Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol

Protargol (silver proteinate) impregnation is a common method used to identify and characterize ciliated protozoa. Unfortunately, chemical companies have stopped producing the ‘strong’ protargol powder used in this method. Based on an in-house protocol for its synthesis published in 2013, more than 10 batches of protargol powder were produced and subsequently applied in taxonomic studies. During these studies, the protocol for protargol powder synthesis was slightly modified and employed a peptone not originally listed in the 2013 protocol. This modification improved the results of the impregnation protocol. Protargol preparations of hypotrichs were optimized by adjusting the pH during staining rather than during the synthesis. The pH was adjusted to 7.5–7.6, and an acetone developer was used. While the conditions used in this study are not completely comparable to those using the commercially produced protargol, access to this information could help researchers investigate the diversity of ciliates, particularly hypotrichs.     

An Optimized Protocol of Protargol Staining for Ciliated Protozoa

Protargol staining is a crucial method to reveal the infraciliature of ciliates, which is the most important morphological character for species identification. In the present study, Wilbert's protocol of protargol staining was emended mainly toward the highly happened improper bleaching. Through reciprocal treatments, both insufficient and excessive bleachings were much eliminated from the protargol protocol and the tests performed with four different species of ciliates established that the stainings were considerably improved and more reliable with optimized bleaching. Compared to the original protocol, the optimized method was proved to be more suitable for the groups difficult to stain, and it is also friendlier for the beginners and researchers in related fields.     

Examination of the Tintinnid Ciliate Parafavella denticulata (Ehrenberg) by Scanning Electron Microscopy and the Bodian Protargol Technique

Scanning electron microscopy and the Bodian protargol technique are applied to the cell body of a tintinnid for the first time. The somatic ciliature, the membranelles, the pulsating vacuole, the preoral cavity and the invagination at the posterior end of the cell body are demonstrated for the species Parafavella denticulata (Ehrenberg 1840). The correlation between SEM and the protargol impregnations is striking, and the presumptive taxonomic and phylogenetic value of these methods for the study of tintinnids is emphasized.     

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.     

Morphogenesis and Cortical Ultrastructure of Brooklynella hostilis,a Dysteriid Ciliate Ectoparasitic on Marine Fishes*

SYNOPSIS. Morphogenesis, and the cortical structures of Brooklynella hostilis, a cyrtophorine gymnostome ciliate ectoparasitic on marine fishes, were studied from protargol silver-impregnated preparations and with the aid of electron microscopy. The pattern of morphogenesis of Brooklynella is close to that found in less differentiated species of the families Chlamydodontidae (e.g., in the genus Trithigmostoma) and Dysteriidae (e.g., in the genus Hartmanula). The full number of kineties in the opisthe is restored after division from a segment of the left one of the 3 kinetics producing the oral rows. The oral rows consist of a double row of kinetosomes arranged in a zig-zag pattern; only the outer row is ciliated, the inner one being barren. However, the positions of the postciliary and transverse fibers indicate that the oral rows are not homologs of an undulating membrane but are akin to a membranelle. In association with the ventral somatic kinetosomes there are 4 postciliary fibers; a rather aberrant, transversally oriented kinetodesma; 2 microtubular, transverse fibers plus a transverse fibrousspur; and one to several ribbons of subkinetal microtubular fibers. Not directly associated with the kinetosomes are fibrous strands running subpellicularly between the kinetosomes and also deep into the cytoplasm. The cortical structures of Brooklynella are compared with those of some other groups of ciliates of about the same phylogenetic level in which the subkinetal microtubules can also be found– rhynchodine, suctorian, and chonotrich ciliates. The nasse consists of 6–8 nematodesmata not closely associated with the microtubular cytopharyngeal tube. The former have a distinctly developed densely fibrous capitulum containing barren kinetosomes which originally produced the nematodesma during stomatogenesis; the capitulum is connected by a fibrous link to the microtubular shaft. Extending from the oral rows to the capitula are fibrous structures strongly reminiscent of filamentous reticulum in hymenostome and peritrich ciliates. The structure of the posterio-ventral glandular organelle is also described and discussed.     

