William A. Bourland

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.

Structured multiple endosymbiosis of bacteria and archaea in a ciliate from marine sulfidic sediments: a survival mechanism in low oxygen, sulfidic sediments?

Marine micro-oxic to sulfidic environments are sites of intensive biogeochemical cycling and elemental sequestration, where prokaryotes are major driving forces mediating carbon, nitrogen, sulfur, phosphorus, and metal cycles, important from both biogeochemical and evolutionary perspectives. Associations between single-celled eukaryotes and bacteria and/or archaea are common in such habitats. Here we describe a ciliate common in the micro-oxic to anoxic, typically sulfidic, sediments of Santa Barbara Basin (CA, USA). The ciliate is 95% similar to Parduzcia orbis (18S rRNA). Transmission electron micrographs reveal clusters of at least three different endobiont types organized within membrane-bound sub-cellular regions. Catalyzed reporter deposition–fluorescent in situ hybridization and 16S rRNA clone libraries confirm the symbionts include up to two sulfate reducers (Desulfobulbaceae, Desulfobacteraceae), a methanogen (Methanobacteriales), and possibly a Bacteroidete (Cytophaga) and a Type I methanotroph, suggesting synergistic metabolisms in this environment. This case study is discussed in terms of implications to biogeochemistry, and benthic ecology.

Morphological and Molecular Phylogeny of Dileptid and Tracheliid Ciliates: Resolution at the Base of the Class Litostomatea (Ciliophora, Rhynchostomatia)

Dileptid and tracheliid ciliates have been traditionally classified within the subclass Haptoria of the class Litostomatea. However, their phylogenetic position among haptorians has been controversial and indicated that they may play a key role in understanding litostomatean evolution. In order to reconstruct the evolutionary history of dileptids and tracheliids, and to unravel their affinity to other haptorians, we have used a cladistic approach based on morphological evidence and a phylogenetic approach based on 18S rRNA gene sequences, including eight new ones. The molecular trees demonstrate that dileptids and tracheliids represent a separate subclass, Rhynchostomatia, that is sister to the subclasses Haptoria and Trichostomatia. The Rhynchostomatia are characterized by a ventrally located oral opening at the base of a proboscis that carries a complex oral ciliature. We have recognized two orders within Rhynchostomatia. The new order Tracheliida is monotypic, while the order Dileptida comprises two families: the new, typically bimacronucleate family Dimacrocaryonidae and the multimacronucleate family Dileptidae. The Haptoria evolved from the last common ancestor of the Litostomatea by polarization of the body, the oral opening locating more or less apically and the oral ciliature simplifying. The Trichostomatia originated from a microaerophylic haptorian by further simplification of the oral ciliature, possibly due to an endosymbiotic lifestyle.

Multigene-based phylogeny of the ciliate families Amphisiellidae and Trachelostylidae (Protozoa: Ciliophora: Hypotrichia).

Phylogenetic analyses of ciliated protozoa have been increasingly relied on multigene information, which was revealed to provide more robust interpretations than single-gene information. Previous studies showed that Amphisiellidae was an extremely divergent group within the order Stichotrichida, with species widely dispersed throughout the stichotrichid assemblage, while Trachelostylidae, excluding gonostomatid species, is a monophyletic group within the order Sporadotrichida. In the present study, we provide 38 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer phylogenetic relationships among all taxa available in Amphisiellidae and Trachelostylidae. The results indicate that: (1) Amphisiellidae is polyphyletic, with Amphisiella, Hemiamphisiella, Orthoamphisiella, Uroleptoides, and Urospinula distributing in different clades; (2) Amphisiella is separated into two clades in phylogenetic trees, corroborated by difference in cortical granule distribution. Thus, cortical granule pattern and distribution may be strong diagnostic features to divide Amphisiella species into two subgenera; (3) the monophyly of Trachelostylidae sensu Berger (2008) is strongly supported, suggesting it is a well-defined family; (4) Gonostomatidae is confirmed to be a valid family.

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea)

Graphical abstract Highlights ► Multiple-locus analyses of litostomatean evolution. ► Unraveling main evolutionary trends in the class Litostomatea. ► Reconciling molecular and morphological evolution of the Litostomatea. ► Defining the main litostomatean lineages by morphological apomorphies.

