Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the generaLeptolyngbya andOscillatoria were cloned and sequenced. For the genusLeptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strainsLeptolyngbya foveolarum Komárek 1964/112,Leptolyngbya sp. VRUC 135 Albertano 1985/1, andLeptolyngbya boryanum PCC 73110 belong to the same cluster. StrainOscillatoria cf.corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria.Oscillatoria agardhii CYA 18, which is a representative of planktonicOscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.     

Stimulation of the brown tide organism, Aureococcus anophagefferens, by selective nutrient additions to in situ mesocosms

The influence of nutrient additions and sediment exchange on Aureococcus anophagefferens growth was studied using 200 l mesocosms deployed in situ at the Southampton College Marine Science Center in Long Island, New York. A. anophagefferens cell density increased in mesocosms with separate additions of the following materials: urea + glucose and desiccation-stressed Enteromorpha tissue. A decrease in A. anophagefferens cell density was observed in mesocosms with either no additions (control) or with added nitrate. A treatment containing a sediment layer exhibited an increase in average cell densities, but the increase was not statistically significant (P = 0.15). In the 9 day experiment, net growth of A. anophagefferens was only observed during the last 3 days, which corresponded to a period of high solar irradiation. Total chlorophyll concentration declined in all treatments during the first 6 days, which corresponded to relatively low daily irradiance, suggesting low-light stress on the phytoplankton assemblage during the initial phase of the experiment. During the ensuing sunny period, a 4–5-fold increase in chlorophyll concentration was observed in the nitrate and urea treatments with lesser increases in the other treatments. A. anophagefferens density increased relative to total phytoplankton biomass (Chl basis) in the urea + glucose and Enteromorpha treatments. Results are consistent with a prevailing hypothesis that organic nitrogen nutrients favor the growth of A. anophagefferens. Specifically, our evidence indicates that A. anophagefferens exhibited net population growth under organic N, but not inorganic N nutrient (specifically NO₃⁻) loading.     

THE EXTRACELLULAR RELEASE Of GLYCOLIC ACID BY A MARINE DIATOM1

The excretion of glycolic acid by the marine diatom Chaetoceros socialis through time was studied. Excretion in axenic cultures was linear for the time intervals used, but for nonaxenic cultures an equilibrium was created, suggesting bacterial uptake of glycolic acid. In studies with an inhibitor of glycolate dehydrogenase, the level of glycolic acid in the medium jumped 15–fold. This shows the presence of this enzyme, and implies the presence of the entire set of enzymes which convert glycolic acid to serine and release carbon dioxide. In both axenic and nonaxenic cultures a steady state was reached. All of the data suggest that at high cell densities glycolic acid is liberated from the cell by a passive mechanism. The effect of such an excretion in natural waters is discussed.     

Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi

Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms.     

PHOTOSYNTHESIS OF ALGAL CULTURES AND PHYTOPLANKTON FOLLOWING AN ACID pH SHOCK1

In acidifying lakes, pH decreases abruptly in response to acid precipitation events. We tested the hypothesis that, in comparison to a circumneutral lake, phytoplankton photosynthesis in an acidifying lake is less sensitive to a rapid decrease in pH (acid pH shock). Phytoplankton in Plastic Lake, which is undergoing acidification, was characterized by a predominance of Pyrrophyta, and phytoplankton photosynthesis decreased to a lesser extent in response to an acid pH shock than the photosynthesis of populations from St. Nora Lake, a circumneutral lake located nearby, in which Pyrrophyta were not abundant. Rates of phytoplankton photosynthesis in acid pH shock experiments were significantly correlated with hydrogen ion but not with dissolved inorganic carbon (DIC) concentrations. Depression of photosynthesis following an acid pH shock occurred in axenic cultures of Chlorella pyrenoidosa Chick but was not observed in axenic cultures of the acidophilic alga Chlorella saccharophila (Krug.) Nadson or in three species isolated from Plastic Lake. However, the three isolates were not acidophilic during growth. We conclude that phytoplankton in acidifying lakes consists predominantly of species which are tolerant to acid pH for short periods (hours) but cannot grow at these pHs.     

