Study of the behavior of Euglena viridis, Euglena gracilis and Lepadella patella cultured in all-glass microaquarium.

In the paper, the microaquarium fabricated in a form of entirely glass lab-on-a-chip for culturing and microscale study of microorganisms has been presented. A new approach towards cellular studies that brings a significant improvement over commonly utilized - polymer-based solutions has been shown. For the first time, all-borosilicate glass chip was applied for the culturing of the selected microorganisms and enabled notable population growth and behaviorism investigation. The chip fabrication method in comparison to typical glass chip technology was notably simplified, including quick patterning and low temperature bonding in 80 °C. In the studies, both a single-cell (Euglena gracilis and Euglena viridis) and multi-cell microorganisms (Lepadella patella) were cultured in the microaquarium. Behaviorism of the selected microorganisms was investigated by supplying various proportions of carbon dioxide, nitrogen and air into the chip. Tests included studies of microorganisms chemotaxis, viability (mostly based on photosynthesis process) and coexistence in the lab-on-a-chip environment. The experiments confirmed that the developed chip is a tool that fits the requirements for the culturing and behavioral studies of microorganisms and constitute ground-works to propel its further application in broadly defined cellular study field.

Higher rates of sex evolve in spatially heterogeneous environments.

The evolution and maintenance of sexual reproduction has puzzled biologists for decades. Although this field is rich in hypotheses, experimental evidence is scarce. Some important experiments have demonstrated differences in evolutionary rates between sexual and asexual populations; other experiments have documented evolutionary changes in phenomena related to genetic mixing, such as recombination and selfing. However, direct experiments of the evolution of sex within populations are extremely rare (but see ref. 12). Here we use the rotifer, Brachionus calyciflorus, which is capable of both sexual and asexual reproduction, to test recent theory predicting that there is more opportunity for sex to evolve in spatially heterogeneous environments. Replicated experimental populations of rotifers were maintained in homogeneous environments, composed of either high- or low-quality food habitats, or in heterogeneous environments that consisted of a mix of the two habitats. For populations maintained in either type of homogeneous environment, the rate of sex evolves rapidly towards zero. In contrast, higher rates of sex evolve in populations experiencing spatially heterogeneous environments. The data indicate that the higher level of sex observed under heterogeneity is not due to sex being less costly or selection against sex being less efficient; rather sex is sufficiently advantageous in heterogeneous environments to overwhelm its inherent costs. Counter to some alternative theories for the evolution of sex, there is no evidence that genetic drift plays any part in the evolution of sex in these populations.

Quantitative phase microscopy: automated background leveling techniques and smart temporal phase unwrapping.

In order for time-dynamic quantitative phase microscopy to yield meaningful data to scientists, raw phase measurements must be converted to sequential time series that are consistently phase unwrapped with minimal residual background shape. Beyond the initial phase unwrapping, additional steps must be taken to convert the phase to time-meaningful data sequences. This consists of two major operations both outlined in this paper and shown to operate robustly on biological datasets. An automated background leveling procedure is introduced that consistently removes background shape and minimizes mean background phase value fluctuations. By creating a background phase value that is stable over time, the phase values of features of interest can be examined as a function of time to draw biologically meaningful conclusions. Residual differences between sequential frames of data can be present due to inconsistent phase unwrapping, causing localized regions to have phase values at similar object locations inconsistently changed by large values between frames, not corresponding to physical changes in the sample being observed. This is overcome by introducing a new method, referred to as smart temporal unwrapping that temporally unwraps and filters the phase data such that small motion between frames is accounted for and phase data are unwrapped consistently between frames. The combination of these methods results in the creation of phase data that is stable over time by minimizing errors introduced within the processing of the raw data.

Comparative transcriptome analysis of obligately asexual and cyclically sexual rotifers reveals genes with putative functions in sexual reproduction, dormancy, and asexual egg production.

