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Phagotrophic algae can consume bacteria that are the predominant microorganisms present in the waste activated sludge (WAS) generated from municipal wastewater treatment processes. In this study, we developed a combined ultrasonication-phagotrophic algal process for WAS conversion. The ultrasonic pretreatment released small volatile solids (VS) including bacteria from WAS flocs. A phagotrophic alga Ochromonas danica then grew by consuming more than 80% of the released VS, with approximately 30% (w/w) algal cell yield. The process reduced the overall WAS VS by 42.4% in 1 day, comparing very favorably with the 27% reduction in 10 days by aerobic digestion. For stabilizing the solids remaining from the ultrasonic step, the total oxygen uptake required was 65%-92% lower than that for the original WAS, indicating substantially reduced aeration cost. Overall, this novel process enhanced the WAS digestion at lower energy requirements and produced microalgae for other potential uses. © 2021 Water Environment Federation PRACTITIONER POINTS: At least 80% of released VS from WAS can be processed by phagotrophic algae. Significant amounts of algae can be produced from WAS. Ultrasonication-phagotrophic algal process can make sludge management more sustainable.

The first step in the evolution of complex multicellular organisms involves single cells forming a cooperative group. Consequently, to understand multicellularity, we need to understand the costs and benefits associated with multicellular group formation. We found that in the facultatively multicellular algae Chlorella sorokiniana: (1) the presence of the flagellate Ochromonas danica or the crustacean Daphnia magna leads to the formation of multicellular groups; (2) the formation of multicellular groups reduces predation by O. danica, but not by the larger predator D. magna; (3) under conditions of relatively low light intensity, where competition for light is greater, multicellular groups grow slower than single cells; (4) in the absence of live predators, the proportion of cells in multicellular groups decreases at a rate that does not vary with light intensity. These results can explain why, in cases such as this algae species, multicellular group formation is facultative, in response to the presence of predators.

Many marine microbial eukaryotes combine photosynthetic with phagotrophic nutrition, but incomplete understanding of such mixotrophic protists, their functional diversity, and underlying physiological mechanisms limits the assessment and modeling of their roles in present and future ocean ecosystems. We developed an experimental system to study responses of mixotrophic protists to availability of living prey and light, and used it to characterize contrasting physiological strategies in two stramenopiles in the genus Ochromonas. We show that oceanic isolate CCMP1393 is an obligate mixotroph, requiring both light and prey as complementary resources. Interdependence of photosynthesis and heterotrophy in CCMP1393 comprises a significant role of mitochondrial respiration in photosynthetic electron transport. In contrast, coastal isolate CCMP2951 is a facultative mixotroph that can substitute photosynthesis by phagotrophy and hence grow purely heterotrophically in darkness. In contrast to CCMP1393, CCMP2951 also exhibits a marked photoprotection response that integrates non-photochemical quenching and mitochondrial respiration as electron sink for photosynthetically produced reducing equivalents. Facultative mixotrophs similar to CCMP2951 might be well adapted to variable environments, while obligate mixotrophs similar to CCMP1393 appear capable of resource efficient growth in oligotrophic ocean environments. Thus, the responses of these phylogenetically close protists to the availability of different resources reveals niche differentiation that influences impacts in food webs and leads to opposing carbon cycle roles.

Summary 1. Toxic compounds produced by many phytoplankton taxa are known to have negative effects on competitors (allelopathy), anti-predatory effects on grazers (mortality or impaired reproduction) or both. Although mixotrophs of the genus Ochromonas are known to be toxic to zooplankton, it has often been assumed in studies of plankton community processes that all flagellates in the size range of this taxon are edible to typical zooplankton grazers (i.e. cells ≤30 μm for Daphnia, ≤6 μm for rotifers). 2. We explored the toxicity of a species of Ochromonas to other planktonic taxa, including its competitors (two species of phytoplankton and protists) and consumers (two species of zooplankton). To test if mode of nutrition by this mixotroph influences its toxicity to other taxa, we exposed each test species to Ochromonas cultured in chemostats under four different nutritional regimes: osmotrophy (labile dissolved organic carbon) and phagotrophy (bacterial prey) in both light and dark conditions (i.e. with or without photosynthesis). 3. Filtrate from osmotrophically fed Ochromonas had a significant negative effect on the population growth rate of two obligate phototrophic phytoplankton, Cryptomonasozolini and Chlamydomonas reinhardtii. The protists Tetrahymena tetrahymena and Paramecium aurelia were also negatively affected by Ochromonas filtrate. Ochromonas cells were toxic to both the rotifer Brachionus calicyflorus and the cladoceran Daphnia pulicaria, with the toxic effects significantly more severe when fed at high cell densities (75 000 cells mL−1) than at low densities (7500 cells mL−1). Ochromonas cultured osmotrophically in the light was more toxic to the Daphnia than cells cultured under other conditions. In contrast, Ochromonas from all nutritional conditions was equally highly toxic to Brachionus. 4. Our findings support the view that Ochromonas can be toxic to other components of the food web with which it interacts. It is especially toxic to zooplankton that directly consume it, although the effect depends upon Ochromonas cell density and whether or not a good food source is simultaneously present. Our results call into question the common practice of pooling flagellates into a single ‘functional group’ included in an ‘edible phytoplankton’ category of cells

SUMMARY A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′-end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E-value

Two-dimensional (2-D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up-regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.

