Cryopreservation of Ochromonas in Liquid Nitrogen with Reproducibility of Thiamin Assay

The first successful cryopreservation of Ochromonas danica and Ochromonas malhamensis is reported. The freezing method was consistently reproducible for the former, but not for the latter. Ochromonas danica cultures established from frozen material still could be used as test organisms for assay of thiamin. This is the first report of a protozoon retaining its assay property after being frozen to -196 C.     

Fine Structure of the Cell Surface and Golgi Apparatus of Ochromonas*

SYNOPSIS. Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340–360 nm long, and small projections, 50–110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthenium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.     

Detecting phenology change in the mayfly Ephemera danica: responses to spatial and temporal water temperature variations

1. Rising water temperatures under climate change are expected to affect the phenology of aquatic insects, including the mayfly Ephemera danica Müller which is widespread throughout Europe. 2. To assess temporal and spatial variability in mayfly emergence, E. danica were monitored at two thermally contrasting reaches in the River Dove, English Peak District over the period 2007–2013. Inter‐annual variations in growing degree days (GDDs) were modelled for an upstream site with intermittent spring flow supplementing main channel flow (Beresford Dale) and downstream site dominated by near constant discharges of cool groundwater (Dovedale). 3. A strong association exists between the emergence cycle of E. danica and GDDs at each site. Beresford Dale accumulated on average 374 more GDDs than Dovedale. After warm summers E. danica emerged after only 1 year in Beresford Dale but began to revert to a bi‐annual cycle after the particularly wet/cool year of 2012. In Dovedale, E. danica maintained a 2‐year cycle throughout the monitoring period in spite of the phenology changes observed 8 km upstream. 4. Data from the present study suggest that habitats near cool groundwater may provide important refugia for populations of insects, potentially delaying permanent shifts in phenology under climate change. However, an ability to detect changes in the thermal triggers and phenological response may be hindered by conventional spot sampling protocols.     

Particulate and Dissolved Organic Carbon Production by the Heterotrophic Nanoflagellate Pteridomonas danica Patterson and Fenchel

Abstract We established a budget of organic carbon utilization of a starved heterotrophic nanoflagellate, Pteridomonas danica, incubated in batch cultures with Escherichia coli as model prey. The cultures were sampled periodically for biomass determinations and total organic carbon dynamics: total organic carbon, total organic carbon <1 μm, and dissolved organic carbon (DOC, <0.2 μm). During the 22 h incubation period, P. danica underwent biovolume variations of 3.2-fold. Gross growth efficiency was 22% and net growth efficiency 40%. P. danica respired 33% and egested 44% of the ingested E. coli carbon during lag and exponential growth phases. The form of the organic carbon egested varied. Of the total ingested carbon, 9% was egested in the form of DOC and occurred mainly during the exponential growth phase; 35% was egested in the form of particulate organic carbon (POC), ranging in size from 0.2 to 1 μm, and took place during the lag phase. P. danica could have reingested as much of 58% of this previously produced POC during the exponential growth phase as food scarcity increased. We concluded that POC egestion by flagellates could represent a significant source of submicrometric particles and colloidal organic matter. In addition, flagellate reingestion of egested POC could play a nonnegligible role in the microbial food web. Finally, the methodology reported in this study has proved to be a useful tool in the study of carbon metabolism in aquatic microorganisms. Received: 31 July 1998; Accepted: 2 March 1999     

Cytogenetic studies in Cochlearia L. (Cruciferae). The chromosomal constitution of C. danica L.

Genome analysis has been used to investigate the relationship of C. danica L. to the diploid and tetraploid species in the genus. The results of this analysis suggest that C. danica has the genomic constitution A¹ ₇ A¹ ₇ A² ₇ A² ₇ T₇ T₇. Both A₇ genomes in C. danica are segmentally differentiated from the A₇ genome in C. groenlandica L. and from the similar A₆ genomes in C. pyrenaica DC. and C. officinalis L. but, in hybrids, are capable of some pairing with these genomes. A² ₇ is more distantly related to A₇ and A₆ than is A¹ ₇ and in A₇ some of the differentiation has taken place by reciprocal translocations. A¹ ₇ and A² ₇ still retain enough homology for pairing between them. The T₇ genome has no homology with any of the A genomes but may be derived from an ancestral T₆ genome.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

The management of metabolic energy storage during the life cycle of mayflies: a comparative field investigation of the collector-gatherer <Emphasis Type="Italic">Ephemera danica</Emphasis> and the scraper <Emphasis Type="Italic">Rhithrogena semicolorata</Emphasis>

