Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 µm × 19.2 µm × 4.2 µm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 µm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.

From nutritional wealth to autophagy: In vivo metabolic dynamics in the cytosol, mitochondrion and shuttles of IgG producing CHO cells.

To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, 13C tracer studies and non-stationary flux calculations revealed that Pyr/H+ symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO42- antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions.

Bacterial Surface Traits Influence Digestion by Tetrahymena pyriformis and Alter Opportunity to Escape from Food Vacuoles.

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide allows any substance carrying amino- or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm.

Small but Manifold - Hidden Diversity in "Spumella-like Flagellates".

Colourless, nonscaled chrysophytes comprise morphologically similar or even indistinguishable flagellates which are important bacterivors in water and soil crucial for ecosystem functioning. However, phylogenetic analyses indicate a multiple origin of such colourless, nonscaled flagellate lineages. These flagellates are often referred to as "Spumella-like flagellates" in ecological and biogeographic studies. Although this denomination reflects an assumed polyphyly, it obscures the phylogenetic and taxonomic diversity of this important flagellate group and, thus, hinders progress in lineage- and taxon-specific ecological surveys. The smallest representatives of colourless chrysophytes have been addressed in very few taxonomic studies although they are among the dominant flagellates in field communities. To overcome the blurred picture and set the field for further investigation in biogeography and ecology of the organisms in question, we studied a set of strains of specifically small, colourless, nonscaled chrysomonad flagellates by means of electron microscopy and molecular analyses. They were isolated by a filtration-acclimatisation approach focusing on flagellates of around 5 µm. We present the phylogenetic position of eight different lineages on both the ordinal and the generic level. Accordingly, we describe the new genera Apoikiospumella, Chromulinospumella, Segregatospumella, Cornospumella and Acrispumella Boenigk et Grossmann n. g. and different species within them.

Sequential healing of the elevated sinus floor after applying autologous bone grafting: an experimental study in minipigs.

AIM: To describe the sequential healing after elevation of the maxillary sinus mucosa applying the lateral access technique with the use of autogenous bone grafting without membrane to occlude the osteotomy access. MATERIAL AND METHODS: Immediately after the elevation of the maxillary sinus Schneiderian membrane, applying the lateral access technique in 10 minipigs, autologous bone was harvested from the lateral aspect of the mandibular molar region and ground into particles with a bone mill. The space under the Schneiderian membrane was filled with this graft. No membranes were placed onto the access osteotomy. The healing was evaluated after 15, 30, 90 and 180 days. Paraffin sections were prepared and analyzed histologically. RESULTS: After 15 days of healing, the elevated area was mainly filled with provisional matrix, newly formed bone and some remnants of bone chips, and appeared reduced in volume compared with that at the time of surgery. After 30 days of healing, further shrinkage of the height of the elevated space was found, with similar percentages of the different tissue components. After 90 and 180 days, the area underneath the Schneiderian membrane appeared reduced in volume and condensed toward the base of the sinus. The bone tissues appeared to be more mature, both for the mineralized and the non-mineralized portions, while connective tissue occupied 20% of the space, most likely related to the lack of the use of a membrane occluding the access at the time of surgery. CONCLUSIONS: Suboptimal healing outcomes with respect to augmentation of the space under the sinus floor membrane were documented when autologous bone chips were used as a filler and no membrane was applied to cover the access.

Healing at implant sites prepared conventionally or by means of Sonosurgery ®. An experimental study in dogs.

OBJECTIVE: To compare peri-implant tissue healing at implants installed in sites prepared with conventional drills or a sonic device. MATERIAL AND METHODS: In six Beagle dogs, the mandibular premolars and first molars were extracted bilaterally. After 3 months, full-thickness muco-periosteal flaps were elevated and recipient sites were prepared in both sides of the mandible. In the right side (control), the osteotomies were prepared using conventional drills, while, at the left side (test), a sonic device (Sonosurgery(®)) was used. Two implants were installed in each side of the mandible. After 8 weeks of non-submerged healing, biopsies were harvested and ground sections prepared for histological evaluation. RESULTS: The time consumed for the osteotomies at the test was more than double compared to the conventional control sites. No statistically significant differences were found for any of the histological variables evaluated for hard and soft tissue dimensions. Although not statistically significant, slightly higher mineralized bone-to-implant contact was found at the test (65.4%) compared to the control (58.1) sites. CONCLUSIONS: Similar healing characteristics in osseointegration and marginal hard tissue remodeling resulted at implants installed into osteotomies prepared with conventional drills or with the sonic instrument (Sonosurgery(®)).