BACKGROUND: Sexual reproduction is a widely studied biological process because it is critically important to the genetics, evolution, and ecology of eukaryotes. Despite decades of study on this topic, no comprehensive explanation has been accepted that explains the evolutionary forces underlying its prevalence and persistence in nature. Monogonont rotifers offer a useful system for experimental studies relating to the evolution of sexual reproduction due to their rapid reproductive rate and close relationship to the putatively ancient asexual bdelloid rotifers. However, little is known about the molecular underpinnings of sex in any rotifer species. RESULTS: We generated mRNA-seq libraries for obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of the monogonont rotifer, Brachionus calyciflorus, to identify genes specific to both modes of reproduction. Our differential expression analysis identified receptors with putative roles in signaling pathways responsible for the transition from asexual to sexual reproduction. Differential expression of a specific copy of the duplicated cell cycle regulatory gene CDC20 and specific copies of histone H2A suggest that such duplications may underlie the phenotypic plasticity required for reproductive mode switch in monogononts. We further identified differential expression of genes involved in the formation of resting eggs, a process linked exclusively to sex in this species. Finally, we identified transcripts from the bdelloid rotifer Adineta ricciae that have significant sequence similarity to genes with higher expression in CP strains of B. calyciflorus. CONCLUSIONS: Our analysis of global gene expression differences between facultatively sexual and exclusively asexual populations of B. calyciflorus provides insights into the molecular nature of sexual reproduction in rotifers. Furthermore, our results offer insight into the evolution of obligate asexuality in bdelloid rotifers and provide indicators important for the use of monogononts as a model system for investigating the evolution of sexual reproduction.

Trafficking of bdelloid rotifer late embryogenesis abundant proteins.

The bdelloid rotifer Adineta ricciae is an asexual microinvertebrate that can survive desiccation by entering an ametabolic state known as anhydrobiosis. Two late embryogenesis abundant (LEA) proteins, ArLEA1A and ArLEA1B, have been hypothesized to contribute to desiccation tolerance in these organisms, since in vitro assays suggest that ArLEA1A and ArLEA1B stabilize desiccation-sensitive proteins and membranes, respectively. To examine their functions in vivo, it is important to analyse the cellular distribution of the bdelloid LEA proteins. Bioinformatics predicted their translocation into the endoplasmic reticulum (ER) via an N-terminal ER translocation signal and persistence in the same compartment via a variant C-terminal retention signal sequence ATEL. We assessed the localization of LEA proteins in bdelloids and in a mammalian cell model. The function of the N-terminal sequence of ArLEA1A and ArLEA1B in mediating ER translocation was verified, but our data showed that, unlike classical ER-retention signals, ATEL allows progression from the ER to the Golgi and limited secretion of the proteins into the extracellular medium. These results suggest that the N-terminal ER translocation signal and C-terminal ATEL sequence act together to regulate the distribution of rotifer LEA proteins within intracellular vesicular compartments, as well as the extracellular space. We speculate that this mechanism allows a small number of LEA proteins to offer protection to a large number of desiccation-sensitive molecules and structures both inside and outside cells in the bdelloid rotifer.

Within-Population Genome Size Variation is Mediated by Multiple Genomic Elements That Segregate Independently during Meiosis.

Within-species variation in genome size has been documented in many animals and plants. Despite its importance for understanding eukaryotic genome diversity, there is only sparse knowledge about how individual-level processes mediate genome size variation in populations. Here, we study a natural population of the rotifer Brachionus asplanchnoidis whose members differ up to 1.9-fold in diploid genome size, but were still able to interbreed and produce viable offspring. We show that genome size is highly heritable and can be artificially selected up or down, but not below a certain basal diploid genome size for this species. Analyses of segregation patterns in haploid males reveal that large genomic elements (several megabases in size) provide the substrate of genome size variation. These elements, and their segregation patterns, explain the generation of new genome size variants, the short-term evolutionary potential of genome size change in populations, and some seemingly paradoxical patterns, like an increase in genome size variation among highly inbred lines. Our study suggests that a conceptual model involving only two variables, 1) a basal genome size of the population, and 2) a vector containing information on additional elements that may increase genome size in this population (size, number, and meiotic segregation behavior), can effectively address most scenarios of short-term evolutionary change of genome size in a population.

Volume and morphology changes of a bdelloid rotifer species (Macrotrachela quadricornifera) during anhydrobiosis.