The concentration and seasonal dynamics of the major energy storage components, triglycerides and glycogen, were measured in two species of mayfly (Rhithrogena semicolorata and Ephemera danica) with contrasting life cycle strategies living in a small mountain stream. E. danica is a burrowing, semivoltine collector-gatherer; R. semicolorata is univoltine and scrapes periphyton from stones. This is the first publication which focuses on the role of metabolic energy sources during the larval life span of two mayfly species until the larvae emerge. Although triglycerides are the major energy reserve in both species (>84% of total energy storage) throughout the whole larval development their seasonal dynamic differed considerably. In R. semicolorata the triglyceride concentration declined during the last weeks prior to emergence in both sexes. The same pattern was found in female larvae of E. danica, but not in male E. danica. It is suggested that females use triglycerides in the last larval stages for egg maturation, which is completed in the last larval instar. In male E. danica the triglyceride concentrations remained high until emergence, presumably due to their high energy demands as adults for their swarming flights. Glycogen concentrations did not show such a difference between species and sexes. Its significance as a storage substrate for energy is rather low; however, concentrations decreased in both species and sexes prior to emergence.     

A note on the fine structure of the Ochromonas danica “tail”

Summary The fine structure of the Ochromonas danica tail is detailed. The microtubules, microtubule anchoring structures, posterior swelling, surface vesicles, and in some instances cup shape of the tail end are described and used to explain the behavior of the organism when attached to surfaces.Aided by a grant GB 20825 from the National Science Foundation.     

A versatile laboratory stream with examples of its use in the investigation of invertebrate behaviour

A versatile and simple laboratory stream was designed and used to investigate the burrowing activity of two insects in response to changes in water velocity and substrate type.Aphelocheirus aestivalis adults were unable to burrow into sand, however, a small proportion of juveniles did burrow in this substrate. The presence of sand in gravel reduced the burrowing success of adults. Steady increases in flow stimulated the burrowing response of both adults and juveniles on gravel and sandy gravel.Ephemera danica was unable to burrow in sand alone at the velocities used in the experiment. The presence of particles greater than 2 mm in diameter in the substrate appeared to be essential for successful burrowing under the test conditions. An increase in flow from 3 to 8 cm s^–1 resulted in an increase in burrowing. The time taken for each specimen to burrow varied widely within replicate tests but most specimens had penetrated the substrate within 150 seconds of introduction. The implications of these observations in influencing the microdistribution of these species are discussed.     

Regulation of nitrous oxide emission associated with benthic invertebrates

1. A number of freshwater invertebrate species emit N₂O, a greenhouse gas that is produced in their gut by denitrifying bacteria (direct N₂O emission). Additionally, benthic invertebrate species may contribute to N₂O emission from sediments by stimulating denitrification because of their bioirrigation behaviour (indirect N₂O emission). 2. Two benthic invertebrate species were studied to determine (i) the dependence of direct N₂O emission on the preferred diet of the animals, (ii) the regulation of direct N₂O emission by seasonally changing factors, such as body size, temperature and availability and (iii) the quantitative relationship between direct and indirect N₂O emission. 3. Larvae of the mayfly Ephemera danica, which prefer a bacteria‐rich detritus diet, emitted N₂O at rates of up to 90 pmol Ind.^?1 h^?1 under in situ conditions and 550 pmol Ind.^?1 h^?1 under laboratory conditions. In contrast, larvae of the alderfly Sialis lutaria, which prefer a bacteria‐poor carnivorous diet, emitted N₂O at invariably low rates of 0–20 pmol Ind.^?1 h^?1. The N₂O emission rate of E. danica larvae was positively correlated with seasonally changing factors (body size, temperature and availability). Direct N₂O emission by E. danica larvae was limited by low temperature in winter, larval development in spring and low availability in summer. 4. Both E. danica and the non‐emitting S. lutaria increased the total N₂O and N₂ emission from sediment in a density‐dependent manner. While N₂O directly emitted by benthic invertebrates can be partially consumed in the sediment (E. danica), non‐emitting species can still indirectly contribute to total N₂O emission from sediment (S. lutaria).     

Research note: Analysis of expressed sequence tags from the chrysophycean alga Ochromonas danica (Heterokontophyta)

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 <10^?4) to sequences in a public database. The annotation of these ESTs was carried out to assess subcellular localization of the putative proteins using several internet‐accessible prediction programs for subcellular localization. These analyses revealed that putative plastid proteins in Ochromonas possess N‐terminal bipartite presequences with a conserved phenylalanine at the N‐terminus of the predicted transit peptide‐like domains, similar to other ‘red‐lineage’ secondary symbiotic organisms. The examination of sequences of 3′‐UTR revealed that, similarly to chlorophyte algae, UGUAA may represent a putative polyadenylation signal in O. danica.     

