Lead and cadmium exposure alters shell morphogenesis in a single-chambered, agglutinated foraminiferan protist.

Changes to calcareous foraminiferal shell morphology are well documented in heavy metal-polluted marine environments, however less is known about how these toxicants affect agglutinated foraminifera, particularly single-chambered (monothalamid) species. Here we used an agglutinated monothalamic foraminifer, Astrammina rara, to study shell morphogenesis during exposure to lead (Pb) and cadmium (Cd). Isolated cell bodies incubated with artificial sediment and control, Pb-, or Cd-spiked artificial seawater constructed new agglutinated shells in four weeks. Time-lapse recordings showed normal motile behavior during treatments. SEM imaging of reconstructed shells, however, revealed dramatic deformations in the bioadhesive of Pb-exposed shells, and less so in Cd-exposed shells. ICP-MS analysis of the isolated shells showed elevated levels of Pb, but not Cd, in the bioadhesive of treated specimens, indicating that the two metals exert their effects differentially. These findings show that certain agglutinated foraminifera may be useful indicator species in studies of heavy metal-polluted benthic marine environments.

Three-dimensional architecture of epithelial primary cilia.

We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron tomography. Our data demonstrate the architecture of primary cilia differs extensively from the commonly acknowledged 9+0 paradigm. The axoneme structure is relatively stable but gradually evolves from base to tip with a decreasing number of microtubule complexes (MtCs) and a reducing diameter. The axonemal MtCs are cross-linked by previously unrecognized fibrous protein networks. Such an architecture explains why primary cilia can elastically withstand liquid flow for mechanosensing. The nine axonemal MtCs in a cilium are found to differ significantly in length indicating intraflagellar transport processes in primary cilia may be more complicated than that reported for motile cilia. The 3D maps of microtubule doublet-singlet transitions generally display longitudinal gaps at the inner junction between the A- and B-tubules, which indicates the inner junction protein is a major player in doublet-singlet transitions. In addition, vesicles releasing from kidney primary cilia were observed in the structural maps, supporting that ciliary vesicles budding may serve as ectosomes for cell-cell communication.

Putative sponge biomarkers in unicellular Rhizaria question an early rise of animals.

The dawn of animals remains one of the most mysterious milestones in the evolution of life. The fossil lipids 24-isopropylcholestane and 26-methylstigmastane are considered diagnostic for demosponges-arguably the oldest group of living animals. The widespread occurrence and high relative abundance of these biomarkers in Ediacaran sediments from 635-541 million years (Myr) ago have been viewed as evidence for the rise of animals to ecological importance approximately 100 Myr before their rapid Cambrian radiation. Here we show that the biosynthesis of 24-isopropylcholestane and 26-methylstigmastane precursors is common among early-branching unicellular Rhizaria-heterotrophic protists that play an important role in trophic cycling and carbon export in the modern ocean. Negating these hydrocarbons as sponge biomarkers, our study places the oldest evidence for animals closer to the Cambrian Explosion. Cambrian silica hexactine spicules that are approximately 535 Myr old now represent the oldest diagnostic sponge remains, whereas approximately 558-Myr-old Dickinsonia and Kimberella (Ediacara biota) provide the most reliable evidence for the emergence of animals. The proliferation of predatory protists may have been responsible for much of the ecological changes during the late Neoproterozoic, including the rise of algae, the establishment of complex trophic relationships and the oxygenation of shallow-water habitats required for the subsequent ascent of macroscopic animals.

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata.

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences (KY468817-KY468826) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).

Sinks and Sources of Intracellular Nitrate in Gromiids.

A substantial nitrate pool is stored within living cells in various benthic marine environments. The fate of this bioavailable nitrogen differs according to the organisms managing the intracellular nitrate (ICN). While some light has been shed on the nitrate carried by diatoms and foraminiferans, no study has so far followed the nitrate kept by gromiids. Gromiids are large protists and their ICN concentration can exceed 1000x the ambient nitrate concentration. In the present study we investigated gromiids from diverse habitats and showed that they contained nitrate at concentrations ranging from 1 to 370 mM. We used 15N tracer techniques to investigate the source of this ICN, and found that it originated both from active nitrate uptake from the environment and from intracellular production, most likely through bacterial nitrification. Microsensor measurements showed that part of the ICN was denitrified to N2 when gromiids were exposed to anoxia. Denitrification seemed to be mediated by endobiotic bacteria because antibiotics inhibited denitrification activity. The active uptake of nitrate suggests that ICN plays a role in gromiid physiology and is not merely a consequence of the gromiid hosting a diverse bacterial community. Measurements of aerobic respiration rates and modeling of oxygen consumption by individual gromiid cells suggested that gromiids may occasionally turn anoxic by their own respiration activity and thus need strategies for coping with this self-inflicted anoxia.