Inter-strain differences in nitrogen use by the coccolithophore Emiliania huxleyi, and consequences for predation by a planktonic ciliate

Four strains of the coccolithophore Emiliania huxleyi (CCMP strains 370, 373, 374, 379) were tested for their ability to grow on various nitrogen sources. All strains grew on ammonium, nitrate, and urea, although growth of CCMP379 on urea was low. Responses to other dissolved organic nitrogen (DON) sources varied. CCMP379 did not grow on any DON source other than urea. All other strains grew on one of the two tested amino acids: CCMP370 and CCMP373 on glutamine, and CCMP374 on alanine. All three of these strains also grew on hypoxanthine; in addition, two grew well on acetamide and one on ethanolamine. E. huxleyi strains also differed in their susceptibility to predation by the ciliate Strobilidium sp. CCMP374 was ingested at substantially higher rates than CCMP373 regardless of E. huxleyi growth condition. Ciliate feeding rates also depended on E. huxleyi growth condition. For CCMP374, feeding rates were 2× higher on growing E. huxleyi cells than on non-growing cells (average 27.5 versus 15.6 cells ciliate⁻¹ h⁻¹, respectively). For CCMP373, a relationship between E. huxleyi growth rate and ciliate feeding rate was not evident, but E. huxleyi grown on some N sources (ammonium, nitrate, urea) were ingested at consistently higher rates than E. huxleyi grown on other sources (ethanolamine, glutamine). Interstrain differences in the ability to utilize DON and resist predation may contribute to maintenance of high genetic diversity within this cosmopolitan, bloom-forming species.     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

ANALYSES OF THE COMPLETE CHLOROPLAST GENOME SEQUENCES OF TWO MEMBERS OF THE PELAGOPHYCEAE: AUREOCOCCUS ANOPHAGEFFERENS CCMP1984 AND AUREOUMBRA LAGUNENSIS CCMP15071

Heterokont members of the Pelagophyceae form the massive brown tides that have continually plagued the coastal regions of the eastern U.S. seaboard and the Gulf of Mexico. To gain a better understanding of the photosynthetic competence that may be linked to their success in forming massive blooms, we sequenced the chloroplast genomes of two pelagophytes: Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis D. A. Stockw., DeYoe, Hargraves et P. W. Johnson. The chloroplast genomes of A. anophagefferens (89,599 bp) and Ar. lagunensis (94,346 bp) are significantly smaller than those of six other stramenopiles sequenced to date. The structure (or configuration) is partially due to the absence of the large inverted repeats common in chloroplast genomes. Eight of 10 small and tandem repeats from the A. anophagefferens and Ar. lagunensis genomes are adjacent to genes coding for photosynthetic or energy production functions, implying that these domains may have functional constraints. High genomic synteny, a multigene phylogenetic analysis, and a synapomorphic change in the form of an attenuated psbA gene confirm that A. anophagefferens and Ar. lagunensis are closely related taxa. Finally, the presence of three light‐independent chl‐biosynthesis genes in the chloroplast of Ar. lagunensis, but absence in the chloroplast and nuclear genomes of A. anophagefferens, suggests the persistence of a more ancient (i.e., dark‐adaptive) potential in Ar. lagunensis but not in A. anophagefferens. Whether the presence of both chl‐biosynthesis pathways in Ar. lagunensis contributes to the ability of this organism to sustain prolonged bloom (continuously for ～8 years) under reduced light conditions, but not A. anophagefferens (a few months), remains an open question.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Microbial herbivory on the brown tide alga, Aureococcus anophagefferens: results from natural ecosystems, mesocosms and laboratory experiments

Experiments were conducted with natural plankton assemblages from two areas in Great South Bay (GSB) and the Peconic Bays Estuary System, NY, to compare the rates of growth and pelagic grazing mortality of Aureococcus anophagefferens with co-occurring phytoplankton. We hypothesized that A. anophagefferens would experience low mortality rates by microbial herbivores (relative to feeding pressure on other algae) thus providing it with a competitive advantage within the phytoplankton community. In fact, substantial rates of mortality were observed in nearly every experiment in our study. However, mortality rates of A. anophagefferens were less than intrinsic growth rates of the alga during late spring and early summer in Great South Bay, resulting in positive net growth rates for the alga during that period. This timing coincided with the development of a brown tide in this estuary. Similarly, growth rates of the alga also exceeded mortality rates during bloom development in natural plankton assemblages from the Peconic Bays Estuary System held in mesocosms. In contrast to the situation for A. anophagefferens, growth rates of the total phytoplankton assemblage, and another common picoplanktonic phytoplankter (Synechococcus spp.), were frequently less than their respective mortality rates. Mortality rates of A. anophagefferens in both systems were similar to growth rates of the alga during later stages of the bloom. Laboratory studies confirmed that species of phagotrophic protists that consume A. anophagefferens (at least in culture) are present during brown tides but preference for or against the alga appears to be species-specific among phagotrophic protists. We conclude that two scenarios may explain our results: (1) protistan species capable of consuming the brown tide alga were present at low abundances during bloom initiation and thus not able to respond rapidly to increases in the intrinsic growth rate of the alga, or (2) the brown tide alga produced substance(s) that inhibited or retarded protistan grazing activities during the period of bloom initiation. The latter scenario seems less likely given that significant mortality of A. anophagefferens was measured during our field study and mesocosm experiment. However, even a minor reduction in mortality rate due to feeding selectivity among herbivores might result in a mismatch between growth and grazing of A. anophagefferens that could give rise to significant net population growth of this HAB species. Either scenario infers an important role for trophic interactions within the plankton as a factor explaining the development of brown tides in natural ecosystems.     

