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.

Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.

The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

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.

Carbon dioxide exchange between air and seawater: no evidence for rate catalysis.

It has been suggested that enzymatic catalysis plays a major role in regulating the mass transport of carbon dioxide from the atmosphere into the oceans. Evidence for this mechanism was not found in a series of gas exchange experiments in which the gas transfer rate coefficients for samples obtained from various natural seawaters, with and without the addition of carbonic anhydrase, were compared with those from artificial seawater. Wind-induced turbulence appears to be the major factor controlling the ocean's response to anthropogenic increases in atmospheric carbon dioxide.

Effect of nitrogen-mediated changes in alkalinity on pH control and CO(2) supply in intensive microalgal cultures.

The freshwater alga Scenedesmus obliquus was grown in continuous culture at a fixed dilution rate of 0.5/day, but at varying pH in the range 4.17-10.67. The pH was regulated in the range 4.17-7.67 by continuously bubbling 1% CO(2)-enriched air into the cultures and by varying the source of nitrogen (NO(3) (-), NH(4) (+), or urea) in the growth medium, which, in turn, led to changes in culture alkalinity. Culture alkalinity and P(CO(2) ) were the sole determinants of pH. A pH-stat system, together with NO(3) (-) in the medium, was used to regulate the pH in the range 7.92-10.67. Maximum productivity, which occurred at pH 6.6, was dependent on N source only to the extent that culture alkalinity was a function of nitrogen uptake. The results demonstrate that the choice of N is a critical factor in controlling the pH of large-scale algal cultures. NH(4) (+) is a poor source of N because it leads to destruction of culture alkalinity and concomitant growth-inhibiting reductions in pH, whereas NO(3) (-) has an opposite effect, although pH is not so severely affected in this case. Urea is, by far, the most suitable N source for maximizing algal yield when it is supplied in combination with the proper amounts of HCO(3) (-) alkalinity in the growth medium and percent CO(2) in the bubbled gas that will lead to an equilibrium pH near the optimum pH.