Maintenance of electrostatic stabilization in altered tubulin lateral contacts may facilitate formation of helical filaments in foraminifera.

Microtubules in foraminiferan protists (forams) can convert into helical filament structures, in which longitudinal intraprotofilament interactions between tubulin heterodimers are thought to be lost, while lateral contacts across protofilaments are still maintained. The coarse geometric features of helical filaments are known through low-resolution negative stain electron microscopy (EM). In this study, geometric restraints derived from these experimental data were used to generate an average atomic-scale helical filament model, which anticipated a modest reorientation in the lateral tubulin heterodimer interface. Restrained molecular dynamics (MD) simulations of the nearest neighbor interactions combined with a Genalized Born implicit solvent model were used to assess the lateral, longitudinal, and seam contacts in 13-3 microtubules and the reoriented lateral contacts in the helical filament model. This electrostatic analysis suggests that the change in the lateral interface in the helical filament does not greatly diminish the lateral electrostatic interaction. After longitudinal dissociation, the 13-3 seam interaction is much weaker than the reoriented lateral interface in the helical filament model, providing a plausible atomic-detail explanation for seam-to-lateral contact transition that enables the transition to a helical filament structure.

Population Dynamics and Parasite Load of a Foraminifer on Its Antarctic Scallop Host with Their Carbonate Biomass Contributions.

We studied the population dynamics and parasite load of the foraminifer Cibicides antarcticus on its host the Antarctic scallop Adamussium colbecki from three localities differing by sea ice cover within western McMurdo Sound, Ross Sea, Antarctica: Explorers Cove, Bay of Sails and Herbertson Glacier. We also estimated CaCO3 biomass and annual production for both species. Cibicides populations varied by locality, valve type, and depth. Explorers Cove with multiannual sea ice had larger populations than the two annual sea ice localities, likely related to differences in nutrients. Populations were higher on Adamussium top valves, a surface that is elevated above the sediment. Depth did not affect Cibicides distributions except at Bay of Sails. Cibicides parasite load (the number of complete boreholes in Adamussium valves) varied by locality between 2% and 50%. For most localities the parasite load was < 20%, contrary to a previous report that ~50% of Cibicides were parasitic. The highest and lowest parasite load occurred at annual sea ice localities, suggesting that sea ice condition is not important. Rather, the number of adults that are parasitic could account for these differences. Cibicides bioerosion traces were categorized into four ontogenetic stages, ranging from newly attached recruits to parasitic adults. These traces provide an excellent proxy for population structure, revealing that Explorers Cove had a younger population than Bay of Sails. Both species are important producers of CaCO3. Cibicides CaCO3 biomass averaged 47-73 kg ha(-1) and Adamussium averaged 4987-6806 kg ha(-1) by locality. Annual production rates were much higher. Moreover, Cibicides represents 1.0-2.3% of the total host-parasite CaCO3 biomass. Despite living in the coldest waters on Earth, these species can contribute a substantial amount of CaCO3 to the Ross Sea and need to be incorporated into food webs, ecosystem models, and carbonate budgets for Antarctica.

Molecular evidence for ß-tubulin neofunctionalization in Retaria (Foraminifera and radiolarians).

Foraminifera and radiolarians are closely related amoeboid protists (i.e., retarians) often characterized by their shells and pseudopodia. Previous studies hypothesized that the unusual "Type 2" ß-tubulin (ß2) is critically involved in forming helical filaments (HFs), a unique microtubule (MT) assembly/disassembly intermediate found in foraminiferan reticulopodia. Such noncanonical ß-tubulin sequences have also been found in two radiolarian species and appear to be closely related to the foraminiferan ß2. In this study, we report 119 new ß-tubulin transcript sequences from six foraminiferans, four radiolarians, and a related non-retarian species. We found that foraminiferan and radiolarian ß2-tubulins share some of the unusual substitutions in the structurally essential and usually conserved domains. In the ß-tubulin phylogeny, retarian ß2-tubulin forms a monophyletic clade, well separated from the canonical ß-tubulin (ß1) ubiquitous in eukaryotes. Furthermore, we found that foraminiferan and radiolarian ß2-tubulin lineages were under positive selection, and used homology models for foraminiferan α- and ß-tubulin hexamers to understand the structural effect of the positively selected substitutions. We suggest that the positively selected substitutions play key roles in the transition of MT to HF by altering the lateral and longitudinal interactions between α- and ß-tubulin heterodimers. Our results indicate that the unusual ß2-tubulin is a molecular synapomorphy of retarians, and the ß-tubulin gene duplication occurred before the divergence of Foraminifera and radiolarians. The duplicates have likely been subjected to neofunctionalization responsible for the unique MT to HF assembly/disassembly dynamics, and/or other unknown physiological processes in retarian protists.