Taxonomic redescriptions of two ciliates, Protogastrostyla pulchra n. g., n. comb. and Hemigastrostyla enigmatica (ciliophora: spirotrichea, stichotrichia), with phylogenetic analyses based on 18S and 28S rRNA gene sequences

The morphology and infraciliature of two stichotrichid ciliates, Gastrostyla pulchra(Perejaslawzewa 1886) Kahl, 1932 and Hemigastrostyla enigmatica(Dragesco and Dragesco-Kernéis 1986) Song & Wilbert, 1997, collected from marine and brackish sediments, were investigated by using living observations and protargol impregnations. Both 18S and 28S rRNA genes of these two species were sequenced. The 18S rDNA show high similarities (98.4%-99.7%) among populations of each species. There is about 94% similarity in 18S rDNA genes between G. pulchra and Gastrostyla steinii, the type species of the genus, which has been confirmed to be an oxytrichid by previous studies. In the phylogenetic trees of 18S, 28S, and combined 18S and 28S rDNA, both G. pulchra and H. enigmatica are consistently placed outside the well-established oxytrichid clade. Based on our analyses and previous ontogenetic data, we conclude that these two species may represent some lower groups in the subclass Stichotrichia, and that G. pulchra should represent a new genus, Protogastrostyla n. g. This new genus, which is morphologically similar to Gastrostyla, differs in its morphogenesis: the apical part of the old AZM is retained combining with the newly built membranelles that develop from the proter's oral primordium; the primary primordia of the dorsal kinety; and marginal primordia commence de novo without a definite contribution from the old structure.     

一种游仆虫无性分裂生殖的研究Ⅰ.形态学和核器的演化

应用光学显微镜和扫描电子显微镜观察了采自上海的游仆虫的形态,描述了它的表面结构以及纤毛器、棘毛下纤维的结构和核器的演化过程。     

A Somatic Kinetid Study of the Pycnotrichid Ciliate Vestibulongum corlissi N. G., N. Sp. (Class: Litostomatea), Symbiont in the Intestines of the Surgeonfish,Acanthurus xanthopterus1

ABSTRACT The ciliate Vestibulongum corlissi n. g., n. sp. was collected from the intestines of surgeonfish, Acanthurus xanthopterus, in the summer of 1986. In has been examined in the light microscope after protargol staining and by scanning and transmission electron microscopy. Its form is distinct from that of other pycnotrichid ciliates at the generic level. Somatic kinetids were examined; these demonstrate that its cytostome is posterior and that the kinetid structures and the presence of a second transverse microtubular ribbon confirm its placement in the class Litostomatea.     

New Observations on the Choreotrich Ciliate Strombidinopsis acuminata Fauré-Fremiet 1924, and Comparison with Strombidinopsis jeokjo Jeong et al., 2004

ABSTRACT. A choreotrich ciliate collected in waters off Geoje Island, on the eastern coast of the South Sea of the southern coastal water of Korea was morphologically and biometrically investigated through observations of permanent preparations after protargol impregnations. Morphological data and ciliary patterns indicated that the ciliate should be identified as Strombidinopsis acuminata based on the number of somatic kineties. The vertical distribution of the population abundance was also examined in relation to environmental conditions: higher abundances occurred in surface water with higher temperature (22–23 °C), lower salinity (32 psu), and higher concentration (ca 10 μg/L) of chlorophyll a . The 18S rRNA gene of specimens from clonal cultures isolated from natural assemblages was sequenced. While this isolate of S. acuminata is closer to Strombidinopsis jeokjo (99.6–99.8%) than other isolates of S. acuminata (<99.0%), monophyly of S. acuminata was not rejected by the AU test. We discuss the discordance between morphological and molecular data for Strombidinopsis . In order to confirm the species level of the Strombidinopsis specimens, additional information is required based on genealogical analyses of multiple loci.     

The edaphic quantitative protargol stain: a sampling protocol for assessing soil ciliate abundance and diversity

It has been suggested that species loss from microbial groups low in diversity that occupy trophic positions close to the base of the detrital food web could be critical for terrestrial ecosystem functioning. Among the protozoans within the soil microbial loop, ciliates are presumably the least abundant and of low diversity. However, the lack of a standardized method to quantitatively enumerate and identify them has hampered our knowledge about the magnitude of their active and potential diversity, and about the interactions in which they are involved. Thus, the Edaphic Quantitative Protargol Staining (EQPS) method is provided to simultaneously account for ciliate species richness and abundance in a quantitative and qualitative way. This direct method allows this rapid and simultaneous assessment by merging the Non-flooded Petri Dish (NFPD) method [Prog. Protistol. 2 (1987) 69] and the Quantitative Protargol Stain (QPS) method [Montagnes, D.J.S., Lynn, D.H., 1993. A quantitative protargol stain (QPS) for ciliates and other protists. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp. 229-240]. The abovementioned protocols were refined by experiments examining the spatial distribution of ciliates under natural field conditions, sampling intensity, the effect of storage, and the use of cytological preparations versus live observations. The EQPS could be useful in ecological studies since it provides both a "snapshot" of the active and effective diversity and a robust estimate of the potential diversity.     