The possible effect of filtered cultures of flagellate Ochromonas sp. on colony formation in M. aeruginosa was investigated in this paper. The results show that filtered cultures of flagellates fed with M. aeruginosa could induce colony formation in M. aeruginosa. Furthermore, induction strength is clearly dependent on the concentration of flagellates and filtered cultures. However, no colonial M. aeruginosa was found in the treatments of filtered cultures of flagellates fed with Microcystis wesenbergii, filtered cultures of flagellate fed with Chlorella pyrenoidosa, and algae homogenates. This suggests that infochemicals released from flagellates fed with M. aeruginosa may be a trigger for colony formation in M. aeruginosa. The clearance rates of flagellates on algae were markedly decreased when they were cultivated with induced colonial M. aeruginosa. These indicate that colony formation in M. aeruginosa is a predator-induced defense which could reduce predation risk from flagellates (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We report the influence of different nutritional modes—autotrophy, mixotrophy, and heterotrophy—on the fatty acid and sterol composition of the freshwater flagellate Ochromonas sp. and discuss the ecological significance of our results with respect to the resource competition theory (rct). Polyunsaturated fatty acids (PUFAs) are the most efficient biochemical variable distinguishing between nutritional modes of Ochromonas sp. Decreasing concentrations of PUFAs were observed in the order autotrophs, mixotrophs, heterotrophs. In mixotrophs and heterotrophs, concentrations of saturated fatty acids were higher than those of monounsaturated fatty acids and PUFAs as a result of bacterivory. Stigmasterol was the main sterol in Ochromonas sp., regardless of nutritional mode. Mixotrophs showed higher growth rates than heterotrophs, which could not be explained by rct. Heterotrophs, in turn, exhibited higher growth rates than autotrophs, which were cultured under the same light conditions as mixotrophs. Mixotrophs can synthesize PUFAs, which are important for many physiological functions such as membrane permeability and growth. Thus, mixotrophy facilitated efficient growth as well as the ability to synthesize complex and essential biomolecules. These strong synergetic effects are due to the combination of biochemical benefits of heterotrophic and autotrophic metabolic pathways and cannot be predicted by rct.

The phylogenetic group stramenopiles refers to the systematic groups that possess tripartite tubular hairs (stramenopiles) on their flagella. There have been a number of studies describing the fine structure of these mastigonemes and a few studies isolating the component proteins; however, these proteins and their gene sequences have not yet been identified. In the present study, we identified a mastigoneme protein (Ocm1) of the chrysophycean alga Ochromonas danica Pringsh. (UTEX LB1298). Its corresponding gene, Ocm1, was identified by using degenerate primers that correspond to the partial amino acid sequences of a protein (85 kDa) obtained from a mastigoneme-rich fraction of isolated flagella. The polypeptide encoded by Ocm1 has four cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interactions. It lacks obvious hydrophobic regions characteristic of transmembrane domains, suggesting that this polypeptide is not likely a protein for anchoring the mastigoneme. In addition, a polyclonal antibody against Ocm1 labeled the area where the tubular shafts of the mastigonemes are located, but not the basal portion or the terminal filaments.

Summary We document a remarkable morphological transformation, attributable to grazing by nanoflagellate Ochromonas sp. DS, of a phycocyanin-rich freshwater Cyanobium sp. (10-NR 98.2% similar 16S rRNA gene sequence to the type species Cyanobium gracile). The single cells aggregated into microcolonies (average size 40 μm) in the presence of the protist. Colonies were characterized by hundreds of tubes (spinae), 100 nm to 1 μm long and 63 ± 6 nm wide, on the surfaces of the Cyanobium cells co-cultured with Ochromonas. Spinae production, previously unknown for the freshwater Cyanobium species, suggests that picocyanobacterial life strategies are more flexible than previously thought.

1. The in situ abundance, biomass and mean cell volume of Actinophrys sol (Sarcodina: Heliozoa), the top predator in an extremely acidic German mining lake (Lake 111; pH 2.65), were determined over three consecutive years (spring to autumn, 2001-03). 2. Actinophrys sol exhibited pronounced temporal and vertical patterns in abundance, biomass and mean cell volume. Increasing from very low spring densities, maxima in abundance and biomass were observed in late June/early July and September. The highest mean abundance recorded during the study was 7 x 10(3) Heliozoa L-1. Heliozoan abundance and biomass were higher in the epilimnion than in the hypolimnion. Actinophrys sol cells from this acidic lake were smaller than individuals of the same species found in other aquatic systems. 3. We determined the growth rate of A. sol using all potential prey items available in, and isolated and cultured from, Lake 111. Prey items included: single-celled and filamentous bacteria of unknown taxonomic affinity, the mixotrophic flagellates Chlamydomonas acidophila and Ochromonas sp., the ciliate Oxytricha sp. and the rotifers Elosa worallii and Cephalodella hoodi. Actinophrys sol fed over a wide-size spectrum from bacteria to metazoans. Positive growth was not supported by all naturally available prey. Actinophrys sol neither increased in cell number (k) nor biomass (k(b)) when starved, with low concentrations of single-celled bacteria or with the alga Ochromonas sp. Positive growth was achieved with single- celled bacteria (k = 0.22 +/- 0.02 d(-1); k(b) = -0.06 +/- 0.02 d(-1)) and filamentous bacteria (k = 0.52 +/- < 0.01 d(- 1); k(b) = 0.66 d(-1)) at concentrations greater than observed in situ, and the alga C. acidophila (up to k = 0.43 +/- 0.03 d(-1); k(b) = 0.44 +/- 0.04 d(-1)), the ciliate Oxytricha sp. (k = 0.34 +/- 0.01 d(-1)) and in mixed cultures containing rotifers and C. acidophila (k = 0.23 +/- 0.02-0.32 +/- 0.02 d(-1); maximum k(b) = 0.42 +/- 0.05 d(-1)). The individual- and biomass-based growth of A. sol was highest when filamentous bacteria were provided. 4. Existing quantitative carbon flux models for the Lake 111 food web can be updated in light of our results. Actinophrys sol are omnivorous predators supported by a mixed diet of filamentous bacteria and C. acidophila in the epilimnion. Heliozoa are important components in the planktonic food webs of 'extreme' environments