Following a study on the changes occurring in a bdelloid species (Macrotrachela quadricornifera, Rotifera, Bdelloidea) when entering anhydrobiosis, we investigated the changes in morphology, including weight and volume during the transition from the active hydrated to the dormant anhydrobiotic state by scanning electron microscopy, confocal microscopy and light microscopy. We compared sizes and morphologies of hydrated extended, hydrated contracted and anhydrobiotic specimens. Bdelloid musculature is defined: longitudinal muscles are contracted in the hydrated contracted animal (head and foot are retracted inside the trunk), but appear loose in the anhydrobiotic animal. When anhydrobiotic, M. quadricornifera appears much smaller in size, with a volume reduction of about 60% of the hydrated volume, and its internal organization undergoes remarkable modifications. Internal body cavities, clearly distinguishable in the hydrated extended and contracted specimens, are no longer visible in the anhydrobiotic specimen. Concomitantly, M. quadricornifera loses more than 95% of its weight when anhydrobiotic; this is more than expected from the volume reduction data and could indicate the presence of space-filling molecular species in the dehydrated animal. We estimate that the majority of body mass loss and volume reduction can be ascribed to the water loss from the body cavity during desiccation.

The impact of cleaner processing on nutrient availability in the bleached kraft industry.

The pulp and paper industry has invested heavily over recent years in cleaner processing, to reduce losses and minimise its impact on the environment. Over the past fifteen years, a New Zealand integrated bleached kraft mill has undergone a comprehensive programme of upgrades to increase production, reduce water consumption and streamline its biological treatment process. Whilst the overall discharge of contaminants from the site decreased, the treatment system performance did not show a concurrent improvement as may have been expected. Reduced BOD removal, low dissolved oxygen levels, and poor solids settlability were symptomatic of phosphorus limitation in the aerated lagoon treatment system. The wastewater entering the system was found to be phosphorus limited at a BOD:P ratio of 100:0.2. Mono-ammonium-phosphate was supplemented, at approximately 30 kg P/d, to raise the phosphorus levels to a BOD:P ratio of 100:0.3. Treatment efficiencies improved very quickly after phosphorus dosage, with a 50% reduction in BOD and TSS discharge, a significant increase in dissolved oxygen levels, and improved BOD removal (85% to 93%). This case study demonstrates that whilst more closed operation can result in reduced discharge of organic loads, there may be negative impacts on the availability of nutrients for balanced biological growth.

Hidden defensive morphology in rotifers: benefits, costs, and fitness consequences.

To cope with predation, many prey species have developed inducible defenses in terms of morphology, behavior, and life history. Rotifers were the first model organisms used to evaluate the ecology and evolution of inducible defenses in aquatic ecosystems. Since the middle of last century, only visible morphological defenses, such as spine development, have been found and only in a few rotifer species. Given the development of ultrastructural defenses is taxonomically widespread in aquatic ecosystems, we hypothesize that rotifer prey, particularly small-sized species, can develop such inducible defenses. We evaluated morphological response of two common Brachionus herbivores (B. calyciflorus and B. angularis) to predatory rotifer Asplanchna brightwellii. Results confirmed existence of predator-induced ultrastructural defenses, which are expressed as increased lorica thickness and enhanced lorica hardness. Such inducible defenses are more evident and effective in the smaller sized B. angularis, leading to higher fitness of B. angularis in predator-prey interactions. As anticipated, development of defenses has inevitable fitness costs manifested as decreased reproduction or reduced sex investment. Our results not only extend understanding of inducible ultrastructural defense to other planktonic taxa that were previously observed only in cladocerans, but also verify effective mechanical protection of such hidden defensive morphology.

Morphological differentiation of Brachionus calyciflorus caused by predation and coal ash pollution.

Different rotifer stains exhibited remarkably morphological differences which could not be eliminated under laboratory conditions. In the present study, we hypothesized that predation pressure and pollution might be two forces driving morphological differentiation of rotifer. To test this hypothesis, rotifers (Brachionus calyciflorus) belonging to two sibling species were collected from three special lakes (with coal ash pollution, high predation pressure or neither) and cultured for more than three months to investigate their potential differentiation in morphology. Twelve morphological parameters were measured and compared among three lakes at four food density (Scenedesmus obliquus). The results showed that most of the tested morphological parameters changed in response to food level and differed among three habitats. Rotifers from the habitat with high predation pressure evolved stable long posterior lateral spine and relatively small body size. Rotifers collected from the polluted habitat was of smaller body size, compared with those from ordinary habitat. Bigger eggs were laid by rotifers from polluted area or lake with high predation pressure, enabling newborns more resistant to pollution or predation, and thus ensuring the survival rate of newborns. Finally, we concluded that both predation and pollution could affect the morphological differentiation and evolution of rotifers.