ELEVATED CARBON DIOXIDE DIFFERENTIALLY ALTERS THE PHOTOPHYSIOLOGY OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) AND EMILIANIA HUXLEYI (HAPTOPHYTA)1

Increasing anthropogenic carbon dioxide is causing changes to ocean chemistry, which will continue in a predictable manner. Dissolution of additional atmospheric carbon dioxide leads to increased concentrations of dissolved carbon dioxide and bicarbonate and decreased pH in ocean water. The concomitant effects on phytoplankton ecophysiology, leading potentially to changes in community structure, are now a focus of concern. Therefore, we grew the coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler and the diatom strains Thalassiosira pseudonana (Hust.) Hasle et Heimdal CCMP 1014 and T. pseudonana CCMP 1335 under low light in turbidostat photobioreactors bubbled with air containing 390 ppmv or 750 ppmv CO₂. Increased pCO₂ led to increased growth rates in all three strains. In addition, protein levels of RUBISCO increased in the coastal strains of both species, showing a larger capacity for CO₂ assimilation at 750 ppmv CO₂. With increased pCO₂, both T. pseudonana strains displayed an increased susceptibility to PSII photoinactivation and, to compensate, an augmented capacity for PSII repair. Consequently, the cost of maintaining PSII function for the diatoms increased at increased pCO₂. In E. huxleyi, PSII photoinactivation and the counter‐acting repair, while both intrinsically larger than in T. pseudonana, did not change between the current and high‐pCO₂ treatments. The content of the photosynthetic electron transport intermediary cytochrome b6/f complex increased significantly in the diatoms under elevated pCO₂, suggesting changes in electron transport function.     

Sterol Chemotaxonomy of Marine Pelagophyte Algae

Several marine algae of the class Pelagophyceae produce the unusual marine sterol 24‐propylidenecholesterol, mainly as the (24E)‐isomer. The (24Z)‐isomer had previously been considered as a specific biomarker for Aureococcus anophagefferens, the ‘brown tide’ alga of the Northeast coast of the USA. To test this hypothesis and to generate chemotaxonomic information, the sterol compositions of 42 strains of pelagophyte algae including 17 strains of Aureococcus anophagefferens were determined by GC analysis. A more comprehensive sterol analysis by HPLC and ¹H‐NMR was obtained for 17 selected pelagophyte strains. All strains analyzed contained 24‐propylidenecholesterol. In all strains belonging to the order Sarcinochrysidales, this sterol was found only as the (E)‐isomer, while all strains in the order Pelagomonadales contained the (Z)‐isomer, either alone or together with the (E)‐isomer. The occurrence of Δ²² and 24α‐sterols was limited to the Sarcinochrysidales. The first occurrence of Δ²²‐24‐propylcholesterol in an alga, CCMP 1410, was reported. Traces of the rare sterol 26,26‐dimethyl‐24‐methylenecholesterol were detected in Aureococcus anophagefferens, and the (25R)‐configuration was proposed, based on biosynthetic considerations. Traces of a novel sterol, 24‐propylidenecholesta‐5,25‐dien‐3β‐ol, were detected in several species.     

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the 3 subdivision of the Proteobacteria.Communicated by K. Horikoshi     

Dissolved Organic Nitrogen Hydrolysis Rates in Axenic Cultures of Aureococcus anophagefferens (Pelagophyceae): Comparison with Heterotrophic Bacteria

The marine autotroph Aureococcus anophagefferens (Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.     