Sub-crestal positioning of implants results in higher bony crest resorption: an experimental study in dogs.

OBJECTIVE: To compare peri-implant soft- and hard-tissue integration at implants installed juxta- or sub-crestally. Furthermore, differences in the hard and soft peri-implant tissue dimensions at sites prepared with drills or sonic instruments were to be evaluated. MATERIAL AND METHODS: Three months after tooth extraction in six dogs, recipient sites were prepared in both sides of the mandible using conventional drills or a sonic device (Sonosurgery(®)). Two implants with a 1.7-mm high-polished neck were installed, one with the rough/smooth surface interface placed at the level of the buccal bony crest (control) and the second placed 1.3 mm deeper (test). After 8 weeks of non-submerged healing, biopsies were harvested and ground sections prepared for histological evaluation. RESULTS: The buccal distances between the abutment/fixture junction (AF) and the most coronal level of osseointegration (B) were 1.6 ± 0.6 and 2.4 ± 0.4 mm; between AF and the top of the bony crest (C), they were 1.4 ± 0.4 and 2.2 ± 0.2 mm at the test and control sites, respectively. The top of the peri-implant mucosa (PM) was located more coronally at the test (1.2 ± 0.6 mm) compared to the control sites (0.6 ± 0.5 mm). However, when the original position of the bony crest was taken into account, a higher bone loss and a more apical position of the peri-implant mucosa resulted at the test sites. CONCLUSIONS: The placement of implants into a sub-crestal location resulted in a higher vertical buccal bone resorption and a more apical position of the peri-implant mucosa in relation to the level of the bony crest at implant installation. Moreover, peri-implant hard-tissue dimensions were similar at sites prepared with either drills or Sonosurgery(®).

Sequential healing of open extraction sockets. An experimental study in monkeys.

AIM: To describe the sequential healing of open extraction sockets at which no attempts to obtain a primary closure of the coronal access to the alveolus have been made. MATERIAL AND METHODS: The third mandibular premolar was extracted bilaterally in 12 monkeys, and no sutures were applied to close the wound. The healing after 4, 10, 20, 30, 90 and 180 days was morphometrically studied. RESULTS: After 4 days of healing, a blood clot mainly occupied the extraction sockets, with the presence of an inflammatory cells' infiltrate. A void was confined in the central zones of the coronal and middle regions, in continuity with the entrance of the alveoli. At 10 days, the alveolus was occupied by a provisional matrix, with new bone formation lining the socket bony walls. At 20 days, the amount of woven bone was sensibly increasing. At 30 days, the alveolar socket was mainly occupied by mineralized immature bone at different stages of healing. At 90 and 180 days, the amount of mineralized bone decreased and substituted by trabecular bone and bone marrow. Bundle bone decreased from 95.5% at 4 days to 7.6% at 180 days, of the whole length of the inner alveolar surface. CONCLUSIONS: Modeling processes start from the lateral and apical walls of the alveolus, leading to the closure of the socket with newly formed bone within a month from extraction. Remodeling processes will follow the previous stages, resulting in trabecular and bone marrow formation and in a corticalization of the socket access.

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea).

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.

Revised systematics of Holospora-like bacteria and characterization of "Candidatus Gortzia infectiva", a novel macronuclear symbiont of Paramecium jenningsi.

The genus Holospora (Rickettsiales) includes highly infectious nuclear symbionts of the ciliate Paramecium with unique morphology and life cycle. To date, nine species have been described, but a molecular characterization is lacking for most of them. In this study, we have characterized a novel Holospora-like bacterium (HLB) living in the macronuclei of a Paramecium jenningsi population. This bacterium was morphologically and ultrastructurally investigated in detail, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and developed a specific probe for fluorescence in situ hybridization experiments. A new taxon, "Candidatus Gortzia infectiva", was established for this HLB according to its unique characteristics and the relatively low DNA sequence similarities shared with other bacteria. The phylogeny of the order Rickettsiales based on 16S rRNA gene sequences has been inferred, adding to the available data the sequence of the novel bacterium and those of two Holospora species (Holospora obtusa and Holospora undulata) characterized for the purpose. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and showed a possible pattern of evolution for some of their features. We suggested to classify inside the family Holosporaceae only HLBs, excluding other more distantly related and phenotypically different Paramecium endosymbionts.

