Protargol Synthesis: An In-House Protocol

The protargol staining method has proved to be indispensable for revealing the cellular structures of a variety of protozoa, especially the flagellates and ciliates. Protargol provides permanent stains of a variety of cellular structures: nuclei, extrusomes, basal bodies, and microfilamentous constituents of cells. Together with the older silver nitrate methods, protargol impregnations have provided the basis for the detailed descriptions of nearly all ciliates to date. The performance of commercially available preparations has varied widely. Recently, suppliers have stopped stocking the effective forms of protargol resulting in a worldwide shortage. Thus, it has become necessary for protistologists to explore on-site synthesis of this critically important agent. An optimum protocol for synthesis of protargol should be rapid, relatively inexpensive, simple enough to be done by non-chemists, and achievable without specialized equipment. In this article, the authors briefly review the interesting history of protargol and describe a protocol, based on the early studies of neuroanatomists, that yields a protargol producing impregnations of ciliates comparable to those obtained with previously available commercial preparations

Assessing Whether Alpha-Tubulin Sequences are Suitable for Phylogenetic Reconstruction of Ciliophora with Insights into Its Evolution in Euplotids

The current understanding of ciliate phylogeny is mainly based on analyses of a single gene, the small subunit ribosomal RNA (SSU-rDNA). However, phylogenetic trees based on single gene sequence are not reliable estimators of species trees, and SSU-rDNA genealogies are not useful for resolution of some branches within Ciliophora. Since congruence between multiple loci is the best tool to determine evolutionary history, we assessed the usefulness of alpha-tubulin gene, a protein-coding gene that is frequently sequenced, for ciliate phylogeny. Here, we generate alpha-tubulin gene sequences of 12 genera and 30 species within the order Euplotida, one of the most frequently encountered ciliate clades with numerous apparently cosmopolitan species, as well as four genera within its putative sister order Discocephalida. Analyses of the resulting data reveal that: 1) the alpha-tubulin gene is suitable phylogenetic marker for euplotids at the family level, since both nucleotide and amino acid phylogenies recover all monophyletic euplotid families as defined by both morphological criteria and SSU-rDNA trees; however, alpha-tubulin gene is not a good marker for defining species, order and subclass; 2) for seven out of nine euplotid species for which paralogs are detected, gene duplication appears recent as paralogs are monophyletic; 3) the order Euplotida is non-monophyletic, and the family Uronychiidae with sequences from four genera, is non-monophyletic; and 4) there is more genetic diversity within the family Euplotidae than is evident from dargyrome (geometrical pattern of dorsal silverline system in ciliates) patterns, habit and SSU-rDNA phylogeny, which indicates the urgent need for taxonomic revision in this area

Genome Structure Drives Patterns of Gene Family Evolution in Ciliates, a Case Study Using \u3ci\u3eChilodonella uncinata\u3c/i\u3e (Protista, Ciliophora, Phyllopharyngea)

In most lineages, diversity among gene family members results from gene duplication followed by sequence divergence. Because of the genome rearrangements during the development of somatic nuclei, gene family evolution in ciliates involves more complex processes. Previous work on the ciliate Chilodonella uncinata revealed that macronuclear β-tubulin gene family members are generated by alternative processing, in which germline regions are alternatively used in multiple macronuclear chromosomes. To further study genome evolution in this ciliate, we analyzed its transcriptome and found that (1) alternative processing is extensive among gene families; and (2) such gene families are likely to be C. uncinata specific. We characterized additional macronuclear and micronuclear copies of one candidate alternatively processed gene family-a protein kinase domain containing protein (PKc)-from two C. uncinata strains. Analysis of the PKc sequences reveals that (1) multiple PKc gene family members in the macronucleus share some identical regions flanked by divergent regions; and (2) the shared identical regions are processed from a single micronuclear chromosome. We discuss analogous processes in lineages across the eukaryotic tree of life to provide further insights on the impact of genome structure on gene family evolution in eukaryotes

Convergence rates of the modified forward reflected backward splitting algorithm in Banach spaces

Consider the problem of minimizing the sum of two convex functions, one being smooth and the other non-smooth in Banach space. In this paper, we introduce a non-traditional forward-backward splitting method for solving such minimization problem. We establish different convergence estimates under different stepsize assumptions