SUMMARY Centrin, the EF-hand Ca2+-binding protein is localized at the basal apparatus of flagella and in centrioles in many eukaryotic cells. In the present study, centrin genes of the heterokont algae have been clarified for the first time. We isolated and analyzed cDNA and genomic DNA of centrin genes from the crysophycean alga Ochromonas danica Prings (UTEX LB1298) and the brown alga Scytosiphon lomentaria (Lyngbye) Link. The centrin gene of Ochromonas contained an open reading frame of 163 amino acids. The deduced protein, named Odcen, exhibited 85%, 78% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. The centrin genes of Scytosiphon contained an open reading frame of 164 amino acids. The deduced protein, named Slcen, exhibited 84%, 77% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. Both Odcen and Slcen possessed N-terminal extensions before the conserved amino acid among various centrins, four EF-hand domains and an aromatic amino acid at the C-terminus. Southern blot hybridization suggested that the centrin gene occurs as a single copy gene in both Ochromonas and Scytosiphon genomes. Comparison of the sequence of the cDNA and the genomic DNA revealed that the Odcen gene was split into three fragments by introns and Slcen gene consisted of five fragments. The junctions of all introns of both genes conformed to the GT–AG rule. The introns of Slcen gene were considerably long and, as a result, the Slcen gene was approximately seven times longer than Odcen gene.

The growth and grazing characteristics of Poterioochromonas malhamensis (Pringsheim) Peterfi (= Ochromonas malhamensis Pringsheim) (ca. 8 μm) feeding on phytoplankton, including the cyanobacteria Synechococcus sp. (ca. 2 μm) and Microcystis viridis (A. Brown) Lemmermann (ca. 6 μm) and the green alga Chlorella pyrenoidosa Chick (ca. 13 μm), were investigated in laboratory experiments involving the following treatments: (1) light without added algal prey (autotrophy), (2) light with added algal prey (mixotrophy), and (3) dark with added algal prey (phagotrophy). There were significantly higher cell numbers under mixotrophic and phagotrophic growth than under autotrophic growth. With phytoplankton as food, growth rates under both mixotrophy and phagotrophy were about two or three times higher than those under autotrophy, indicating that the algal diets were readily able to support the population growth of P. malhamensis. There were no significant differences in growth rate between mixotrophic and phagotrophic cultures during exponential growth. The ingestion rate of P. malhamensis with algal prey was also similar under both continuous light and dark. Poterioochromonas malhamensis ingested on average 0.27 M. viridis cells·flagellate−1·h−1 and 0.18 C. pyrenoidosa cells·flagellate−1·h−1 in continuous light and 0.25 M. viridis cells·flagellate−1·h−1 and 0.18 C. pyrenoidosa cells·flagellate−1·h−1 in continuous dark during exponential growth. The results showed that light had no effect on the growth and ingestion rates of P. malhamensis for phagotrophy during exponential growth. However, phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days. Populations of P. malhamensis showed no increase when prey was added again after 4 days in continuous darkness, indicating that light is necessary for sustained phagotrophic growth of P. malhamensis. The study suggests that P. malhamensis, which has strong tolerance for light, is light dependent for phagotrophy.

High resolution video-microscopy was used to observe grazing patterns of the heterotrophic nanoflagellates Cafeteria roenbergensis, Bodo saltans, Spumella sp., and Ochromonas sp. Spumella and Ochromonas enclose food particles with pseudopodia while Cafeteria and Bodo engulf particles by invagination of the cell surface. The following parameters of the feeding process were quantified: frequency of flagellar beating, speed of particles in different positions of the feeding current, food size selection, feeding rate, and the time budget for the handling of particles. The mean handling times differed between 94 s for Cafeteria and 4 s for Ochromonas for ingested particles. Handling times for ingested particles were significantly longer than for non-captured particles. Long handling times were calculated to be disadvantageous only for flagellates which propel a high water volume per hour (esp. Ochromonas) or live in a bacteria-rich environment. Our model calculations may provide a reasonable theoretical explanation for a concentration-dependent behavioural variability of the feeding strategy of different heterotrophic nanoflagellates (HNF) species.

Epifluorescence microscopy reveals the presence of fluorescence in the living cells of at least three classes of flagellates. In Ochromonas cells, the fluorescence is blue-green in color and is found only in the short flagellum, both in the flagellar swelling and throughout the length of the flagellum. As recognized by the locale and color of the flagellar fluorescence, the same fluorescence is observed in only certain other heterokont algal groups but is also found in one of the two isokont flagella of the prymnesiophyte Prymnesium parvum.

Mixotrophic flagellates can compete with obligately heterotrophic flagellates for the uptake of food particles, whereas mixotrophs and obligately phototrophic phytoplankton can compete for soluble nutrients. Competition for soluble nutrients is expected when photosynthesis covers a significant portion of the mixotrophs' carbon metabolism. When heterotrophy predominates, however, mixotrophs may release soluble nutrients and facilitate phototrophs. Mechanistic resource competition theory predicts that, due to their ability to utilize substitutable C and P sources, mixotrophs should be able to coexist with their more specialized competitors under certain conditions of resource supply. In laboratory experiments, the mixotrophic flagellate Ochromonas excluded heterotrophic flagellates when only phototrophic growth was possible. However, Ochromonas was excluded by heterotrophic flagellates when only phagotrophic growth was possible. Both coexisted when food bacteria and light were supplied simultaneously. Ochromonas coexisted with a P-limited phytoplankter, Cryptomonas sp., when soluble reactive phosphorus (SRP) and bacterial phosphorus were available as alternative P sources. In nature, the mixotrophic strategy may be successful when resources are limiting. This is supported by published data on the occurrence of mixotrophic chrysophytes.