Notes on Soil Ciliates (Protozoa, Ciliophora) from The Netherlands, with Description of Keronopsis schminkei nov. spec. and Apobryophyllum schmidingeri nov. spec

Fifty-six ciliate taxa, including three new species, were found in two soil samples from the Hoge Veluwe National Park in The Netherlands. A literature search showed that The Netherlands, Belgium and Denmark are terra incognita with respect to soil ciliates: only about 100 species have been recorded. Likely, a much greater number, including many undescribed species, can be found on more detailed investigations. Two of the three new species are described in detail. Keronopsis schminkei nov. spec. differs from the congeners by the nuclear apparatus and cirral pattern. Keronopsis wetzeli?Wenzel, 1953 is neotypified with the Austrian population described by Berger and Foissner (1987). Apobryophyllum schmidingeri differs from the congeners by body shape, the macronucleus pattern (many nodules), the arrangement of the extrusomes, and various morphometric features. Scanning electron micrographs and protargol preparations of Enchelys polynucleata confirm earlier transmission electron microscopic data on the occurrence of oralized somatic monokinetids and the lack of a circumoral kinety. Extrusome length and morphology of the resting cysts are rather different in various populations of E. polynucleata, indicating high genetic diversity (subspecies?).     

四种淡水缘毛类纤毛虫的形态学研究

通过活体显微观察及银染法对采自武汉东湖的4种固着缘毛类纤毛虫: 粗茎睫纤虫Ophrydium crassicaule Penard, 1922, 念珠伪钟虫Pseudovorticella monilata (Tatem, 1870) Foissner & Schiffmann, 1974, 垂盖虫Opercularia nutans (Ehrenberg, 1831) Stein, 1854和一个鞘居虫未定种Vaginicola sp.的活体、纤毛图式、银线系以及细胞核的形态学特征进行详细的描述, 补充和完善了缘毛类纤毛虫的形态学资料, 为纤毛虫分类和系统发育学的研究提供重要信息。此外, 首次在伪钟虫中观察到有性生殖现象, 为纤毛虫生殖进化的研究提供了基础资料。     

Morphological descriptions of new and little-known benthic ciliates from Ganghwa tidal flat, Korea

Ciliates are highly diverse in the benthos where there are rare species to be unrevealed and described. By isolating species during successive and diversified cultivations, we discovered several new and interesting taxa from the top layer sediment of a muddy site in the Ganghwa tidal flat. These include three new species Spirodysteria ganghwaensis n. sp., Uronemella parafilificum n. sp., Zosterodasys minuta n. sp., and one poorly known form Loxophyllum chaetonotumBorror 1965. The morphology of live cells and infraciliature of these four species are described based on living observations, protargol impregnations, and morphometrics. Diagnoses and improved definitions are also provided. The newly established genus Spirodysteria n. g. differs from Dysteria mainly in its spirally twisted body shape. Spirodysteria kahli (Tucolesco 1962) n. comb. (formerly Dysteria kahliTucolesco 1962) has been included in this new genus.     

A morphogram for silica‐witherite biomorphs and its application to microfossil identification in the early earth rock record