Comparative study on hydrolases in five species of Ochromonas (Chrysomonadina)

Acid phosphatase and β-glucosidase were shown to be present in five species of Ochromonas grown in organic media (O. danica, O. malhamensis, O. minuta, O. sociabilis and Ochromonas sp. 933/4). Acid phosphatase was found to have a pH optimum at 4.0 in O. danica, and at 5.1 in the four other species. No alkaline phosphatase was found in any of the above mentioned species. β-glucosidase in the species studied has a pH optimum at 4.6 Low α-glucosidase activity was found only in O. danica. Acid phosphatase in all the five species shows an increase in activity during the logarithmic phase of growth and a decrease during the early stationary phase. β-glucosidase shows a similar behavior only in O. danica.     

Dynamics and Nutritional Ecology of a Nanoflagellate Preying Upon Bacteria

Ingestion and growth rates of the nanoflagellate predator Ochromonas danica feeding on the bacterium Pseudomonas fluorescens were quantified in laboratory cultures. Bacterial prey were grown under four nutritional conditions with respect to macronutrient elements: C-limited, N-limited, P-limited, and balanced. Ingestion and growth rates were saturating functions of prey abundance when preying upon nutritionally balanced, C-limited, and P-limited bacteria but were unimodal functions of abundance when preying on N-limited bacteria. At saturating prey concentrations, the ingestion rate of C-limited prey was about twice that of prey in other nutritional states, while at subsaturating prey concentrations, the ingestion rates of both C- and N-limited prey were higher than those of prey in other nutritional states. Over all prey concentrations, growth was most rapid on balanced and C-limited prey and generally lowest for P-limited prey. Due to the unimodal response of growth rate to abundance of N-limited prey, growth rate on N-limited prey approached that obtained on balanced and C-limited prey when prey were available at intermediate abundances. The accumulation of recycled N increased with the growth rate of O. danica. Recycling of N was highest when O. danica was feeding upon P-limited prey. The accumulation of recycled P increased with growth rate for balanced and N-limited prey, but not for P-limited prey, which consistently had low accumulation of recycled P. The low growth rate and negligible recycling of P for O. danica preying on P-limited prey is consistent with the theory of ecological stoichiometry and resembles results found for crustacean zooplankton, especially in the genus Daphnia. Potentially, the major predators of bacterioplankton and a major predator of phytoplankton play analogous roles in the trophic dynamics and biogeochemistry of aquatic ecosystems.     

The costs and benefits of multicellular group formation in algae*

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.     

Disproving the hypothesis of a common ancestry for the Ochromonas danica chrysoplast and Heliobacterium chlorum

The phylogenetic position of the golden-yellow alga Ochromonas danica chrysoplast was investigated by comparison of the 16S rRNA catalogue and two long 16S rRNA stretches (804 and 454 bases) with catalogues from eubacteria and chloroplasts and with homologoes 16S rRNA regions from Escherichia coli, Bacillus subtilis, Heliobacterium chlorum, Anacystis nidulans and chloroplasts from Zea mays, Nicotiana tabacum, Euglena gracilis and Chlamydomonas reinhardii, respectively. Both approaches indicate a closer relationship of the chrysoplast to chloroplasts and cyanobacteria than to the brownish photoheterotrophic Heliobacterium chlorum for which a common ancestry has recently been hypothesized.     

Ultrastructural Observations on Aging of Stationary Cultures and Feeding in Ochromonas*

SYNOPSIS. Changes accompanying aging of stationary cultures of Ochromonas danica were examined with the electron microscope. The cultures included light- and dark-grown populations ranging in age from 3 days to 5 weeks. Cells from the youngest cultures contained minimal amounts of lipid and a distinct leucosin vacuole. After 1 week, the number of lipid globules in the cytoplasm increased. The amount of lipid increased progressively in cells from older cultures until the leucosin vacuole was obliterated by the coalescing spheres. Cells from cultures older than 3 weeks showed a general breakdown of cytoplasmic integrity. An area of pinocytotic activity was also present; a relationship between this anterior region and blebs arising from the cell membrane is suggested.     

The structure of some Middle Cambrian conodonts, and the early evolution of conodont structure and function

Elements presumed to belong to conodontophorids from the lower Middle Cambrian of Scania, Sweden, are investigated with emphasis on histology. The new genus Amphigeisina is described, with A. danica (V. Poulsen) as type species. The material also includes Hertzina? bisulcata Miiller and Proconodontus? sp. These forms, here termed proto-conodonts, appear to have grown by basal-internal accretion, unlike elements of the essentially post-Cambrian Conodontiformes, but partly similar to the Late Cambrian para-conodonts. A proposed model of the early evolution of conodonts involves the following points: (1) Conodonts were primarily external elements, secreted by an ectodermal epithelium. (2) An evolutionary trend during the Cambrian involved gradual retraction of the elements into pockets of the epithelium. (3) Elements of the Conodontiformes, herein termed euconodonts, were completely engulfed in epithelial pockets, which enabled them to grow holoperipherally and thus to assume more complex shapes that could be retained during growth. (4) The secreting epithelium adhered only to the basal body of the euconodont, the conodont proper being extruded from the pockets when the apparatus was in function.     