In laboratory experiments, I studied the influences of bacterial density and light on the ingestion and growth rates, pigment contents, and the carbon and phosphorus turnover rates of the mixotrophic flagellate Ochromonas sp. The investigated strain is a bacterivorous flagellate that can enhance its photosynthetic apparatus and grow phototrophically when bacterial densities are low. This was also evident from significantly higher chlorophyll a contents during active photosynthetic growth phases. Moderate phototrophic growth should be possible even if bacteria were absent. Bacterial ingestion rates increased hyperbolically with bacterial density, and there was no difference between light- and darkadapted cells. Ochromonas released soluble reactive phosphorus (SRP) when growth was predominantly phagotrophic, but it took up SRP when growth was phototrophic. The mixotrophic strategy in Ochromonas appears to be bound up with costs and trade-offs : Ochromonas needs high bacterial densities to reach maximum growth rates, its basic metabolic costs are higher than for obligately phagotrophic flagellates, and its phototrophic growth rates are lower than for obligately phototrophic phytoplankton of comparable size.

The total loss of plastid DNA has never been reported for any alga or plant cell line, with the sole exception of the protozoan Euglena, yet plastid distribution at mitosis is apparently stochastric (Birky and Skavaril, Journal of Theoretical Biology, vol. 106, pp. 441-447, 1984) and accidental loss might be expected. It is not obvious how stem cells of photosynthetic eukaryotes avoid this problem. The chrysophyte alga Ochromonas danica, described as having but one or two plastids, can proliferate indefinitely without the benefit of photosynthesis. Under such conditions its plastid genome copy number per cell might drop to the absolute minimum compatible with maintaining its inheritance. In situ quantitation of Ochromonas plastid DNA in both photosynthetic and enriched mixotrophic growth, and in heterotrophic growth in prolonged darkness, suggests that plastids are capable of very wide variation (7 to >200 genomes/plastid) in their DNA content, and likewise, cells can vary from one to >8 plastids per cell, with total genomes numbers from 7 to >1,000 per cell. Among many growth conditions tested, the smallest plastids were found in rapidly dividing cells grown in the dark, many of which contained but oine plastid. The inability to find plastids with fewer than seven plastid genome equivalents of DNA, even in these rapidly multiplying cells grown in total darkness for months, suggests that multiple copies of the plastid genome may be very carefully maintained, even in the prolonged absence of photosynthesis. This implies that multiple copies are important for reasons other than photosynthetic capability; two possibilities are the biosynthetic steps necessary for eukaryote cell survival known to occur solely within a plastid, and/or the potential that multiple plastid genome copies provide to escape the effects of Muller's ratchet.

SUMMARY Sulcochrysis biplastida gen. et sp. nov., a golden, marine, mixotrophic flagellate is described. Cells resemble Ochromonas in light microscopic features, but they are distinct at the electron microscopic level from Ochromonas or any other typical chrysophyte. Ultrastructural features that discriminate Sulcochrysis from the Chrysophyceae are: (i) a proximal helix in the flagellar transition region; (ii) basal bodies situated in the anterior depression of the nucleus; (iii) the lack of the rhizoplast; and (iv) simple flagellar hairs lacking lateral filaments. These features suggest that Sulcochrysis is a relative of the Pedinellophyceae, Dictyochophyceae and Pelagophyceae. However, Sulcochrysis has a flagellar root system similar to that of the Ochromonas-type cell and it may use the R3 root for prey capture, as do ochromonadalean algae. The R3 root and the phagotrophic mechanism using the R3 root are interpreted as a plesiomorphy, because these are also distributed in primitive heterokonts such as the bicosoecids. Sulcochrysis has one microtubule probably homologous with the x-fibre of Bicosoeca maris Picken. Based on these features it is suggested that Sulcochrysis is an organism that links bicosoecids (Bicosoeco-phyceae), Pedinellophyceae, Dictyochophyceae and Pelagophyceae.

Bacterivory in obligate phototrophic algal flagellates may be an important strategy for acquiring nutrients during periods of inorganic nutrient limitation. Several marine algal flagellates were shown to increase bactivory when phosphate was limited. Grazing on bacteria by algal flagellates was found during blooms of Prymnesium parvum in Sandsljorden, western Norway, in 1989 and Chrysochromulina polylepis on the south and west coast of Norway in 1988. Dissolved phosphate was not detectable in these situations. Algal flagellates may graze bacteria to obtain phosphate, which may permit these algal flagellates to develop blooms when phosphate becomes limited. True mixotrophy, defined as use of particulate organic matter for cellular growth, has been conclusively demonstrated for only a few species in the genus Ochromonas (Fenchel 1982) and for Poteiroochromonas malhamensis (Caron et al. 1990). Much more widespread is the ability to use a restricted range of organic substances, available at high concentrations, as a dietary supplement or sole C source in the dark (Antia 1980). Mixotrophy in algal flagellates has been considered mainly a strategy for gaining C during low light (Bird and Kalff 1987; Sanders and Porter 1988) and therefore has been studied in algae that use bacteria as their primary C source. Doddema and Van der Veer ( 198 3) suggested that phagocytosis might permit utilization of particulate organic N and P when inorganic nutrients are in limited supply. Obligate phototrophs are the appropriate test organisms to evaluate this hypothesis. Bacteria have a high P content even when phosphate is limited (Andersen et al. 1986), although their P content can be reduced when they are drastically starved (Tezuka 1990). Bacteria are therefore a potential source of P when obligate phototrophic algal flagellates are subjected to

2-Methylpentan-3-ol stereospecifically labelled with deuterium on one of the isopropyl methyl groups has been synthesized, thus allowing the unequivocal assignment of their 13C NMR resonances. 13C NMR assignments for these groups proposed earlier are shown to be incorrect. Involvement in the biosynthesis of the 24β-ethylsterol poriferasterol in the crysophyte Ochromonas malhamensis is discussed.