Phylogenetic Positions of Aspidisca steini and Euplotes vannus within the Order Euplotida (Hypotrichia: Ciliophora) Inferred from Complete Small Subunit Ribosomal RNA Gene Sequences

Summary. The small subunit rRNA (SSrRNA) genes were sequenced for the hypotrichous ciliates, Aspidisca steini and Euplotes vannus. These two genera form a monophyletic clade and branch first in the euplotid clade at a long level with strong bootstrap support in both distance matrix and maximum parsimony tree construction methods. The phylogenetic trees further suggest the postulated relationships among families within the order Euplotida that (1) the order Euplotida, represented by Uronychia, Diophrys, Euplotidium, Euplotes and Aspidisca, forms a paraphyletic group; (2) the families Euplotidae and Aspidiscidae, likely as a monophyletic clade, share a common ancestor; (3) two other related genera, Uronychia and Diophrys, which were usually placed in the family Uronychiidae, branch later and share closer relationship each other than they are to other euplotids. On the contrary, Euplotidium arenarium, placed in the family Gastrocirrhidae, might be more closely related to Uronychia-Diophrys than to the Aspidisca-Euplotes group

Genome Analyses of the New Model Protist \u3ci\u3eEuplotes vannus\u3c/i\u3e Focusing on Genome Rearrangement and Resistance to Environmental Stressors

As a model organism for studies of cell and environmental biology, the free-living and cosmopolitan ciliate Euplotes vannus shows intriguing features like dual genome architecture (i.e., separate germline and somatic nuclei in each cell/organism), “gene-sized” chromosomes, stop codon reassignment, programmed ribosomal frameshifting (PRF) and strong resistance to environmental stressors. However, the molecular mechanisms that account for these remarkable traits remain largely unknown. Here we report a combined analysis of de novo assembled high-quality macronuclear (MAC; i.e., somatic) and partial micronuclear (MIC; i.e., germline) genome sequences for E. vannus, and transcriptome profiling data under varying conditions. The results demonstrate that: (a) the MAC genome contains more than 25,000 complete “gene-sized” nanochromosomes (~85 Mb haploid genome size) with the N50 ~2.7 kb; (b) although there is a high frequency of frameshifting at stop codons UAA and UAG, we did not observe impaired transcript abundance as a result of PRF in this species as has been reported for other euplotids; (c) the sequence motif 5′-TA-3′ is conserved at nearly all internally-eliminated sequence (IES) boundaries in the MIC genome, and chromosome breakage sites (CBSs) are duplicated and retained in the MAC genome; (d) by profiling the weighted correlation network of genes in the MAC under different environmental stressors, including nutrient scarcity, extreme temperature, salinity and the presence of ammonia, we identified gene clusters that respond to these external physical or chemical stimulations, and (e) we observed a dramatic increase in HSP70 gene transcription under salinity and chemical stresses but surprisingly, not under temperature changes; we link this temperature-resistance to the evolved loss of temperature stress-sensitive elements in regulatory regions. Together with the genome resources generated in this study, which are available online at Euplotes vannus Genome Database (http://evan.ciliate.org), these data provide molecular evidence for understanding the unique biology of highly adaptable microorganisms

Two New and Two Poorly Known Species of Ancistrum (Ciliophora, Scuticociliatia, Thigmotrichida) Parasitizing Marine Molluscs from Chinese Coastal Waters of the Yellow Sea