Influence of buccal bony crest width on marginal dimensions of peri-implant hard and soft tissues after implant installation. An experimental study in dogs.

AIM: To evaluate the influence of the width of the buccal bony wall on hard and soft tissue dimensions following implant installation. MATERIAL AND METHODS: Mandibular premolars and first molars of six Labrador dogs were extracted bilaterally. After 3 months of healing, two recipient sites, one on each side of the mandible, were prepared in such a way as to obtain a buccal bony ridge width of about 2 mm in the right (control) and 1 mm in the left sides (test), respectively. Implants were installed with the coronal margin flush with the buccal alveolar bony crest. Abutments were placed and the flaps were sutured to allow a non-submerged healing. After 3 months, the animals were euthanized and ground sections obtained. RESULTS: All implants were completely osseointegrated. In respect to the coronal rough margin of the implant, the most coronal bone-to-implant contact was apically located 1.04 ± 0.91 and 0.94 ± 0.87 mm at the test and control sites, respectively, whereas the top of the bony crest was located 0.30 ± 0.40 mm at the test and 0.57 ± 0.49 mm at the control sites. No statistically significant differences were found. A larger horizontal bone resorption, however, evaluated 1 mm apically to the rough margin, was found at the control (1.1 ± 0.7 mm) compared to the test (0.3 ± 0.3 mm) sites, the difference being statistically significant. A thin peri-implant mucosa (2.4-2.6 mm) was found at implant installation while, after 3 months of healing, a biological width of 3.90-4.40 mm was observed with no statistically significant differences between control and test sites. CONCLUSIONS: A width of the buccal bony wall of 1or 2 mm at implant sites yielded similar results after 3 months of healing in relation of hard tissue and soft tissues dimensions after implant installation.

Ridge preservation at implants installed immediately after molar extraction. An experimental study in the dog.

AIM: To evaluate the influence of deproteinized bovine bone mineral (DBBM), in conjunction with a collagen membrane, on bone resorption at implants installed in a lingual position immediately into extraction sockets with horizontal residual buccal defects >2.0 mm. MATERIAL & METHODS: The pulp tissue of the mesial roots of (1) M(1) was removed in six Labrador dogs, and the root canals were filled with gutta-percha and cement. Flaps were elevated. The molars were hemi-sectioned and the distal roots removed. Implants were installed in a lingual position and with the shoulder flush with the buccal bony crest. After installation, defects of about 2.5 and 2.7 mm in width resulted at the buccal aspects of the test and control sites, respectively. Only in the left site (test), deproteinized bovine bone mineral (DBBM) particles were placed into the defect concomitantly with the placement of a collagen membrane. On the control sites, no biomaterials were applied. A non-submerged healing was allowed. RESULTS: After 3 months of healing, one control implant was not integrated and was excluded from the analysis, together with the contralateral test implant. All remaining implants were integrated into mature bone. The buccal alveolar bony crest was resorbed more at the test compared with the control sites, 2.2 ± 0.9 mm and 1.5 ± 1.3 mm, respectively. The vertical resorption of the lingual plate was 1.6 ± 1.5 mm and 1.5 ± 1.1 mm at the test and control sites, respectively. Only small residual DBBM particles were found at the test sites (1.4%). CONCLUSION: The use of DBBM particles to fill buccal defects of ≥2.5 mm at implants installed immediately into alveolar extraction sockets did not preserve the buccal bony wall.

Intracellular bioaccumulation of the rare earth element Gadolinium in ciliate cells resulting in biogenic particle formation and excretion.