The 16S-like ribosomal RNA genes from Mallomonas papillosa Harris et Bradley (Synurophyceae) and Tribonema aequale Pascher (Xanthophyceae) were sequenced and compared to those of other eukaryotes. Mallomonas is closely related to Ochromonas (Chrysophyceae) and supports the general hypothesis of a close phylogenetic relationship between the Synurophyceae and Chrysophyceae. Tribonema is specifically related to Costaria costata (C. A. Agardh) Saunders (Phaeophyceae) demonstrating an unexpected phylogenetic relationship between the Xanthophyceae and Phaeophyceae. Distance and parsimony analysis place these four chromophyte genera in a complex evolutionary assemblage that includes the Bacillariophyceae and Oomycetes but excludes the Dinophyceae. The close relationship between the chromophyte algae and the Oomycete fungi supports the hypothesis that protists with tripartite hairs form a natural assemblage.

Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (˜120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.

Colorado and Wyoming lakes with high pH values generally ranging from 8 to 10 are dominated by phytoplankton belonging to the chrysophytes, diatoms and cryptomonads. Our studies on phytoplankton diversity in Dowdy, South Delaney Buttes and Cowdrey Lakes in Colorado and Diamond and Twin Buttes Lakes in Wyoming have yielded several isolates of rare or new chrysophytes. These isolates were examined with light microscopy, SEM, TEM, and novel information regarding their structure is presented. Proposed new species belong to the genera Saccochrysis, Prymnesium and Chrysochromulina. Chrysophytes that were examined for the first time include Monochrysis aphanaster, Uroglenopsis, and a mixotrophic species of Ochromonas, which ingests small diatoms. Probable new species are the following: Saccochrysis sp. nov. differs in chloroplast structure, cell shape and cell number/colony, and absence of a posterior vacuole; Prymnesium sp. nov. is the first freshwater form examined with EM and differs in scale morphology; and Chrysochromulina sp. nov. is similar to Chrysochromulina parva but lacks scales. The ultrastructure of M. aphanaster is unique and appears to be a member of the Chrysophyceae. It has two flagella, one of which is emergent, while the other is hidden within an invagination of the cytoplasm. Diacronema and Sarcinochrysis spp. from these lakes also were isolated and reinvestigated, and some unique cellular features will be discussed.

Ochromonas sensu lato is the largest genus in the Chrysophyceae, containing over 100 names. Ochromonas species are biflagellate, naked, plastid-bearing single cells, distinguished from loricate, scaled, colonial and colorless genera. Most, if not all, species of Ochromonas are mixotrophic, i.e., they photosynthesize but they also engulf bacteria and other small prey. Preliminary evidence from SSU rRNA sequences show that Ochromonas is a polyphyletic genus. Ochromonas tuberculata is the most distinct from all other Ochromonas species. The other Ochromonas species (examined thus far) are scattered in three clades. For example, O. danica and O. sphaerocystis are sister to Poterioochromonas stipitata and P. malhamensis. Four additional species (identified by light microscopy as O. elegans Doflein, O. globosa Skuja, O. ovalis Dolfein, O. sociabilis Pringheim) have SSU rRNA sequences identical to P. malhamensis. Of these, only O. sociabilis has been transferred to Poterioochromonas. Thus, at least some species may be synonymous with others. Two clades of marine species are also known, one containing coastal species and the other containing open ocean species. A number of genera (some also polyphyletic) are interspersed amongst the Ochromonas species (e.g., Chrysolepidomonas, Chrysonephele, Chrysoxys, Cyclonexis, Dinobryon, Epipyxis, Uroglena, Uroglenopsis). The goal of this research (just beginning) is to establish a monophyletic Ochromonas, probably by assigning some species to other genera (existing or new). One major problem is that the type species, O. triangulata Vysotskii, hasn't been observed in over 100 years, and it is unclear which of several clades of Ochromonas contains the type. Results will be discussed.

Simultaneous measurements of inorganic carbon fixation and phagotrophic particle uptake by Dinobryon in a metalimnetic algal abundance peak showed that this alga depended more strongly on ingested bacteria for nutrition than on photosynthesis. Measurements of the grazing rate at different depths in Lac Gilbert, Quebec, showed that the particle ingestion rate depends on water temperature rather than light availability. Phagocytosis of bacteria proceeded at a similar rate both day and night in most lakes where Dinobryon was found. Since other chrysomonad genera (Chlsophaerella, Uroglena, Catenochrysis, Ochromonas, Chromulina, and Chrysococcus) were also found to ingest particles, it will not be possible to estimate grazing on bacteria by counting nonpigmented cells and ignoring those containing chlorophyll. In oligotrophic Lac Bowker, 30% of the phytoplankton cells were actively ingesting small particles, and it was these phytoplankton and not the less numerous zooplankton that were responsible for most bacterial grazing in the lake. Phagotrophy by algae appears to be an important but unexpected pathway for energy flow in lakes. The flagellated Chrysophyceae, or chrysomonads, bear affinities to both plants and animals and have been claimed for study by zoologists as well as by botanists (Allen 1969). Coexistence of autoand heterotrophy in the chrysomonads and the widespread tendency toward complete loss of chlorophyll have fostered the suggestion that these algae may model progenitors of the Metazoa (Provasoli and Pintner 1953). The diversity and versatility of the group are strongly expressed in the genus Uchromonas, which is capable of phagotrophy, heterotrophy, photoheterotrophy, and photoautotrophy (Pringsheim 19 5 5; Aaronson and Baker 1959). However, Ochromonas is ’ This work was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to J. Kalff and an NSERC postgraduate scholarship to D. Bird. A contribution to the McGill Limnology Research Centre, Lake Memphremagog Project. one of only a few chrysomonads to have been studied extensively in the laboratory, and the nutritional capabilities of other chrysomonads are virtually unknown, especially in their natural setting. Pascher (1943) observed under the microscope that the colonial chrysophycean alga Dinobryon could capture and digest small particles, and thus concluded that at least some part of this alga’s nutrition was heterotrophic. This unusual phagotrophic ability was not studied further until the recent discovery that several Dinobryon spp. are not only phagotrophic in nature, but can be major grazers of bacteria in lakes (Bird and Kalff 1986). Here we present further work on four different aspects of the capabilities of this alga: first, the relative importance of bacterivory and photosynthetic carbon fixation to Dinobryon’s nutrition; second, a comparison of day and night grazing rates; third, the relationship of grazing