The morphology and taxonomy of two new and two poorly known ciliate species of Ancistrum, found in the mantle cavity (mainly on gills) of marine molluscs from culture beds and pools along the Chinese coast of the Yellow Sea, were investigated using living observation and silver impregnation. Ancistrum haliotis n. sp. was isolated from the abalone Haliotis discus hannai Ino, A. crassum Fenchel, 1965 from the purple clam Saxidomus purpuratus (Sowerby), A. acutum n. sp. from the surf clam Mactra veneriformis Reeve, and A. japonicum Uyemura, 1937 from both the venus clam Cyclina sinensis (Gmelin) and from Dosinia japonica (Reeve). Ancistrum haliotis differs from its most similar relative A. mytili (Quennerstedt, 1867) by the body outline (anterior portion narrower vs. wider than the posterior portion), the macronuclear shape (broadly ellipsoidal vs. reniform or sausage-like), and by having fewer somatic kineties (28–32 vs. usually more than >40). Ancistrum crassum is characterized by the naked area at the apical end of the cell, the relatively short buccal field occupying about two thirds of the body length, and the posterior-dorsal cone-shaped prolongation. Ancistrum acutum n. sp. and A. japonicum are almost identical in morphometry, but differ distinctly in the live morphology (anterior end pointed and posterior end rounded vs. anterior end narrowly rounded and posterior-dorsal end protruded) and ciliary pattern (all right-side kineties extend to posterior body end vs. all right-side kineties excluding somatic kinety 1 distinctly shortened posteriad, forming a glabrous zone). We neotypify Ancistrum japonicum and discuss the taxonomic status of the four species. Based on an evaluation of all nominal species of Ancistrum and Ancistrumina, we recognize nine valid species of Ancistrum and provide a tabular guide to their identification. Fenchelia Raabe, 1970 is regarded as a junior synonym of Ancistrum Maupas, 1883. We synonymize Ancistrumina nucellae Khan, 1970 with Ancistrum japonicum Uyemura, 1937 and Ancistrum edajimanum Oishi, 1978 with A. crassum Fenchel, 1965

Phylogeny of the Ciliate Family Psilotrichidae (Protista, Ciliophora), a Curious and Poorly-Known Taxon, with Notes on Two Algae-Bearing Psilotrichids from Guam, USA

Background: The classification of the family Psilotrichidae, a curious group of ciliated protists with unique morphological and ontogenetic features, is ambiguous and poorly understood particularly due to the lack of molecular data. Hence, the systematic relationship between this group and other taxa in the subclass Hypotrichia remains unresolved. In this paper the morphology and phylogenetics of species from two genera of Psilotrichida are studied to shed new light on the phylogeny and species diversity of this group of ciliates. Results: The 18S rRNA gene sequences of species from two psilotrichid genera were obtained. In the phylogenetic trees, the available psilotrichid sequences are placed in a highly supported clade, justifying the establishment of the family Psilotrichidae. The morphology of two little-known species, packed with green algae, including a new species, Hemiholosticha kahli nov. spec., and Psilotrichides hawaiiensis Heber et al., 2018, is studied based on live observation, protargol impregnation, and scanning electron microscopy. Both species are easily recognized by their green coloration due to the intracellular algae, and a comprehensive discussion as to the possible roles of the intracellular algae is provided. Conclusions: The 18S rRNA gene phylogeny supports the morphological argument that Hemiholosticha, Psilotrichides and Urospinula belong to the same family, Psilotrichidae. However, the single-gene analysis, not surprisingly, does not resolve the deeper relationships of Psilotrichidae within the subclass Hypotrichia. Two littleknown psilotrichid genera with green algae were collected from the same puddle on the island of Guam, indicating a high species diversity and broader geographic distribution of this group of ciliates than previously supposed. Phylogenetic inferences from transcriptomic and/or genomic data will likely be necessary to better define the systematic position and evolution of the family Psilotrichidae. Further studies are also needed to clarify the role of the intracellular eyespot-bearing algae in these ciliates

Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154538/1/fsb2028004032.pd

Species Separation and Identification of Uronychia spp. (Hypotrichia: Ciliophora) using RAPD Fingerprinting and ARDRA Riboprinting

Summary. The 3 most common morphospecies of Uronychia, i.e. U. setigera, U. transfuga and U. binucleata, were examined in vivo and following protargol impregnation. Among these, U. transfuga is morphologically different to the others (large cell size, more macronuclear segments etc.). By contrast, U. setigera and U. binucleata are very similar and difficult to separate based only on their morphologies. Random amplified polymorphic DNA (RAPD) fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA riboprinting) were therefore performed in order to confirm the division between them and to aid species identification. Using 4 different random primers the RAPD fingerprinting revealed 3 distinct patterns. Thus, 7 strains could be separated into 3 species with a similarity index of over 82% between different strains of the same species and only 30% to 40% between strains of different species. The unique restriction pattern of highly-conserved rDNA fragments (ARDRA) of different strains of U. setigera and U. binucleata using the enzyme Msp I was found to be species-specific and could be applicable for both species identification and species separation. According to our molecular analyses, the Uronychia-populations comprised at least 3 taxa (morphospecies). Moreover, the morphologically similar U. setigera and U. binucleata could be reliably separated and identified at the molecular level