At 17°C, the maximum division rates of the two species were statistically indistinguishable. The waste water-seawater mixture used proved to have insufficient Si, relative to N and P, for the growth of T. pseudonana, which requires approximately 5x10^-14 g-at Si cell^-1 to divide at a maximum rate. P. tricornutum, on the other hand, although capable of taking up nearly 9x10^-15 g-at Si cell^-1, could sustain maximum rates of division with 4.3x10^-18 g-at Si cell^-1 or less. No allelopathic interaction between the two species could be detected. We conclude that P. tricornutum enjoys a considerable competitive advantage over T. pseudonana in a waste water-seawater-based mariculture system that is not supplemented with Si. Although Si proved necessary for T. pseudonana to complete more successfully with the other diatom, the presence of excess amounts of Si is not necessarily sufficient for the maintenance of T. pseudonana in mixed continuous culture with P. tricornutum: other factors, such as light-related or photoperiod-related growth response, are believed to determine the ultimate outcome of competition between these algae in light-limited continuous culture.Contribution No. 3999, from the Woods Hole Oceanographic Institution.Communicated by M.R. Tripp, Newark     

Host feeding and nutrient sufficiency for zooxanthellae in the sea anemone Aiptasia pallida

Nutrient sufficiency of zooxanthellae in the sea anemone Aiptasia pallida cultured in low nutrient seawater depends on the availability of particulate food to the host. Zooxanthellae in anemones unfed for 20 to 30 d exhibited the following characteristics of nutrient deficiency: cell division rates decreased; chlorophyll a content gradually decreased from 2 to <1 pg cell^–1; and C:N ratios increased from 7.5 to 16. Over a 3-mo period, algal populations in unfed anemones gradually decreased, indicating that zooxanthellae were lost faster than they were replaced by division. The mitotic index of zooxanthellae in unfed anemones was stimulated either by feeding the host or by the addition of inorganic N and P to the medium. Whether algae are nutrient-limited in hosts under field conditions has not been examined fully; however, C:N ratios in zooxanthellae from field-collected hosts are slightly higher (9.4 vs 7.5) than in hosts fed to repletion in laboratory cultures. This observation might indicate N limitation in the field.