Oceanic protists with different forms of acquired phototrophy display contrasting biogeographies and abundance.

This first comprehensive analysis of the global biogeography of marine protistan plankton with acquired phototrophy shows these mixotrophic organisms to be ubiquitous and abundant; however, their biogeography differs markedly between different functional groups. These mixotrophs, lacking a constitutive capacity for photosynthesis (i.e. non-constitutive mixotrophs, NCMs), acquire their phototrophic potential through either integration of prey-plastids or through endosymbiotic associations with photosynthetic microbes. Analysis of field data reveals that 40-60% of plankton traditionally labelled as (non-phototrophic) microzooplankton are actually NCMs, employing acquired phototrophy in addition to phagotrophy. Specialist NCMs acquire chloroplasts or endosymbionts from specific prey, while generalist NCMs obtain chloroplasts from a variety of prey. These contrasting functional types of NCMs exhibit distinct seasonal and spatial global distribution patterns. Mixotrophs reliant on 'stolen' chloroplasts, controlled by prey diversity and abundance, dominate in high-biomass areas. Mixotrophs harbouring intact symbionts are present in all waters and dominate particularly in oligotrophic open ocean systems. The contrasting temporal and spatial patterns of distribution of different mixotroph functional types across the oceanic provinces, as revealed in this study, challenges traditional interpretations of marine food web structures. Mixotrophs with acquired phototrophy (NCMs) warrant greater recognition in marine research.

Phase I study of recombinant human CD40 ligand in cancer patients.

PURPOSE: To determine the toxicity, maximum-tolerated dose (MTD), and pharmacokinetics of recombinant human CD40 ligand (rhuCD40L) (Avrend; Immunex Corp, Seattle, WA), suggested in preclinical studies to mediate cytotoxicity against CD40-expressing tumors and immune stimulation. PATIENTS AND METHODS: Patients with advanced solid tumors or intermediate- or high-grade non-Hodgkin's lymphoma (NHL) received rhuCD40L subcutaneously daily for 5 days in a phase I dose-escalation study. Subsequent courses were given until disease progression. RESULTS: Thirty-two patients received rhuCD40L at three dose levels. A total of 65 courses were administered. The MTD was 0.1 mg/kg/d based on dose-related but transient elevations of serum liver transaminases. Grade 3 or 4 transaminase elevations occurred in 14%, 28%, and 57% of patients treated at 0.05, 0.10, and 0.15 mg/kg/d, respectively. Other toxicities were mild to moderate. At the MTD, the half-life of rhuCD40L was calculated at 24.8 +/- 22.8 hours. Two patients (6%) had a partial response on study (one patient with laryngeal carcinoma and one with NHL). For the patient with laryngeal cancer, a partial response was sustained for 12 months before the patient was taken off therapy and observed on no additional therapy. Three months later, the patient was found to have a complete response and remains biopsy-proven free of disease at 24 months. Twelve patients (38%) had stable disease after one course, which was sustained in four patients through four courses. CONCLUSION: The MTD of rhuCD40L when administered subcutaneously daily for 5 days was defined by transient serum elevations in hepatic transaminases. Encouraging antitumor activity, including a long-term complete remission, was observed. Phase II studies are warranted.

Identification of heterotrophic nanoflagellates by restriction fragment length polymorphism analysis of small subunit ribosomal DNA.

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures.

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.

Insights on the diversity within a "species" of Thalassicolla (Spumellarida) based on 16S-like ribosomal RNA gene sequencing.

We compared 16S-like ribosomal RNA (rRNA) coding regions of samples of the solitary spumellarian radiolarian Thalassicolla nucleata collected from the Sargasso Sea and the Pacific Ocean. Sequences derived from these locations showed variability in both length and base-pair composition. This level of sequence variability is similar to the degree of variability reported in the literature for species- or even genus-level distinctions. Explanations for our results include multiple alleles for the rRNA gene, or the existence of multiple species of Thalassicolla that are morphologically indistinguishable. The seven existing descriptions of Thalassicolla species, including T. nucleata, are discussed in view of these molecular findings and with reference to our current understanding of the physiology and life cycle of the spumellarian radiolaria.

Phylogenetic relationships between the Acantharea and the Polycystinea: a molecular perspective on Haeckel's Radiolaria.

