Light and electron microscopic observations of the reproductive swarmer cells of nassellarian and spumellarian polycystines (Radiolaria)

We observed reproductive swarmer cells of the nassellarian and spumellarian polycystine radiolarians Didymocyrtis ceratospyris, Pterocanium praetextum, Tetrapyle sp., and Triastrum aurivillii using light, scanning and transmission electron microscopy. The swarmer cells had subspherical to ovoid or spindle shapes with two unequal flagella tapered to whip-like ends. The cell size was approximately 2.5–5.5 μm long and 1.6–2.2 μm wide, which is significantly smaller than that of the collodarian (colonial or naked) polycystine radiolarians. Transmission electron microscopy revealed that the swarmer cells possessed a nucleus, mitochondria with tubular cristae, Golgi body, and small lipid droplets in the cytoplasm; they also had a large vacuole in which a single crystalline inclusion (approx. 1.0–1.5 μm) that was probably celestite (SrSO₄) was enclosed. The swarmer cells were released directly from the parent cells. At that time, morphological change such as encystment was not observed in the parent cells, and the axopodia remained extended in a period of swarmer reproduction for floating existence. This may have prevented the polycystine swarmers from rapidly sinking down to great depths. Thus, we concluded that the polycystine radiolarians release the swarmer cells into the photic layer in the same way as the symbiotic acantharians.     

A cell length‐dependent transition in MinD‐dynamics promotes a switch in division‐site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length‐dependent; short swarmers divide at mid‐cell, while long swarmers switch to a specific non‐mid‐cell placement of the division site. Transition to non‐mid‐cell positioning of the Z‐ring is promoted by a cell length‐dependent switch in the localization‐dynamics of the division regulator MinD from a pole‐to‐pole oscillation in short swarmers to a multi‐node standing‐wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z‐ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division‐events to one per cell at a specific non‐mid‐cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Phylogenetic affinities of an enigmatic nannoplankton,Braarudosphaera bigelowii based on the SSU rDNA sequences

The phylogenetic position of an enigmatic calcareous nannoplankton Braarudosphaera bigelowii, whose taxonomic position has long been questioned, was investigated using molecular genetic methods. The SSU rDNA sequences of B. bigelowii were obtained using a single cell PCR technique independently from two single cells isolated from field samples. A BLAST search revealed that the SSU rDNA of B. bigelowii was most similar to those of the division Haptophyta. Subsequent phylogenetic analyses showed that B. bigelowii was included in a clade comprising members of the orders Isochrysidales and Coccolithales, the genus Chrysoculter and two unidentified haptophytes. Morphometric measurements of extant B. bigelowii populations were made to obtain precise information on their intraspecific size variation. The results showed that these populations could be subdivided into a small form (< 2.4 μm), an intermediate form A (4.0–5.3 μm), and an intermediate form B (5.3–7.2 μm), although the borders of the latter two forms overlapped. The SSU rDNA sequences showed the presence of 10-bp substitutions plus six insertions/deletions between specimens of intermediate forms A and B. The levels of DNA variation between them suggested that these two forms are genetically distinct from each other.     

The Density of a Homogeneous Population of Cells Controls Resetting of the Program for Swarmer Formation in the Unicellular Marine MicroorganismNoctiluca scintillans

Noctiluca scintillansis a luminescent marine dinoflagellate. The life cycle ofNoctilucaconsists of a vegetative stage and a swarmer stage. The swarmer stage ofNoctilucais initiated by formation of a swarmer-mother cell instead of binary fission of vegetative cells. We studied the formation of swarmers under various conditions and became convinced that the cells have a strict program for the formation of swarmers which starts to operate in every cell after a defined number of cell cleavages. The probability that the program will be executed appeared to be affected by the presence of other cells. In other words, a high density of cells suppressed the expression of the program. Suppression was achieved by resetting the mechanism and was related to the number of cell divisions. Our findings provide one of the simplest examples of a mechanism by which a large population produces individuality in a group of genetically homogeneous organisms.     

Adhesion ofEnteromorpha swarmers to microbial films

Laboratory experiments were conducted to determine the effect of bacterial films on adhesion ofEnteromorpha sp. reproductive swarmer cells. Swarmers always attached in greater numbers to filmed than to unfilmed polystyrene surfaces. Surface energy measurements produced higher values on filmed surfaces than on unfilmed surfaces. Our data indicate that this higher surface energy may contribute to the increased adhesion by the algal swarmers.     

Expanded host and geographic range of tadpole associations with the Severe Perkinsea Infection group

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

Molecular,biochemical and morphological data suggest an affiliation of Spongiochrysis hawaiiensis with the Trentepohliales (Ulvophyceae,Chlorophyta)