Morphologic and Molecular Description of Metopus fuscus Kahl from North America and New rDNA Sequences from Seven Metopids (Armophorea, Metopidae)

Most species in the large ciliate genus Metopus Claparède & Lachmann, 1858 lack detailed descriptions based on modern morphologic and molecular methods. This lack of data for the vast majority of species hampers application of a morphospecies approach to the taxonomy of Metopus and other armophorids. In this report we redescribe the large species, Metopus fuscusKahl, 1927 based on in vivo observation, silver impregnation, scanning electron microscopy, and single-cell 18S rDNA sequencing of a freshwater North American (Idaho) population. Metopus fuscus invariably has a perinuclear envelope of endosymbiotic bacteria not found in other species. Unlike the original description of a single row of coarse granules between ciliary rows, the Idaho population has five loose rows of small interkinetal granules. We discuss the possible importance of this character in metopids. We also provide a phylogenetic analysis including seven other new metopid 18S rDNA sequences: Brachonella spiralis, B. galeata, Metopus laminarius, M. setosus, M. striatus, M. violaceus, Palmarella lata. Metopus fuscus and M. setosus form a fully supported clade, challenging previous morphospecies groupings. We discuss some ambiguities of armophorid morphologic terminology in the earlier literature. Our phylogenetic analysis of Idaho metopids indicates that the genera Metopus and Brachonella are both nonmonophyletic.

Morphology and Phylogeny of a New Woodruffiid Ciliate, Etoschophrya inornata sp. n. (Ciliophora, Colpodea, Platyophryida), with an Account on Evolution of Platyophryids

Bourland, W. A., Hampikian, G. & Vďačný, P. (2012). Morphology and phylogeny of a new woodruffiid ciliate, Etoschophrya inornata sp. n. (Ciliophora, Colpodea, Platyophryida), with an account on evolution of platyophryids. —Zoologica Scripta, 41, 400–416.

Nullomer Derived Anticancer Peptides (NulloPs): Differential Lethal Effects on Normal and Cancer Cells in vitro

We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in 1M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time.

Redescription of Atopospira galeata (Kahl, 1927) nov. comb. and A. violacea (Kahl, 1926) nov. comb. with redefinition of Atopospira Jankowski, 1964 nov. stat. and Brachonella Jankowski, 1964 (Ciliophora, Armophorida).

The taxonomy of the Metopidae (Ciliophora, Armophorida) remains poorly understood since most of its members have not been studied by modern morphologic and molecular methods. Recent molecular investigations have indicated that the two most species-rich genera, Metopus and Brachonella, are likely nonmonophyletic with at least one well-supported 18S rDNA clade comprised of a species from each of these genera (Brachonella galeata and Metopus violaceus). We investigated these two species with silver impregnation and scanning electron microscopy. Both taxa share important morphologic characteristics not described in other species of Metopus or Brachonella. These synapomorphies include: (1) a diplostichomonad paroral membrane, (2) a bipartite adoral zone with a short buccal part composed of ordinary membranelles and a longer distal part composed of much smaller membranelles bearing a single cilium or none and extending the same length as the perzonal ciliary stripe. We transfer Brachonella galeata (Kahl, 1927) Jankowski, 1964 and Metopus violaceus Kahl, 1926 to genus Atopospira Jankowski, 1964 nov. stat. Pending detailed morphologic and molecular characterization, Brachonella campanula, B. cydonia and B. pyriforma, B. intercedens, and B. lemani remain in Brachonella Jankowski 1964.

Morphology, Morphometrics, and Molecular Characterization of Bryophrya gemmea n. sp. (Ciliophora, Colpodea): Implications for the Phylogeny and Evolutionary Scenario for the Formation of Oral Ciliature in the Order Colpodida

ABSTRACT. We studied the morphology, morphometry, resting, and reproductive cysts, as well as the molecular phylogeny of Bryophrya gemmea n. sp., a colpodid ciliate that was discovered in ephemeral puddles in Idaho, northwest United States. This new species is distinguished from congeners by the irregularly pentagonal adoral organelles, four to five vestibular kineties, the single micronucleus, and one to three rows of brightly refractive protuberant interkinetal cortical granules to the right of the preoral suture. Resting cysts have two distinct membranes and an outer mucous coat. As typical for most colpodids, reproduction occurs in division cysts but details of ontogenesis are unknown. The 18S rRNA gene sequence shows only weak support for the phylogenetic relationship between Bryophrya and the bryophryid genus Notoxoma previously inferred from morphologic characters. Further, our molecular phylogenies classify bryophryids rather basal within the order Colpodida, not supporting ordinal status suggested by morphologists. Based on molecular data and morphologic characters, the colpodid genus Ilsiella is removed from the family Marynidae and placed in a new family, Ilsiellidae. Considering the molecular data, an evolutionary scenario for the formation of colpodid oral structures from a cyrtolophosidid ancestor through a bryophryid intermediate is proposed.