Polycystine radiolaria are among few protistan groups that possess a comprehensive fossil record available for study by micropaleontologists. The Polycystinea and the Acantharea, whose skeletons do not become fossilized, were once members of the class "Radiolaria" ("Radiolaria" sensu lato: Polycystinea, Phaeodarea, and Acantharea) originally proposed by Haeckel but are now included in the superclass Actinopoda. Phylogenetic relationships within this superclass remain largely enigmatic. We investigated the evolutionary relationship of the Acantharea and the Polycystinea to other protists using phylogenetic analyses of 16S-like ribosomal RNA (rRNA) coding regions. We circumvented the need to culture these organisms by collecting and maintaining reproductive stages that contain many copies of their genomic DNA. This strategy facilitated extraction of genomic DNA and its purification from symbiont and prey DNA. Phylogenetic trees inferred from comparisons of 16S-like coding regions do not support a shared history between the Acantharea and the Polycystinea. However, the monophyly of the Acantharea and the separate monophyly of the Polycystinea (Spumellarida) are well supported by our molecular-based trees. The acantharian lineage branches among crown organisms whereas the polycystine lineage diverges before the radiation of the crown groups. We conclude that the Actinopoda does not represent a monophyletic evolutionary assemblage and recommend that this taxonomic designation be discarded.

Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria.

Recent analyses of the small subunit ribosomal DNA (srDNA) from dinoflagellate symbionts of cnidaria have confirmed historical descriptions of a diverse but well-defined clade, Symbiodinium, as well as several other independent symbiont lineages (Rowan 1991; Rowan and Powers 1992; Sadler et al. 1992; McNally et al 1994). Dinoflagellates also occur as intracellular symbionts in a number of pelagic protistan taxa, but the srDNA of these symbionts has not been examined. We analyzed the srDNA sequences of the symbiotic dinoflagellates from four planktonic foraminiferal species and six radiolarian species. The symbionts from these sarcodines formed two distinct lineages within the dinoflagellates. Within each lineage, symbionts obtained from different host species showed few, if any, srDNA sequence differences. The planktonic foraminiferal symbionts were most closely related to Gymnodinium simplex and the Symbiodinium clade, whereas the radiolarian symbionts were most closely related to the dinoflagellate symbiont from the oceanic chondrophore, Velella velella. Therefore, although the dinoflagellate symbionts of foraminifera appear to be a sister taxon of the symbionts from benthic foraminifera and invertebrates, the symbionts of radiolaria are distinct and arose from an independent lineage of dinoflagellate symbionts that shares common ancestry with the symbiont of at least one pelagic metazoan. The lack of srDNA variability within the sarcodine symbiont lineages suggests that coevolution of host and symbiont has not occurred.

Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes.

A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described. Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.). Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates. We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.

Inorganic nutrients, bacteria, and the microbial loop.

The realization that natural assemblages of planktonic bacteria may acquire a significant fraction of their nitrogen and phosphorus via the uptake of dissolved inorganic nutrients has modified our traditional view of these microorganisms as nutrient remineralizers in plankton communities. Bacterial uptake of inorganic nitrogen and phosphorus may place bacteria and phytoplankton in competition for growth-limiting nutrients, rather than in their traditional roles as the respective "source" and "sink" for these nutrients in the plankton. Bacterial nutrient uptake also implies that bacterivorous protozoa may play a pivotal role in the remineralization of these elements in the microbial loop. The overall contribution of bacterial utilization of inorganic nutrients to total nutrient uptake in the ocean is still poorly understood, but some generalizations are emerging with respect to the geographical areas and community physiological conditions that might elicit this behavior.

Application of rRNA-based probes for observing marine nanoplanktonic protists.

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.

Light-dependent phagotrophy in the freshwater mixotrophic chrysophyte Dinobryon cylindricum.

The mixotrophic (bacterivorous), freshwater chrysophyte Dinobryon cylindricum was cultured under a variety of light regimes and in bacterized and axenic cultures to investigate the role of phototrophy and phagotrophy for the growth of this alga. D. cylindricum was found to be an obligate phototroph. The alga was unable to survive in continuous darkness even when cultures were supplemented with high concentrations of bacteria, and bacterivory ceased in cultures placed in the dark for a period longer than one day. Axenic growth of the alga was poor even in an optimal light regime. Live bacteria were required for sustained, vigorous growth of the alga in the light. Carbon (C), nitrogen (N), and phosphorus (P) budgets determined for the alga during growth in bacterized cultures indicated that bacterial biomass ingested by the alga may have contributed up to 25% of the organic carbon budget of the alga. Photosynthesis was the source of most ([Symbol: see text]75%) of the organic carbon of the alga. D. cylindricum populations survived but did not grow when cultured in a continuous low light intensity (30 µE m(-2) sec(-1)), or in a light intensity of 150 µE m(-2) sec(-1) for only two hours each day. Net efficiency of incorporation of bacterial C, N, and P into algal biomass under these two conditions was zero (i.e., no net algal population growth). We conclude that the primary function of bacterivorous behavior in D. cylindricum may be to provide essential growth factor(s) or major nutrients for photosynthetic growth, or to allow for the survival of individuals during periods of very low light intensity or short photoperiod.