Ecology of phytoplankton communities dominated by Aureococcus anophagefferens: the role of viruses, nutrients, and microzooplankton grazing

We investigated the impact of viruses, nutrient loading, and microzooplankon grazing on phytoplankton communities in two New York estuaries that hosted blooms of the brown tide alga Aureococcus anophagefferens during 2000 and 2002. The absence of a bloom at one location during 2002 allowed for the fortuitous comparison of a bloom and non-bloom year at the same location as well as a comparison of two sites experiencing bloom and non-bloom conditions during the same year. During the study, blooms were found at locations with high levels of dissolved organic nitrogen and lower nitrate concentrations compared to a non-bloom location. Experimental additions of inorganic nitrogen and phosphorus yielded growth rates within the total phytoplankton community which significantly exceeded control treatments in 83% of experiments, while A. anophagefferens experienced significantly increased growth during only 20% of experimental inorganic nutrient additions. Consistent with prior research, these results suggest brown tides are not caused by eutrophication, but instead are more likely to occur when sources of labile DOM are readily available. Microzooplankton grazing rates on the total phytoplankton community during a bloom were lower than grazing rates at a non-bloom site, and grazing rates on A. anophagefferens were lower than grazing rates on the total community on some dates, suggesting that reduced grazing mortality may also promote brown tides. Mean densities of viruses during blooms (3 × 10⁸ ml⁻¹) were elevated compared to most estuarine environments and were twice the levels found at a non-bloom site. Experimental enrichment of the natural viral densities yielded a significant increase in A. anophagefferens growth rates relative to control treatments when background levels of viruses were low (<1.7 × 10⁸ ml⁻¹), suggesting that viruses may promote bloom occurrence by regenerating DOM or altering the composition of microbial communities.     

Emergence of brown tides caused by Aureococcus anophagefferens Hargraves et Sieburth in China

Large-scale blooms suspected to be “brown tides” occurred in early summer for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, and had significant negative impacts on the shellfish mariculture industry. To identify the causative species of the blooms, phytoplankton samples were collected from regions with and without bloom in the coastal waters of Qinhuangdao in 2011, and clone libraries were built using eukaryote-specific 18S ribosomal RNA gene (18S rDNA). Altogether 50 clones, including 17 clones from bloom area and 33 clones from nearby regions without bloom were amplified. Blasted in GenBank, 17 clones amplified from the bloom area were assigned to Pelagophyceae (8 clones), Mediophyceae (2 clones), Cryptophyta (2 clones), Dinophyceae (2 clones) and unidentified eukaryotic species (3 clones). Those from the non-bloom site were assigned to Cryptophyta, Eustigmatophyceae, Prasinophyceae, Coscinodiscophyceae, Mediophyceae, Raphidophyceae and Dinophyceae, but not Pelagophyceae. All 8 pelagophyte clones from the bloom area were 99.7–100% similar to a single species, Aureococcus anophagefferens Hargraves et Sieburth, the causative species of brown tides on the east coast of USA. For nearly the entire length of the 18S rDNA, there were 0–6 base pair differences between the 8 amplicons and those of A. anophagefferens from USA. Furthermore, all of the 8 clones were clustered into the same well-supported clade with A. anophagefferens (posterior probability = 0.99) in a phylogenetic tree established for pelagophytes and other related microalgae. In our previous studies, the causative species of the bloom was tentatively identified as a pelagophyte, haptophyte or silicoflagellate, based on the pigment profile of the size-fractioned phytoplankton samples. Based on this study, we conclude that blooms in the coastal waters of Qinhuangdao of the Bohai Sea were brown tides caused by A. anophagefferens. As far as we know, this is the first report of brown tide events caused by A. anophagefferens in China, which is the third country in the world reporting A. anophagefferens blooms in addition to USA and South Africa.     

INTERACTIONS BETWEEN PULSED NUTRIENT SUPPLIES AND A PHOTOCYCLE AFFECT PHYTOPLANKTON COMPETITION FOR LIMITING NUTRIENTS IN LONG-TERM CULTURE1