Magnetococcus marinus gen. nov., sp. nov., a marine, magnetotactic bacterium that represents a novel lineage (Magnetococcaceae fam. nov., Magnetococcales ord. nov.) at the base of the Alphaproteobacteria.

Magnetotactic bacteria are a morphologically, metabolically and phylogenetically disparate array of bacteria united by the ability to biomineralize membrane-encased, single-magnetic-domain mineral crystals (magnetosomes) that cause the cell to orientate along the Earth's geomagnetic field. The most commonly observed type of magnetotactic bacteria is the ubiquitous magnetotactic cocci, which comprise their own phylogenetic group. Strain MC-1(T), a member of this group, was isolated from water collected from the oxic-anoxic interface of the Pettaquamscutt Estuary in Rhode Island, USA, and cultivated in axenic culture. Cells of strain MC-1(T) are roughly spherical, with two sheathed bundles of flagella at a single pole (bilophotrichous). Strain MC-1(T) uses polar magnetotaxis, and has a single chain of magnetite crystals per cell. Cells grow chemolithoautotrophically with thiosulfate or sulfide as the electron donors, and chemo-organoheterotrophically on acetate. During autotrophic growth, strain MC-1(T) relies on the reductive tricarboxylic acid cycle for CO2 fixation. The DNA G+C content is 54.2 mol%. The new genus and species Magnetococcus marinus gen. nov., sp. nov. are proposed to accommodate strain MC-1(T) ( = ATCC BAA-1437(T)  = JCM 17883(T)), which is nominated as the type strain of Magnetococcus marinus. A new order (Magnetococcales ord. nov.) and family (Magnetococcaceae fam. nov.) are proposed for the reception of Magnetococcus and related magnetotactic cocci, which are provisionally included in the Alphaproteobacteria as the most basal known lineage of this class.

The revised classification of eukaryotes.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information.

High-throughput sequencing of Astrammina rara: sampling the giant genome of a giant foraminiferan protist.

BACKGROUND: Foraminiferan protists, which are significant players in most marine ecosystems, are also genetic innovators, harboring unique modifications to proteins that make up the basic eukaryotic cell machinery. Despite their ecological and evolutionary importance, foraminiferan genomes are poorly understood due to the extreme sequence divergence of many genes and the difficulty of obtaining pure samples: exogenous DNA from ingested food or ecto/endo symbionts often vastly exceed the amount of "native" DNA, and foraminiferans cannot be cultured axenically. Few foraminiferal genes have been sequenced from genomic material, although partial sequences of coding regions have been determined by EST studies and mass spectroscopy. The lack of genomic data has impeded evolutionary and cell-biology studies and has also hindered our ability to test ecological hypotheses using genetic tools. RESULTS: 454 sequence analysis was performed on a library derived from whole genome amplification of microdissected nuclei of the Antarctic foraminiferan Astrammina rara. Xenogenomic sequence, which was shown not to be of eukaryotic origin, represented only 12% of the sample. The first foraminiferal examples of important classes of genes, such as tRNA genes, are reported, and we present evidence that sequences of mitochondrial origin have been translocated to the nucleus. The recovery of a 3' UTR and downstream sequence from an actin gene suggests that foraminiferal mRNA processing may have some unusual features. Finally, the presence of a co-purified bacterial genome in the library also permitted the first calculation of the size of a foraminiferal genome by molecular methods, and statistical analysis of sequence from different genomic sources indicates that low-complexity tracts of the genome may be endoreplicated in some stages of the foraminiferal life cycle. CONCLUSIONS: These data provide the first window into genomic organization and genetic control in these organisms, and also complement and expands upon information about foraminiferal genes based on EST projects. The genomic data obtained are informative for environmental and cell-biological studies, and will also be useful for efforts to understand relationships between foraminiferans and other protists.

Extracellular fibrils of pathogenic yeast Cryptococcus gattii are important for ecological niche, murine virulence and human neutrophil interactions.

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40-100 nm diameter x500-3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12alpha mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN- mediated killing.

An ectobiont-bearing foraminiferan, Bolivina pacifica, that inhabits microxic pore waters: cell-biological and paleoceanographic insights.