Licht- und elektronenmikroskopische Untersuchung des Corpus cardiacum der Eintagsfliege Ephemera danica Müll. (Ephemeroptera: Ephemeridae) während der Metamorphose*

Kaiser, H. 1980. Licht- und elektronenmikroskopische Untersuchung des Corpus cardiacum des Eintagsfliege Ephemera danica Müll. (Ephemeroptera: Ephemeridae) während der Metamorphose. [Light- and electron microscopic investigation on the corpus cardiacum in the mayfly Ephemera danica Müll. (Ephemeroptera: Ephemeridae) during metamorphosis.] (Zoologisches Institut der Universität Basel, Schweiz.) — Acta zool. (Stockh.) 61(2): 93–103. The corpus cardiacum (cc) of Ephemera danica is an unpaired organ in the dorsal wall of the aorta. During the last four instars there has been no significant change in the volume of the cc of the males. In the females, however, the cc enlarges in the final nymphal instar due to a thickening of the organ. Owing to a shortening prior to the subimaginal moult the volume has diminished in the subimagines and imagines. In the electron microscope different types of axons and secretory granules could be observed. The type a axons contain electron opaque granules with a mean diameter of 125 nm. The granules in the axons of type b are smaller (mean diameter 85 nm) and are more or less electron opaque, and the type c axons are filled with less electron opaque secretory droplets with mean diameters of 170 nm. In the fourth type of axons (d) the granules are approximately the same size as those of the type a but are more irregular in shape. In addition to the different axons two cell types—interstitial and secretory cells—are described, and the probable mechanism of the release of neurosecretory material is discussed. During metamorphosis only minor changes were apparent at the ultrastructural level.     

THE FINE STRUCTURE OF MITOSIS AND CELL DIVISION IN THE CHRYSOPHYCEAN ALGA OCHROMONAS DANICA1

At the ultrastructural level, cell division in Ochromonas danica exhibits several unusual features. During interphase, the basal bodies of the 2 flagella replicate and the chloroplast divides by constriction between its 2 lobes. The rhizoplast, which is a fibrous striated root attached to the basal body of the long flagellum, extends under the Golgi body to the surface of the nucleus in interphase cells. During proprophase, the Golgi body replicates, apparently by division, and a daughter rhizoplast, appears. During prophase, the 2 pairs of flagellar basal bodies, each with their accompanying rhizoplast and Golgi body, begin to separate. Three or 4 flagella are already present at this stage. At the same time, there is a proliferation of microtubules outside the nuclear envelope. Gaps then appear in the nuclear envelope, admitting the microtubules into the nucleus, where they form a spindle. A unique feature of mitosis in O. danica is that the 2 rhizoplasts form the poles of the spindle, spindle microtubules inserting directly onto the rhizoplasts. Some of the spindle microtubules extend from pole to pole; others appear to attach to the chromosomes. Kinetochores, however, are not present. The nuclear envelope breaks down, except, in the regions adjacent, to the chloroplasts; chloroplast ER remains intact throughout mitosis. At late anaphase the chromosomes come to lie against part of the chloroplast ER. This segment of the chloroplast ER appears to be incorporated as part of the reforming nuclear envelope, thus reestablishing the characteristic nuclear envelope—chloroplast ER association of the interphase cell.     

Element Stoichiometry of a Mixotrophic Protist Grown Under Varying Resource Conditions

ABSTRACT. The balance of essential elements (e.g. carbon [C], nitrogen [N], and phosphorus [P]) between consumers and their resources influences not only the growth and reproduction of the consumers but also the nutrients they regenerate. Flagellate protists are significant predators of aquatic bacteria and directly influence nutrient flow to higher trophic levels and, through excretion, influence the mineral element composition of dissolved nutrients. Because the element stoichiometry of protists is poorly characterized, we varied the resource composition of the bacterium Pseudomonas fluorescens and used it to grow the mixotrophic bacterivorous flagellate Ochromonas danica. Using a mass balance approach, the element composition of O. danica was found to vary depending upon the nutrient composition of the prey and ranged between 482:36:1 and 80:12:1 (C:N:P molar). Homeostasis plots suggested that flagellate protists weakly regulate their element composition and are likely to regenerate different elements depending upon the nature of the element limiting growth of their prey.