The cysts (statospores) of Ochromonas tuberculata Hibberd are produced within a cytoplasmic silica deposition vesicle (SDV) whose membrane (silicalemma) appears to be formed by the coalescence of golgi vesicles. Silica is first deposited as small nodules and the collar and spines develop by centrifugal growth only after a complete but still thin wall has been laid down. Small vesicles appear to be attached to the SDV only in the region overlying the developing collar; a cap of radially arranged, moderately electron-dense material occurs at the tip of the growing spines. The cyst pore is formed at the anterior end of the flagellate cell, by lack of silica deposition over a small region of the SDV and rupture of the SDV and other membranes crossing this region. When the cyst wall is complete, an extracystic plug is formed in the pore, resulting in the loss of some extracystic cytoplasm and the plasmalemma, and the inner SDV membrane becomes the functional plasmalemma. The plug develops first by coalescence with the cell membrane of golgi-derived vesicles containing dense but apparently nonsiliceous spicules surrounded by amorphous material. During later stages of plug formation only fibrous material is deposited, some of which may be extruded through the pore forcing out some of the spiculate component. Scanning electron micrographs of the mature wall show it is smooth except for the concentrically wrinkled inner face of the flared collar and that the real pore diameter is only ca. half that of the collar. At germination the plug completely disappears in an unknown way and a single cell, similar to a normal vegetative cell emerges through the pore. Chrysophycean cyst formation generally resembles cell wall formation in diatoms, but differs in some details.

Evidence is provided for the importance of phagocytosis to mixotrophic chrysophycean algae and to the phytoplankton community as a whole. First, we confirmed the major assumption of past work that the constituents of ingested particles are incorporated efficiently into algal tissue. Cells of the phagotrophic chrysomonad Dinobryon sertularia collected from Lac Gilbert, Quebec, incorporated carbon from radiolabeled bacterial prey with 54% efficiency (95% C.L. = 40-79%) over a 5-h period. When removed from labeled bacteria, the loss of previously incorporated label from Dinobryon was undetectable over a 4-h period. We conclude that this alga’s quantitative utilization of prey tissue is indistinguishable from that of purely heterotrophic organisms. Second, simultaneous measurements were made of phagotrophic feeding and photosynthesis within a highly concentrated layer of chrysomonads (Dinobryon and Ochromonas) in the metalimnion of Lac Gilbert on 3-4 July 1986. Chrysomonad abundance ranged from 300 cells ml-’ at 6 m, to 9,000 at 7 m, and back to < 100 at 9 m. At the 7-m peak, phagotrophy accounted for 79% of algal community carbon assimilation on this overcast day.

SYNOPSIS Ochromonas malhamensis cells were allowed to feed on autoclaved Aerobacter aerogenes, biochemically inert polystyrene latex particles of comparable size, shape and density, or a combination of these to determine if this protozoan is capable of phagotrophic selectivity under defined experimental conditions. Electron microscopic examination of Gomoristained food vacuoles indicated that the type of particle ingested was determined entirely by the type available. Apparently the biochemical or physical configuration of the particle surface is not decisive in particle selection. Gomori acid-phosphatase reaction product, which indicates digestive enzyme activity, was present in all particle-containing vacuoles, suggesting that digestive activity in O. malhamensis is due to the presence of particulate material in the vacuole, but not necessarily to the nature of the particle.

Data obtained from commonly used methods of assaying for riboflavin, vitamin B6, niacin, pantothenic acid, thiamine, biotin and vitamm B1 2 were compared to data obtained from vitamin analysis procedures employing protozoa as the test organisms. The protozoa were of the genera Tetrahymena and Ochromonas. Generally, the procedures employing Tetrahymena indicated higher levels of the vitamins in the foods analyzed, while the procedures employing Ochromonas indicated lower vitamin levels than the corresponding commonly used methods of vitamin analysis. Both protozoan and the commonly used methods for analysis of vitamins indicated higher levels of vitamins in food samples when the sample extracts received enzymatic as well as acid hydrolysis treatments. Neutralization salts formed during vitamin extraction procedures were found to interfere with both the conventional and the protozoan methods. High-acid foods (e.g., orange and tomato juices) were found to inhibit the growth of Tetrahymena. Further studies are required to develop optimal methods of vitamin extraction and analysis in order to quantify the biologically available vitamin contents of foods.

Open ocean chloroplast-containing chrysomonads have been observed to prey on bacteria. Predominant among these forms are new species of Ochromonas and Chrysamoeba. These algae coexist with nearly identical apochlorotic forms, are abundant in oceanic nanoplankton (averaging 100 cells ml−1), will not grow axenically, can be maintained for extended periods with endogenous bacteria enriched by rice grain culture in the light, and contain bacteria within their food vacuoles. The results demonstrate that protists with and without chloroplasts can occupy overlapping trophic roles and should therefore be studied together. They also challenge recent estimates of nanoflagellate predation on bacteria, as these estimates have assumed that only apochlorotic forms are predatory. If other groups of pelagic nanoflagellates with chloroplasts also prey on bacteria, the transfer of material from bacteria to protists in the open ocean could approach twice previous estimates.