Ciliates are abundant unicellular organisms capable of resisting high concentrations of metal ions in the environment caused by various anthropogenic activities. Understanding the cellular pathways involved in resistance to and detoxification of elements is required to predict the impact of ciliates on environmental element cycles. Here, we investigated the so far unknown process of tolerance, cellular uptake and bioaccumulation of the emerging rare earth element gadolinium (Gd) in the common ciliate Tetrahymena pyriformis. Gd treatment results in the intracellular formation and excretion of biogenic Gd-containing particles. This cellular process effectively removes dissolved Gd from the organic growth medium by 53.37% within 72 h. Based on light and electron microscopic observations, we postulate a detoxification pathway: Cells take up toxic Gd3+ ions from the medium by endocytosis, process them into stable Gd-containing particles within food vacuoles, and exocytose them. Stable biogenic particles can be isolated, which are relatively homogeneous and have a diameter of about 3 µm. They consist of the elements Gd, C, O, P, Na, Mg, K, and Ca. These findings broaden the view of metal ion accumulation by protists and are of relevance to understand environmental elemental cycles and may inspire approaches for metal recovery or bioremediation.

Use of a titanium device in lateral sinus floor elevation: an experimental study in monkeys.

AIM: To evaluate the effect of a space-maintaining device fixed to the lateral wall of the maxillary sinus after the elevation of the sinus mucosa on bone filling of the sinus cavity. MATERIAL AND METHODS: Immediately after the elevation of the maxillary sinus Schneiderian membrane accomplished through lateral antrostomy in four monkeys, a titanium device was affixed to the lateral sinus wall protruding into the sinus cavity to maintain the mucosa elevated without the use of grafting material. The healing of the tissue around the implants was evaluated after 3 and 6 months. Ground sections were prepared and analyzed histologically. RESULTS: The void under the elevated sinus membrane, originally filled with the blood clot, was reduced after 3 as well as after 6 months of healing of about 56% and 40.5%, respectively. In seven out of eight cases, the devices had perforated the sinus mucosa. The formation of mineralized bone and bone marrow amounted to about 42% and 69% after 3 and 6 months, respectively. The connective tissue represented about 53% and 23% of the newly formed tissue after 3 and 6 months, respectively. CONCLUSIONS: New bone formation was found below the devices. However, shrinkage of the newly formed tissue was observed both after 3 and 6 months of healing. Hence, the space-maintaining function of the devices used in the present study has to be questioned.

Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi.

Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as "Candidatus F. noatunensis subsp. endociliophora" subsp. nov.

Influence of various implant platform configurations on peri-implant tissue dimensions: an experimental study in dog.

AIM: To evaluate the influence (i) of various implant platform configurations and (ii) of implant surface characteristics on peri-implant tissue dimensions in a dog model. MATERIAL AND METHODS: Mandibular premolars and first molars were extracted bilaterally in six Labrador dogs. After 3 months of healing, two implants, one with a turned and a second with a moderately rough surface, were installed on each side of the mandible in the premolar region. On the right side of the mandible, implants with a tapered and enlarged platform were used, while standard cylindrical implants were installed in the left side of the mandible. Abutments with the diameter of the cylindrical implants were used resulting in a mismatch of 0.25 mm at the tapered implant sites. The flaps were sutured to allow a non-submerged healing. After 4 months, the animals were sacrificed and ground sections were obtained for histometric assessment. RESULTS: All implants were completely osseointegrated. A minimal buccal bone resorption was observed for both implant configurations and surface topographies. Considering the animals as the statistical unit, no significant differences were found at the buccal aspect in relation to bone levels and soft tissue dimensions. The surface topographies did not influence the outcomes either. CONCLUSIONS: The present study failed to show differences in peri-implant tissue dimensions when a mismatch of 0.25 mm from a tapered platform to an abutment was applied. The surface topographies influence a neither marginal bone resorption or peri-implant soft tissue dimension.

Exploring flow-biofilm-sediment interactions: Assessment of current status and future challenges.