The aeroterrestrial, unicellular green alga Spongiochrysis hawaiiensis had been included in the ulvophycean order Cladophorales based on small subunit (SSU) rDNA sequence data, and represents so far the only fully terrestrial member of this order. Other characteristics of S. hawaiiensis that are atypical for Cladophorales include the presence of large amounts of carotenoids and a budding‐like mode of cell division. As the position of this terrestrial, unicellular alga in an order of aquatic, multicellular green algae is unusual, we re‐evaluated the phylogenetic relationships of this enigmatic organism based on supplementary SSU rDNA sequences as well as novel large ribosomal subunit (LSU) rDNA and internal transcribed spacer (ITS rDNA) sequences. Additionally, we examined several morphological characters of S. hawaiiensis, as well as low molecular weight carbohydrate (LMWC) patterns of S. hawaiiensis and members of the Cladophorales and Trentepohliales as potential chemotaxonomic markers. We found S. hawaiiensis to be uninucleate. The analysis of the LMWC content detected the presence of the polyol erythritol in S. hawaiiensis and in the Trentepohliales, while this compound was missing in the Cladophorales. The phylogenetic analyses of the novel sequences placed S. hawaiiensis in the terrestrial Trentepohliales. This placement is supported by the aeroterrestrial habitat, the presence of large amounts of carotenoids, the uninucleate cells, and the presence of the polyol erythritol as a protective compound against water loss.     

Molecular diversity of alveolates associated with neritic north atlantic radiolarians

Seven species of polycystine radiolarians and one phaeodarian species were investigated in order to determine the diversity of their associate organisms and their species specificity. Twelve partial 18S ribosomal DNA (rDNA) sequences were obtained showing a high diversity of associates, both within spumellarian and nassellarian radiolarians and among species. Two of the sequences obtained are highly similar to Scrippsiella, a dinoflagellate genus already reported as a symbiont of polycystine radiolarians. Nine of the new 18S rDNA sequences group with various alveolates. Some of these groups include parasites, such as the lethal endoparasite Amoebophrya, while others consist of non-annotated novel organisms found worldwide in various types of marine environments. We also obtained a sequence from a bacillariophytan highly similar to the 18S rDNA of the diatom species Diatoma tenue, which may derive from radiolarian food. Additionally, this is the first study to report on a phaeodarian associate.     

Phylogenetic affinity of Mycochaetophora gentianae, the causal fungus of brown leaf spot on gentian (Gentiana triflora), to Pseudocercosporella-like hyphomycetes in Helotiales

Mycochaetophora gentianae, the causal agent of brown leaf spot on gentian (Gentiana scabra), is characterized by its hyaline besom-like sporophore, although its conidiogenesis and phylogenetic position have so far remained unknown. We isolated the causal fungus from a new host, G. triflora, in Iwate, Japan. Both the G. triflora isolate and the ex-type M. gentianae isolate produced symptoms on G. triflora but not on G. scabra. Microscopic observations of the diseased leaves indicated that conidiogenesis was blastic from short conidiophores, and schizolytic secession of conidia left unthickened and inconspicuous conidial scars on the conidiogenous cells. Conidia were catenate, in branched acropetalous chains; secondary conidia were blastically produced from the first or second cell at the base of primary conidium. The G. triflora isolate was identified as M. gentianae because of its identity to the ex-type in characteristics of culture, pathogenicity, and conidia. Phylogenetic analyses using three ribosomal DNA (rDNA) sequences combined [small subunit (SSU) + large subunit (LSU) + 5.8S rDNA] indicated that both isolates clustered with Rhexocercosporidium carotae, and the cluster was placed within Helotiales–Rhytismatales. Additional analyses using internal transcribed spacers including 5.8S rDNA sequences revealed that both isolates were monophyletic and that they were closely related to three helotialean Pseudocercosporella-like hyphomycetous genera: Helgardia, Rhexocercosporidium and Rhynchosporium.     

New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes

Abstract:Primers for amplification and sequencing of partial glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were designed for lichenized fungi. The 5′ gpd primer is most probably fungal specific, since a BLAST search in GenBank found identical sequences only from ascomycetous taxa, whereas the 3′ gpd primer was more universal. Utility of the gpd primers and previously designed beta-tubulin primers was tested in nine lichen taxa. Both the gpd and beta-tubulin primer pairs amplified in most of the taxa examined: the gpd primers generated a c. 1100 nucleotide fragment, whereas the PCR product obtained from the beta-tubulin primers was c. 900 nucleotides long. The gpd amplification products of Cladonia arbuscula and C. rangiferina were sequenced and both were found to contain three introns, the length of which varied between 49 to 83 nucleotides. To examine the applicability of gpd sequences in resolving relationships within Ascomycota, trees were calculated from 22 fungal gpd sequences obtained from GenBank together with the twoCladonia sequences using parsimony jackknifing. The gpd tree was compared with the SSU rDNA tree of the respective species (or genera). A similar analysis of the beta-tubulin gene was not performed, because only a few beta-tublin sequences from the same taxa were available in GenBank. The gpd tree was well resolved but in conflict with the SSU rDNA tree. In contrast to the SSU rDNA tree, the gpd tree did not support the monophyly of the Ascomycota. Analysis of the combined data set produced a tree very similar to that of the SSU rDNA data. However, the relationship of Lecanorales to the other orders remained unresolved. Even though gpd and beta-tubulin are highly conserved proteins, the third codon positions and introns are variable and both genes have the potential for inferring phylogenetic relationships at the lower taxonomic levels in the lichenized fungi. The two genes may be useful even below species level, depending on the species investigated.     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.     

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov,Nassonova et Cavalier-Smith, 2008

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.     

Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn

A two-stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary-otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto-fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.