Vitamin analyses using protozoa (Tetrahymena pyriformis, Ochromonas danica and Ochromonas malhamensis) as test organisms were found to he affected by lower concentrations of neutralization salts than were the vitamin analyses which used bacteria and yeast as test organisms. Neutralization salts are formed during vitamin extraction procedures. Growth of protozoa and yeast was affected by the food preservatives-sodium sorbate, sodium propionate and sodium benzoate. Except for sodium benzoate inhibition of Lactobacillus leichmannii, the growth of bacterial test cultures was not affected by these chemicals. Levels of the neutralization salts and food preservatives in vitamin assays that are required to affect the growth of the test organisms were calculated. These levels were compared to the concentrations of salts formed by accepted vitamin extraction procedures to determine the dilutions of sample extracts necessary to avoid interference. Similar calculations were completed to determine minimal dilutions of foods necessary to avoid the effects of food preservatives on the test organisms. Acids and bases that neutralized to form salts that had the least effect on the growth of the test cultures were determined.

SUMMARY Ochromonas danica Prings., a chrysomonad alga which demonstrates a high degree of nutritional versatility, is capable of feeding on the toxic blue-green alga Microcystis aeruginosa Kuetz. In this paper light microscopic, electron microscopic, and cytochemical examinations of endocytosis in O. danica are reported, with particular emphasis on the vicissitudes of endocytic and lysosomal activities during intra-cellular digestion. An interpretation of the function of organelles associated with endocytosis is presented.

SYNOPSIS Tryptophan enhanced the growth of Ochromonas malhamensis at concentrations up to 0.4 mg/ml; higher concentrations inhibited, the growth inhibition being reversible by tyrosine and adenine. The presence of a tryptophan synthetase system in vitro was demonstrated. Tyrosine and phenylalanine stimulated the activity of this enzyme. The uptake of exogenous tryptophan was accompanied by an increase in the free tryptophan pool which in turn suppressed the tryptophan synthetase system, thus pointing to a controlled mechanism. Incorporation of tryptophan in the growth medium enhanced the biosynthesis of folate-active compounds. An elucidation of the mode of action of tryptophan is attempted on the basis of known metabolic pathways.

The effects were studied of extracts from the chrysophyte Ochromonas malhamensis on Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis and Escherichia coli. The extracts contained three different antibiotically active substances, one of which was produced during the extraction. The other two had no effect on E. coli, but inhibited the growth of the three other bacteria. The present experiments show that one of the products increases its antibiotic activity when exposed to visible light, and that its activity is enhanced by boiling the extracts for 5 minutes. The other active substance is not affected by light, but shows increased activity after being boilt for 5 minutes.

SYNOPSIS. Five- to 6-day-old resting cells of Ochromonas malhamensis were incubated at pH 6.5 with glucose and appropriate C14 precursors of the methyl groups of phospholipid-choline. Under the experimental conditions L-methionine-C14H3 was the most efficient source of choline-methyl groups, followed by formate-C14, formaldehyde-C14 and DL-serine-3-C14, respectively. Glycine-2-C14 was not incorporated into choline. Both L-methionine-C14H3 and formate-C14 served as precursors for the methyl groups of monomethylethanolamine, dimethylethanolamine and choline. Addition of non-radio-active L-methionine depressed the incorporation of formate-C14 into choline-methyl groups by 50%. The results support the hypothesis that methionine can be the source of all 3 methyl groups of choline, and that formate is probably converted to the methyl group of methionine before transmethylation to choline. However, an alternate pathway from single-carbon sources cannot be excluded.

SUMMARY The following marine chrysomonads, originally described by Carter, are redescribed: Pseudopedinella pyriforme, Ochromonas oblonga, and Pyrmnesium parvum. Parachrysiclalis n.g. is set up for P. estuariale, with a flattened body and 2 equal flagella inserted in the middle of one side. Redescribed Xanthophy-cean species include Nephrochloris salina and Olis-thocliscus luteus. Olisthodiscus carterae and O. magnus are new species, the first distinguished by the position of the flagella and large number of chromatophores, O. magnus by its size (30-18 μ X 15-25 μ) and anteriorly inserted but posteriorly trailing flagellum. Among Cryptophyceae, Chroomonas vectensis Carter, Rhodomonas amphioxeia Conrad, and Cryptomonas salina and C. stigmatica Wislouch are redescribed along with Rhodomonas minuta v. apyrenoidosa var. nov. Redescribed Chlorophyta are Bipedinomonas pyriformis Carter, Pyramimonas obovata Carter, P. amylifera Conrad, and what may be Tetra-selmis suecica (Kylin) Butcher. The loricate, probably colorless Calycomonas gracilis (Lohmann) Wulff is redescribed.

SUMMARY Ochromonas danica grown on a chemically defined medium under controlled conditions in the light synthesized the following vitamins: ascorbate, B6, N5-methyltetrahydrofolate, tetrahydrofolate polyglutamates, oxidized folate monoglutamates, nicotinate, pantothenate, riboflavin, vitamin A, β-carotene, and vitamin E but no vitamin. B12. The cells also secreted molecules into their growth medium including the vitamins ascorbate, B6, the above folates, nicotinate, pantothenate, riboflavin, vitamin E, and the amino acids alanine, aspartic acid, leucine, and valine. The role of such secretions in nature is discussed.