Biofilm activities and their interactions with physical, chemical and biological processes are of great importance for a variety of ecosystem functions, impacting hydrogeomorphology, water quality and aquatic ecosystem health. Effective management of water bodies requires advancing our understanding of how flow influences biofilm-bound sediment and ecosystem processes and vice-versa. However, research on this triangle of flow-biofilm-sediment is still at its infancy. In this Review, we summarize the current state of the art and methodological approaches in the flow-biofilm-sediment research with an emphasis on biostabilization and fine sediment dynamics mainly in the benthic zone of lotic and lentic environments. Example studies of this three-way interaction across a range of spatial scales from cell (nm - µm) to patch scale (mm - dm) are highlighted in view of the urgent need for interdisciplinary approaches. As a contribution to the review, we combine a literature survey with results of a pilot experiment that was conducted in the framework of a joint workshop to explore the feasibility of asking interdisciplinary questions. Further, within this workshop various observation and measuring approaches were tested and the quality of the achieved results was evaluated individually and in combination. Accordingly, the paper concludes by highlighting the following research challenges to be considered within the forthcoming years in the triangle of flow-biofilm-sediment: i) Establish a collaborative work among hydraulic and sedimentation engineers as well as ecologists to study mutual goals with appropriate methods. Perform realistic experimental studies to test hypotheses on flow-biofilm-sediment interactions as well as structural and mechanical characteristics of the bed. ii) Consider spatially varying characteristics of flow at the sediment-water interface. Utilize combinations of microsensors and non-intrusive optical methods, such as particle image velocimetry and laser scanner to elucidate the mechanism behind biofilm growth as well as mass and momentum flux exchanges between biofilm and water. Use molecular approaches (DNA, pigments, staining, microscopy) for sophisticated community analyses. Link varying flow regimes to microbial communities (and processes) and fine sediment properties to explore the role of key microbial players and functions in enhancing sediment stability (biostabilization). iii) Link laboratory-scale observations to larger scales relevant for management of water bodies. Conduct field experiments to better understand the complex effects of variable flow and sediment regimes on biostabilization. Employ scalable and informative observation techniques (e.g., hyperspectral imaging, particle tracking) that can support predictions on the functional aspects, such as metabolic activity, bed stability, nutrient fluxes under variable regimes of flow-biofilm-sediment.

Distinguishing Activities in the Photodynamic Arsenals of the Pigmented Ciliates Blepharisma sinuosum Sawaya, 1940 and Blepharisma japonicum Suzuki, 1954 (Ciliophora: Heterotrichea).

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50  ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

Complex life cycle, broad host range and adaptation strategy of the intranuclear Paramecium symbiont Preeria caryophila comb. nov.

Holospora and related bacteria are a group of obligate Paramecium symbionts. Characteristic features are their infectivity, the presence of two distinct morphotypes, and usually a strict specialization for a single Paramecium species as host and for a nuclear compartment (either somatic or generative nucleus) for reproduction. Holospora caryophila steps out of line, naturally occurring in Paramecium biaurelia and Paramecium caudatum. This study addresses the phylogenetic relationship among H. caryophila and other Holospora species based on 16S rRNA gene sequence comparison analyzing the type strain and seven new macronuclear symbionts. Key aspects of Holospora physiology such as infectivity, symbiosis establishment and host range were determined by comprehensive infection assays. Detailed morphological investigations and sequence-based phylogeny confirmed a high similarity between the type strain of H. caryophila and the novel strains. Surprisingly, they are only distantly related to other Holospora species suggesting that they belong to a new genus within the family Holosporaceae, here described as Preeria caryophila comb. nov. Adding to this phylogenetic distance, we also observed a much broader host range, comprising at least eleven Paramecium species. As these potential host species exhibit substantial differences in frequency of sexual processes, P. caryophila demonstrates which adaptations are crucial for macronuclear symbionts facing regular destruction of their habitat.

Discomorphella pedroeneasi sp. nov. (Ciliophora, Odontostomatida): An anaerobic ciliate hosting multiple cytoplasmic and macronuclear endocytobionts.

Odontostomatids are among the less studied representatives of the Ciliophora. They are anaerobic microeukaryotes usually occurring as rare species in sapropelic environments. Here we describe a novel species of Discomorphella, named Discomorphella pedroeneasi sp. nov., using light and electron microscopy observations. Discomorphella pedroeneasi displays many complex morphological features, for which new terms are introduced, such as the auricules, epistomial fringe spacer, frontal awning, odontostomatid ciliary sockets, oral lips and ventral flap. Remarkably, D. pedroeneasi has at least six types of endocytobionts, including intramacronuclear ones, and organelles ultrastructurally similar to the M/H bodies of the odontostomatid Saprodinium dentatum. Based on the new data and revision of the literature, we propose two new diagnostic characters for species separation within Discomorphella: the fringe spacer ratio and the posterior fringe ratio. The taxonomy of Discomorphella is revised and an identification key is provided.