Interspecies Interactions Reverse the Hazard of Antibiotics Exposure: A Plankton Community Study on Responses to Ciprofloxacin hydrochloride.

The ecotoxicological effects of Ciprofloxacin hydrochloride (CIP) were tested on population densities of plankton assemblages consisting of two algae (Isochrysis galbana and Platymonas subcordiformis) and a rotifer (Brachionus plicatilis). The I. galbana showed a significant decrease in densities when concentrations of CIP were above 2.0 mg L-1 in single-species tests, while P. subcordiformis and B. plicatilis were stable in densities when CIP were less than10.0 mg L-1. The equilibrium densities of I. galbana in community test increased with CIP concentrations after falling to a trough at 5.0 mg L-1, showed a completely different pattern of P. subcordiformis which decreased with CIP concentrations after reaching a peak at 30.0 mg L-1. The observed beneficial effect was a result of interspecies interactions of trophic cascade that buffered for more severe direct effects of toxicants. The community test-based NOEC of CIP (2.0 mg L-1), embodying the indirect effects, was different from the extrapolated one derived by single-species tests (0.5 mg L-1), but all lacked confidence interval. A CIP threshold concentration of obvious relevance to ecological interaction was calculated with a simplified plankton ecological model, achieving a value of 1.26 mg L-1 with a 95% bootstrapping confidence interval from 1.18 to 1.31 mg L-1.

Spermatozoon of the freshwater rotifer Brachionus calyciflorus (Rotifera, Monogononta): Advances in morphological and ultrastructural studies.

The morphological and ultrastructural features of the spermatozoon in Brachionus calyciflorus are described using light, fluorescence and transmission electron microscopy (TEM). The mature spermatozoon, which appears to be thread-like, is composed of a slightly expanded anterior of cell body region and a flagellum region without acrosome. The cell body region and flagellum region are respectively 16-27µm and 20-33µm in length (n=60). The spermatozoon is characterized by a mass of dense tubular materials, which occupy most of the cell. Some mitochondria are distributed around the nuclear region in the anterior of the cell body region, while in the posterior portion of cell body, the chromatin often contains a single lobated nucleus arranged at the center of cell. The flagellum contains the classic axoneme (9×2+2) and possesses lateral undulating membrane. Mature B. calyciflorus males have no germ cell stages earlier than the spermatids in the testis. TEM examination reveals rigid rods as well as predominant typical spermatozoon in the testis. Observations, based on successive photographs and videos, enabled a first-time recording of the unique inverted movement of the spermatozoon, which indicated that the movement of the spermatozoon is driven by the flagellum. Our study also provides further supplementary insights into the phylogenetic systematics of the Rotifera.

The effect of dietary restriction on cell division potential, DNA content and enzyme levels in the rotifer Asplanchna brightwelli.

Dietary restriction in the rotifer Asplanchna brightwelli, carried out by lengthening the interval between feedings, causes an increase both in mean lifespan and in the length of the reproductive period. In the present study, we found that the rate of nuclear division in the gastric glands and vitellarium of the rotifer, as determined by daily nuclear counts, was retarded by dietary restriction. Spectro-fluorimetric measurements show that total DNA content remains constant from the beginning to the end of the lifespan, and is unaffected by dietary restriction. Lactic dehydrogenase and malic dehydrogenase activities were also measured throughout the lifespan of the rotifer, and were not affected by dietary restriction.

Cytological examination of Leeuwenhoek's first microbial specimens.

Of the nine packets of specimens sent by van Leeuwenhoek to London in the seventeenth century, and which were recently rediscovered in their original condition, three contained dried aquatic microorganisms. The author has reconstituted portions of this material. Several recognizable organisms have been noted, including portions of this material. Several recognizable organisms have been noted, including water-fleas, chlorophyte and cyanophyte algae, desmids, diatoms and rotifers. These provide evidence for the formation of the 'heavenly paper', which was believed at the time to represent charred notepaper dropped from the skies, but which Leeuwenhoek rightly assumed to be dried algal felts. Photomicrographs reveal many cytological details in the material, and this has been correlated with Leeuwenhoek's descriptions, extracted from the original correspondence.

Musculature of an illoricate predatory rotifer Asplanchnopus multiceps as revealed by phalloidin fluorescence and confocal microscopy.