The presence of tests (shells) in foraminifera could be taken as an indicator that this protist taxon is unlikely to possess ectosymbionts. Here, however, we describe an association between Bolivina pacifica, a foraminiferan with a calcareous test, and a rod-shaped microbe (bacterium or archaeon) that is directly associated with the pores of the foraminiferan's test. In addition to these putative ectosymbionts, B. pacifica has previously undescribed cytoplasmic plasma membrane invaginations (PMIs). These adaptations (i.e. PMIs, ectobionts), along with the clustering of mitochondria under the pores and at the cell periphery, suggest active exchange between the host and ectobiont. The B. pacifica specimens examined were collected from sediments overlain by oxygen-depleted bottom waters (0.7 µM) of the Santa Barbara Basin (California, USA). An ultrastructural comparison between B. pacifica from the Santa Barbara Basin and a congener (Bolivina cf. B. lanceolata) collected from well-oxygenated sediments (Florida Keys) suggests that PMIs, ectobionts and peripherally distributed mitochondria are all factors that promote inhabitation of microxic environments by B. pacifica. The calcitic δ(13)C signatures of B. pacifica and of a co-occurring congener (B. argentea) that lacks ectobionts differ by > 1.5‰, raising the possibility that the presence of ectobionts can affect incorporation of paleoceanographic proxies.

Peroxisome proliferation in Foraminifera inhabiting the chemocline: an adaptation to reactive oxygen species exposure?

Certain foraminiferal species are abundant within the chemocline of marine sediments. Ultrastructurally, most of these species possess numerous peroxisomes complexed with the endoplasmic reticulum (ER); mitochondria are often interspersed among these complexes. In the Santa Barbara Basin, pore-water bathing Foraminifera and co-occurring sulfur-oxidizing microbial mats had micromolar levels of hydrogen peroxide (H(2)O(2)), a reactive oxygen species that can be detrimental to biological membranes. Experimental results indicate that adenosine triphosphate concentrations are significantly higher in Foraminifera incubated in 16 microM H(2)O(2) than in specimens incubated in the absence of H(2)O(2). New ultrastructural and experimental observations, together with published results, lead us to propose that foraminiferans can utilize oxygen derived from the breakdown of environmentally and metabolically produced H(2)O(2). Such a capability could explain foraminiferal adaptation to certain chemically inhospitable environments; it would also force us to reassess the role of protists in biogeochemistry, especially with respect to hydrogen and iron. The ecology of these protists also appears to be tightly linked to the sulfur cycle. Finally, given that some Foraminifera bearing peroxisome-ER complexes belong to evolutionarily basal groups, an early acquisition of the capability to use environmental H(2)O(2) could have facilitated diversification of foraminiferans during the Neoproterozoic.

Phylogeny and ultrastructure of Miliammina fusca: evidence for secondary loss of calcification in a Miliolid Foraminifer.

The classification of the Foraminifera, a widely distributed group of largely marine protists, has traditionally been based on morphological characters. The most important of these are the composition and structure of the shell or "test." Here, we use both phylogenetic analysis of the genes for small subunit rRNA and beta-tubulin and ultrastructural analysis to document a reversion in wall type from more derived calcareous tests to an agglutinated test. These data indicate that the genus Miliammina, and possibly other members of the Rzehakinidae, should be placed in the Order Miliolida as opposed to their current assignment in Order Textulariida. We also address the effects this reversion may have had on the ability of rzehakinacids to effectively colonize marginal marine environments. Finally, the hypothesis that some multilocular agglutinated foraminiferans descended from calcareous lineages has implications for interpretation of the foraminiferal fossil record.

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

The second species of Gromia (Protista) from the deep sea: its natural history and association with the Pakistan margin oxygen minimum zone.

We describe a gromiid protist Gromia pyriformis sp. nov., from bathyal depths on the Pakistan margin (NE Arabian Sea), an area characterised by a well-developed Oxygen Minimum Zone (OMZ). The new species is smaller (length usually <1 mm) than the only other described deep-sea gromiid species (Gromia sphaerica) or the well-known coastal species Gromia oviformis. Its identification as a gromiid is based on the test-wall ultrastructure. This includes (i) an outer wall (165-300 nm thick) limited by an electron-opaque layer and perforated by pore structures which typically extend through its entire thickness, and (ii) inner "honeycomb membrane" structures which form a discontinuous sheet (18-20 nm thick) lying parallel to the outer wall. An outermost glycocalyx (approximately 75 nm thick), not observed in other gromiid species, is also present and imparts a finely granular appearance to the outer test surface, as seen by Scanning Electron Microscopy (SEM). Numerous rod-shaped prokaryotes are attached to the exterior of the glycocalyx. Gromia pyriformis sp. nov. typically occurs above the sediment-water interface, attached to the large arborescent foraminiferan Pelosina sp. It is confined to a very narrow bathymetric zone (approximately 1000 m water depth) in the lower portion of the OMZ, where bottom-water oxygen concentrations are approximately 0.2 ml l(-1).