Relationship between phototrophy and phagotrophy in the mixotrophic chrysophytePoterioochromonas malhamensis.

The time scales involved in the transition between phototrophic and phagotrophic modes of nutrition were examined in the mixotrophic chrysophytePoterioochromonas malhamensis. Phagotrophy began almost immediately when bacteria were added to phototrophically growing cultures of the alga, and chlorophylla concentration per cell in these cultures decreased over a 24-hour period. Chlorophyll concentrations per cell began to increase when bacteria were grazed to a density of approximately 10(6) ml(-1), but after more than 24 hours they had not returned to the higher chlorophyll concentrations observed in the phototrophically grown cultures. Bacterivory was the dominant mode of nutrition in all cultures containing heat-killed bacteria. Photosynthesis did not contribute more than ≈7% of the total carbon budget of the alga when in the presence of abundant heat-killed bacteria. Bacterial density was the primary factor influencing the ability ofP. malhamensis to feed phagotrophically, while light intensity, pH, and the presence of dissolved organic matter had no effect on phagotrophy. We conclude thatP. malhamensis is capable of phagotrophy at all times. In contrast, phototrophy is inducible in the light during starvation and is a long-term survival strategy for this mixotrophic alga (i.e., it operates on time scales greater than a diel cycle).

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates.

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged ≈ 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPN∶DC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.

Grazing of attached bacteria by heterotrophic microflagellates.

Four species of heterotrophic microflagellates were examined for their ability to graze attached and unattached bacteria. The species tested displayed pronounced differences in their ability to graze the bacteriumPseudomonas halodurans attached to chitin particles. Two species of microflagellates (Monas andCryptobia sp.) efficiently grazed unattached bacteria but showed little or no ability to graze attached or aggregated cells. In contrast,Rhynchomonas nasuta andBodo sp. showed marked preferences for attached and aggregated bacteria and a limited ability to graze unattached cells. The density of attached bacteria was reduced by an order of magnitude due to grazing byBodo andR. nasuta, even though the density of unattached bacteria was ∼5-90× the density of attached cells. The maximum densities attained by microflagellates in the cultures were related to the density of unattached bacteria forMonas andCryptobia but not forBodo andR. nasuta. Growth of the latter two species appeared to be related to the density of attached or aggregated bacteria. Based on the results of these experiments, it is concluded that the pelagic existence of microflagellates that graze attached bacteria may be strongly linked to the distribution of suspended particles and their associated bacteria. In addition, the removal of attached bacteria by microflagellates can significantly affect the density of bacteria attached to particles in the plankton. This activity may have important implications for the controversy concerning the relative importance of attached and free-living bacteria in the plankton.

Effect of temperature on growth, respiration, and nutrient regeneration by an omnivorous microflagellate.

The effect of temperature on the rates and extent of carbon and nitrogen cycling by the heterotrophic microflagellate Paraphysomonas imperforata (diameter, 7 to 12 mum) fed with the diatom Phaeodactylum tricornutum was investigated over an ecologically pertinent temperature range (14 to 26 degrees C). All physiological rates investigated increased with increasing temperature. Q(10) values were similar for all rate changes and were comparable to those which have been reported for other protozoa. In contrast to all rates, microflagellate gross growth efficiency and cell volume were unaffected by temperature. Decreases in the concentrations of particulate carbon and particulate nitrogen from grazed diatom cultures also were similar when summed over the entire growth phase of the microflagellate population. Therefore, the proportions of ingested carbon and nitrogen which were incorporated or remineralized by the microflagellate were independent of temperature between 14 and 26 degrees C. At temperatures above 18 degrees C, growth rates of P. imperforata were greater than the maximum growth rates reported for most phytoplankton. We conclude that the impact of P. imperforata on natural phytoplankton communities is not controlled by temperature above 18 degrees C but may be affected by the rate at which zooplankton or microzooplankton prey on the microflagellate, as well as the inability of the microflagellate to graze efficiently when phytoplankton are present at low cell densities.