Archean hydrothermal environments formed a likely site for the origin and early evolution of life. These are also the settings, however, were complex abiologic structures can form. Low‐temperature serpentinization of ultramafic crust can generate alkaline, silica‐saturated fluids in which carbonate–silica crystalline aggregates with life‐like morphologies can self‐assemble. These “biomorphs” could have adsorbed hydrocarbons from Fischer–Tropsch type synthesis processes, leading to metamorphosed structures that resemble carbonaceous microfossils. Although this abiogenic process has been extensively cited in the literature and has generated important controversy, so far only one specific biomorph type with a filamentous shape has been discussed for the interpretation of Archean microfossils. It is therefore critical to precisely determine the full distribution in morphology and size of these biomorphs, and to study the range of plausible geochemical conditions under which these microstructures can form. Here, a set of witherite‐silica biomorph synthesis experiments in silica‐saturated solutions is presented, for a range of pH values (from 9 to 11.5) and barium ion concentrations (from 0.6 to 40 mmol/L BaCl₂). Under these varying conditions, a wide range of life‐like structures is found, from fractal dendrites to complex shapes with continuous curvature. The size, spatial concentration, and morphology of the biomorphs are strongly controlled by environmental parameters, among which pH is the most important. This potentially limits the diversity of environments in which the growth of biomorphs could have occurred on Early Earth. Given the variety of the observed biomorph morphologies, our results show that the morphology of an individual microstructure is a poor criterion for biogenicity. However, biomorphs may be distinguished from actual populations of cellular microfossils by their wide, unimodal size distribution. Biomorphs grown by diffusion in silica gel can be differentiated by their continuous gradient in size, spatial density, and morphology along the direction of diffusion.     

Morphology and Morphogenesis of Fuscheria terricola n. sp. and Spathidium muscorum (Ciliophora: Kinetofragminophora)1

The morphology and morphogenesis of the kinetofragminophoran soil ciliates, Fuscheria terricola n. sp. and Spathidium muscorum Dragesco & Dragesco-Kerneis, 1979, are described. Stained specimens (protargol) are characterized biometrically. The new species differs from the other species of the genus in its body size, body shape, number of kineties, length of extrusomes, and habitat. Both species have telokinetal stomatogenesis, which commences with a proliferation of kinetosomes at those kineties which bear the brosse. Fuscheria terricola does not have a complex perioral ciliature; indeed, it might be that this species has only monokinetids. Thus only a proliferation of kinetosomes and the separation of the kineties takes place in the prospective division furrow. In contrast, S. muscorum differentiates short dikinetid kinetofragments in the region of the division furrow, which are arranged to form the perioral kinety of the opisthe in the intermediate and late stages of the stomatogenesis. The right part of the perioral kinety develops first. This and other studies show that telokinetal stomatogenesis proceeds very differently depending on the differentiation of the oral ciliature; however, detailed studies on the morphogenesis of kinetofragminophoran ciliates are still too few in number for subtypes to be defined.     

Light microscopical observations on two marine dysteriid ciliates from Japan,including a description of Dysteria yagiui nov. spec. (Ciliophora,Cyrtophorida)

Two cyrtophorid ciliates, Dysteria yagiui nov. spec. and D. derouxi, were isolated from fish-farming waters at the New Fishing Port of Nagasaki, Japan. Their morphology and infraciliature were investigated using observation of living cells and protargol impregnation. D. yagiui nov. spec. is a large marine species distinguished from its congeners by the presence of 13 ventral kineties in the right field, of which six extend apically and 6–8 short fragments of kineties in the left equatorial field. D. derouxi has the characteristics previously described except that ciliates of this population possess nine ventral kineties in the right field, of which four extend apically.     

西藏三种苔藓栖生纤毛虫的形态和系统发育分析

本研究利用活体观察和蛋白银染色方法对采自西藏日喀则和那曲的3种苔藓栖生纤毛虫,鬃异源棘尾虫(Tetmemena pustulata)、棘毛虫未定种(Sterkiella sp.)和殖口虫未定种(Gonostomum sp.)进行了形态学研究,描述了活体、核器以及纤毛图式等形态学特征.同时,测定了这3种纤毛虫的SSU r...     

Redescription of Ellobiophrya brevipes (Laird, 1959) N. Comb. (Ciliophora, Peritrichia) and the Fine Structure of its Pellicle and Cinctum

ABSTRACT. A species of peritrich that attaches to gills of the skate, Raja erinacea , was identified by its original describer as a member of Caliperia , a genus characterized by having a noncontractile skeletal rod within the arms of its cinctum and by not having the cinctal arms bonded to one another at their tips. Our observations of the living ciliates confirmed by protargol impregnation and electron microscopy revealed that their cinctal arms are linked by a bouton and that the cytoskeletal structure within them has the fine structure of a myoneme. These characteristics place this peritrich unequivocally in the genus Ellobiophrya and it is thus renamed Ellobiophrya brevipes (Laird, 1959) n. comb. Clumps of epithelial cells clasped by the cincta of E. brevipes show damage at their bases but not on their luminal surfaces. The known species of Ellobiophrya are compared for significant structural differences that separate species of this genus.