Total lipids of several protozoa were determined and found higher than in most other microorganisms. The lipid content in a phytoflagellate, Ochromonas malhamensis, could be increased by excess nicotinic acid or deficiency in nitrogen supply. Cell multiplication and lipid synthesis were unaffected by inhibitors of vitamin synthesis such as penicillamine, pan-toyltaurine, or pyridine-3-sulfonic acid, up to 100 mg%. The phytoflagellates, O. danica, O. malhamensis, Euglena gracilis, and the photosynthetic bacterium Chlorobium limkola contained large quantities of non-saponifiable lipids: ergesterol was identified as a major component of this lipid fraction. Sterols were not found in the bacteria, Streptococcus faecalis, Rhodopseudomonas palustris; the algae. Cyanidium calidarnm. Anacystis nidulans, and Porphyridium cruentum; and the protozoan Tetrahymena pyriformis.

SYNOPSIS Anacystis nidulans disappeared rapidly from culture in the presence of an unidentified species of Ochromonas. Disappearance was light-independent and could be induced neither by bacteria associated with, nor by soluble products released from the flagellate. Electronmicrographs of mixed cultures revealed numerous A. nidulans cells in various stages of digestion within vacuoles of Ochromonas. Evidently the disappearance of the alga from culture resulted from phagotrophy by the chrysomonad. A 2-stage digestive process is suggested whereby A. nidulans cells are initially sequestered in the posterior “leucosin” vacuole and then undergo the terminal stages of digestion and elimination in smaller, peripheral vacuoles.

SUMMARY. Ochromonas malhamensis (Pringsheim strain) can be grown above 35.5°C.; below 35°, the previous chemically defined medium supports dense growth. The B12 and thiamine requirements rise steeply with temperature, and growth promotion by folic acid emerges; folic acid spares the enhanced B12 requirement. B12 is spared also, perhaps wholly bypassed, by purines + pyrimidines + amino acids (below 35°, exogenous purines, pyrimidines, and folic acid have little effect). Requirements also emerge for glycine (spared by serine), valine and isoleucine (their ratio is critical; leucine and threonine assist in maintaining a good balance), and, at very slightly higher temperatures, phenylalanine, tryptophan, cystine, and lysine. Requirements for Mg, Fe, Zn, and Mn appear to rise steeply with temperature; metal toxicities have to be circumvented carefully. The proportion of histidine + arginine to carbohydrate has to be increased, and a Krebs-cycle component such as succinic acid becomes stimulatory. At 36.3–36.7°, a further supplement of crude natural materials such as an autoclaved suspension of Ochromonas cells is needed. Relevance of these findings to fever stress in vertebrates, general mitochondrial function, and repair of radiation damage, is discussed.

Summary Ochromonas ostreaeformis nov. sp. is described from a pool in the English Lake District. The motile cells have been recorded each spring for 4 years, although the formation of cysts was first observed in 1965. Possible factors influencing periodicity are discussed, especially variation in the phosphate phosphorus content of the water, which tended to show an inverse relationship with the number of cells.

SYNOPSIS. Mastigonemes occur on flagella of many species of Protozoa, but the only function so far proposed for the mastigonemes is to increase the effective surface of the flagellum. Locomotion of Ochromonas malhamensis, O. danica, and an unidentified marine chrysomonad was examined by means of high speed microcinematography up to 600 pictures per second. In addition, analyses were performed on the movement of the organism in a suspension of polystyrene spheres (1.17 μ in diameter). The films clearly show that the organisms move by means of a planar sine wave in the long anteriorly-directed flagellum. The wave starts at the basal end of the flagellum and progresses distally. It is apparent that the organisms are pulled forward by the action of the long flagellum, and that the flagellum does not push backward as would ordinarily be expected with a forwardly-directed sine wave. It is well known that flagellar sine waves usually start at the base, move distally, and produce a push toward the point of attachment. It also is known (2) that in Mastigamoeba a helical wave may start at the tip, progress proximally toward the base, and pull the organism. However, in the present organisms the wave begins at the base, moves distally (i.e., forward), and pulls the organism. The flagellum is a true tractellum. The theoretical calculations of Taylor (1952, Proc. Roy. Soc., 214A, 158) show that if a thin cylinder, undergoing sinuous movements produced by a traveling sine wave of active bending, has a roughness of surface which is sufficiently great and has a certain directional character, propulsion may be achieved in the direction opposite to that expected of a smooth cylinder bending in exactly the same manner. It is known that the long flagellum of the chrysomonads is pantonematic, i.e., has numerous stiff lateral mastigonemes on at least two and probably all sides. It is proposed that one function of these mastigonemes is to provide a roughness of surface which brings about this type of reversal.

SUMMARY. A new growth factor which is more active than folic acid for the trypanosomid parasite of mosquitoes, Crithidia fasciculata, was partially purified. This factor is synthesized in relatively high concentration by Ochromonas and is an impurity in some commercial preparations of purine ribosides. Crithidia factor is tentatively identified as a polysubstituted pteridine which may be the pteridine actually involved in the biosynthesis of functional folic acid.

SYNOPSIS. Ochromonas malhamensis contains folate derivatives active for Streptococcus faecalis, Pediococcus cerevisiae and Lactobacillus casei. These activities increase about 6-, 4- and 3-fold, respectively, on treatment with chicken liver conjugase, establishing the presence of polyglutamyl folates in the organism. The nature of the folate derivatives as well as their functional groups has been ascertained by chromatography on DEAE cellulose columns and assay of the activities of the eluted fractions, before and after the conjugase treatment, using differential microbiologic assay technic. The folate complex has been resolved into 7 fractions corresponding to N10-formyltetrahydrofolic acid, N5-formyltetrahydrofolic acid, unsubstituted tetrahydrofolic acid, and 4 polyglutamyl folates. The implications of these findings are indicated.