Technique for enumeration of heterotrophic and phototrophic nanoplankton, using epifluorescence microscopy, and comparison with other procedures.

A new method is described that uses the fluorochrome primulin and epifluorescence microscopy for the enumeration of heterotrophic and phototrophic nanoplankton (2 to 20 mum). Phototrophic microorganisms are distinguished from heterotrophs by the red autofluorescence of chlorophyll a. Separate filter sets are used which allow visualization of the primulin-stained nanoplankton without masking chlorophyll a fluorescence, thus allowing easy recognition of phototrophic cells. Comparison with existing epifluorescence techniques for counting heterotrophic and phototrophic nanoplankton shows that primulin provides more accurate counts of these populations than the fluorescein isothiocyanate or proflavine techniques. Accuracy is comparable to that with the acridine orange technique, but this method requires only one filter preparation for the enumeration of both phototrophic and heterotrophic populations.

Heterotrophic bacteria and bacterivorous protozoa in oceanic macroaggregates.

Oceanic macroaggregates (marine snow and Rhizosolenia mats) sampled from the Sargasso Sea are associated with bacterial and protozoan populations up to four orders of magnitude greater than those present in samples from the surrounding water. Filamentous, curved, and spiral bacteria constituted a higher proportion of the bacteria associated with the particles than were found among bacteria in the surrounding water. Protozoan populations were dominated numerically by heterotrophic microflagellates, but ciliates and amoebas were also observed. Macroaggregates are highly enriched heterotrophic microenvironments in the oceans and may be significant for the cycling of particulate organic matter in planktonic food chains.

Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant.

The Pseudomonas aeruginosa mutant Z61 has been shown to be highly supersusceptible to a wide range of antibiotics, including beta-lactams, aminoglycosides, rifampin, tetracycline, and chloramphenicol (W. Zimmerman, Int. J. Clin. Pharmacol. Biopharm. 17:131-134, 1979). Spontaneous revertants were isolated, using gentamicin or carbenicillin as selective agents, and shown to have two patterns of susceptibility to a group of 12 antibiotics. Partial revertants had 2- to 10-fold greater resistance to these antibiotics than mutant Z61, whereas full revertants had antibiotic susceptibilities indistinguishable from those of the wild-type strain K799, from which mutant Z61 had been derived. Uptake of a chromogenic beta-lactam nitrocefin was studied in both uninduced and induced cells of all strains by measuring the steady-state rate of nitrocefin hydrolysis by the inducible, periplasmic beta-lactamase in both whole and broken cells. This demonstrated that outer membrane permeability decreased as antibiotic resistance increased in the series mutant Z61, partial revertants, wild type, and full revertants. The data were consistent with the idea of low outer membrane permeability being caused by a low proportion of open functional porins in the outer membrane as the reason for the high natural antibiotic resistance of wild-type P, aeruginosa strains. In addition, it was observed that levels of benzylpenicillin below the minimal inhibitory concentration for mutant Z61 failed to induce beta-lactamase production. The possibility that this was related to the observed increase in outer membrane permeability is discussed. Preliminary evidence is presented that the pore-forming outer membrane porin protein F is not altered in mutant Z61.

Disruption of the primary fouling sequence on fiber glass-reinforced plastic submerged in the marine environment.

Fiber glass-reinforced plastic immersed in an experimental estuarine mesocosm fouled at estimated rates of 0.5, 5.5, and 18.8 ng (wet weight) mm day over days 0 to 2, 2 to 6, and 6 to 14, respectively. Protists, dominated by diatoms, which developed between days 3 and 6 and covered 90% of the undisturbed surface in 2 weeks, were effectively removed by twice-weekly brushing of the surface to maintain an immature 3-day bacterial film which covered 12% or less of the surface and had a biomass 3 orders of magnitude smaller than surfaces with 2 weeks' unrestricted fouling. Direct brushing of the fiber glass-reinforced plastic tank walls of experimental estuarine mesocosms minimized the "wall effect" by keeping a surface that maintained a low biomass of a slowly accumulating bacterial film rather than a surface which supported the more rapid accumulation of protists which in turn may induce the settlement of invertebrates and macrophytes.