The influence of periodic nutrient supplies and a photocycle on phytoplankton competition for limiting nutrients was examined using the diatoms Thalassiosira rotula Meunier (clone 411) and Chaetoceros sp. cf. vixvisibilis Schiller (clone 847). Chaetoceros sp. cf. vixvisibilis displaced T. rotula from ammonium-limited cultures under constant light irrespective of whether ammonium was supplied continuously, in 6 pulses.day^?1 or in a single daily pulse. In contrast, the species coexisted under the 14:10 h LD photocycle under either continuous or pulsed ammonium supplies with the relative abundance of C. sp. cf. vixvisibilis increasing as the interval between ammonium additions lengthened. Coexistence was not observed with either silicic acid or nitrate limitation. Chaetoceros sp. cf. vixvisibilis displaced T. rotula from both nitrate- and silicic acid-limited chemostat cultures and from semi-continuous cultures grown under the same photoperiod that produced coexistence with a daily pulse of ammonium. The presence of a photocycle was both necessary and sufficient to permit coexistence with ammonium limitation. Under continuous ammonium supply the photocycle may have induced a temporal separation of ammonium uptake between species, permitting sharing of the limiting nutrient and coexistence. In contrast, the species were shown to be in direct competition for the daily ammonium pulse. A competition model suggested that coexistence in this case arose from a balance between the species’ammonium uptake rates and their nitrogen demands for steady-state growth induced by the photocycle. The results indicate that variations in nutrient supply rates may contribute to the coexistence of phytoplankton species in the sea, but that the identity of the limiting nutrient and the influence of variations in other non-limiting resources play important roles in affecting the outcome of nutrient competition among planktonic algae.     

OBSERVATIONS SUR LES BESOINS EN VITAMINES DES DESMIDIÉES (CHLOROPHYCÉES-ZYGNEMATALES)1

The minimal nutrient requirements of 61 strains of desmids belonging to 18 different genera were investigated within 16 axenic cultures and 45 contaminated cultures. Thirty-nine strains were autotrophic: they included all 30 strains belonging to the genera Cosmarium, Staurastrum, and Micrasterias. Twenty-two strains were auxotrophic; they included all 14 strains belonging to the genus Closterium. Of the 22 auxotrophic strains, 13 required only vitamin B₁₂; the specific requirements of the other 9 strains were not determined.     

Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles

Amoebae of Dictyostelium discoideum produce tracks with two distinct morphologies on gold-coated coverslips. The wild-type strain and other strains that feed only by phagocytosis produced indistinct, fuzzy tracks, whereas mutants capable of axenic growth produced clear, sharp tracks. The sharp track morphology was found to be a recessive phenotype that segregates with axenicity and probably requires a previously unidentified axenic mutation. Axenic and nonaxenic strains also differed in their ability to pinocytose. When the two types of cells were shifted from bacterial growth plates to nutrient media, within 24 h the axenic strain established a rapid rate of pinocytosis, approximately 100-fold higher than the low rate detectable for the nonaxenic strain. However, track formation did not appear to be directly related to endocytosis. Electron microscopic examination of cells during track formation showed that both axenic and nonaxenic strains accumulated gold particles on their surfaces, but neither strain internalized the gold to any significant degree. Observation of living cells revealed that axenic strains collected all particles that they contacted, whereas wild-type strains left many particles undisturbed. The size of the gold particle clusters discarded by the cells also contributed to track morphology.     

VARIABILITY IN RATIOS OF PHYTOPLANKTON CARBON AND RNA TO ATP AND CHLOROPHYLL A IN BATCH AND CONTINUOUS CULTURES1,2

The feasibility of estimating phytoplankton carbon and RNA concentrations from measurements of ATP and chlorophyll a (chl a) concentrations was studied using chemostat populations of the marine diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle (= T. fluviatilis Hustedt). C:ATP and RNA:ATP ratios were studied for six additional marine species in batch culture representing five classes of phytoplankton. Statistical analyses revealed that both the growth rate and the factor limiting growth (NO₃^-, NH₄⁺, PO₄^3- or light) could alter C:ATP, RNA: ATP, C:chl a and RNA:chl a ratios by amounts which were large compared to measurement error. An analysis of variance of the batch culture results indicated that both species and the source of inorganic nitrogen (NO₃^-, or NH₄⁺) had a significant effect on C:ATP and RNA:ATP ratios. Light had less of an influence on C:ATP and RNA:ATP ratios than on C:chl a and RNA:chl a ratios, and for this reason we feel that phytoplankton C and RNA concentrations can be estimated with greater reliability from ATP than from chl a measurements. The range of C:ATP and RNA:ATP values found for T. weissflogii under a variety of growth conditions was similar to that for the six additional species grown in batch culture, suggesting that this range of values is indicative of the extremes likely to occur in living cells. Our results and additional data in the literature indicate that phytoplankton C and RNA concentrations can be estimated to within a factor of two by multiplying ATP concentrations by 311 and 35, respectively, in N limited systems, and by 341 and 36, respectively in PO₄^3- limited systems.