The pattern of muscles in the actively swimming predatory rotifer Asplanchnopus multiceps is revealed by staining with tetramethyl-rhodamine isothiocyanate (TRITC)-labelled phalloidin and confocal scanning laser microscopy (CSLM). The major components of the musculature are: prominent semicircular muscles of the corona; paired lateral, dorsal and ventral retractors in the trunk; a network of six seemingly complete circular muscles and anastomosing longitudinal muscles in the trunk; two short foot retractors, originating from a transverse muscle in the lower third of the trunk. The sphincter of the corona marks the boundary between the head and the trunk. The muscular patterns in rotifers with different lifestyles differ clearly, therefore, the muscular patterns seem to be determined by the mode of locomotion and feeding behaviour.

Preservation, development, and hatching of resting eggs in the freshwater rotifer Brachionus calyciflorus pallas.

The resting eggs of Brachionus calyciflorus produced under laboratory by manipulating various environmental conditions were subjected to preservation for long-term storage. Preservation strategies included as storing freshly collected resting eggs, dry resting eggs, and storing at 4°C. Results showed that the resting eggs preserved by drying as they were able to maintain the viability without much variation in the hatchability. The scanning electron micrographs clearly depicted the surface damage as well as change in the shape and surface topography due to the storage. The resting eggs thus stored were traced for their normal embryonic development, during which as many as 11 stages were traced and time taken for each stage of development was observed. Microscopic observations revealed that the first four stages represented the development of embryo and thereafter around 16-18 h extrution of neonate from egg occurred. At 18-19 h of development the characteristic of anterior and posterior spines appeared in the neonate. Just released neonate, after 4-5 h of development reached adult stage. Overall the hatching and embryonic development of the resting eggs preserved by "drying" were normal and hence this method could be adopted for long-term storage.

Genomic clues to an ancient asexual scandal.

Despite abandoning meiosis, the bdelloid rotifers have persisted for millions of years and given rise to hundreds of species. Several mechanisms--allelic variants with different functions, high effective population size, and resistance to radiation--may contribute to their success.

Use of confocal linescan to document ciliary beat frequency.

We present a method to document ciliary beat frequency with the linescan function of a scanning confocal microscope, using ciliated tracheal cells and free-swimming rotifers as examples. Depending on the clarity of the original data, the ciliary beat frequency can be determined from the confocal linescan directly or from an intensity linescan analysis of the original data. Fast Fourier transform treatment of the data can be used to verify the derived ciliary beat frequency. The linescan approach allows analysis of simple ciliary movements displayed by the ciliated tracheal cells, as well as complex movements performed by free-swimming rotifers while feeding.

Characterization of an extremely motile cellular network in the rotifer Asplanchna spp. Structure, kinetics, and the cytoskeleton.

The pseudocoelomic body cavity of the rotifer Asplanchna spp. contains free cells that form a highly dynamic, three-dimensional polygonal network of filopodia. Using video-enhanced differential interference contrast microscopy, we have qualitatively and quantitatively characterized the motion types involved with network motility: (1) filopodial junctions are displaced laterally at 10.52 +/- 0.46 microns/s; (2) free-ending filopodia form and extend at rates of 8.77 +/- 0.40 microns/s, until they retract again at 7.23 +/- 0.87 microns/s; (3) filopodial strands fuse either laterally or tip to the lateral side. The combination of these motion types results in enlargements, diminutions, and extinctions of filopodial polygons, and in the formation of new polygons. Moreover, there is intense and fast (5.11 +/- 0.28 microns/s) particle transport within the filopodial strands. The organization of the cytoskeleton in filopodia was examined by electron microscopy and by labeling with fluorescent-tagged phalloidin. Filopodia contain several microtubules that are often organized in a bundle. Moreover, F-actin is present within the filopodia. To characterize which of these cytoskeletal systems is involved with cell and organelle motility, we have examined cell dynamics after incubations with colchicine or cytochalasin D. The results of these pharmacological experiments provide evidence that microtubules are required for both cell and organelle motility, but that actin filaments contribute to these phenomena and are required for the structural maintenance of slender filopodia.

Diatoms and rotifers in cytological smears.

We describe several uncommon contaminants presumably derived from the tap water used in the staining procedure of cytological specimens. We would like to draw attention to the occasional presence of diatoms and fragments of rotifers in cytological specimens. Whilst most of these entities are harmless curiosities, they may cause concern as to their nature and significance.