Ultrastructure of feeding in the karyorelictean ciliate Tracheloraphis examined by scanning electron microscopy

The karyorelictean ciliate Tracheloraphis is considered to be among the most primitive of the extant ciliates based on both nuclear and somatic characters. These organisms lack the elaborate oral ciliation present in most ciliates. Their mode of ingestion is a type of phagocytosis through a non-ciliated region, the glabrous stripe, which runs the length of the cell. This type of ingestion is reminiscent of feeding in amoebae and some flagellate groups. It is possible that ciliate oral structures evolved within the karyorelictean ciliates from an ancestor resembling Tracheloraphis. We studied the ingestion process in a Tracheloraphis species from the Chesapeake Bay using scanning electron microscopy. The results indicate that the anterior terminus of the organism is not involved in the actual ingestion process, only the glabrous stripe. There is some interaction between the food particle and the surface of the stripe, possibly mediated by a substance secreted by the underlying extrusomes. The somatic cilia do not appear to be involved. The stripe invaginates at the ingestion site engulfing the particle. The cell becomes greatly distended at this site, but neither the anterior nor posterior terminus is affected.     

The quest for the ancestor of the Ciliophora: a brief review of the continuing problem

Although the assumption has long been made that the Ciliophora arose from flagellates, limited progress has been made in determining exactly what extant group - flagellate or other - may best resemble the ancestral "preciliate" assemblage. The distinctiveness of the ciliates and of the many protist phyla containing flagellated stages in their life cycles makes the recognition of potentially homologous features difficult. The suggestion that there may be a phylogenetic relationship between dinoflagellates and ciliates, while seeming unlikely from consideration of a number of specialized characters, is attracting increased attention. This is because some basic similarities (possibly shared derived characters) are characteristic of many species from both groups: cortical alveoli, tubular mitochondrial cristae, kinetidal systems, acentric mitoses with persisting nuclear envelope, functional cytostome, extrusomes (mucocytsts and explosive trichocysts), locomotory organelles, and small subunit rRNAs (close structural similarity values). But many questions remain unanalyzed or unanswered, and caution is still advisable in drawing any firm conclusions about the evolutionary closeness of ciliates and dinoflagellates.     

纤毛虫念珠异列虫射出胞器的超微结构观察

应用扫描电镜术和透射电镜术显示,纤毛虫念珠异列虫(Anteholosticha monilata)的射出胞器早期发生在细胞质深处,附近有不同类型的囊泡结构。成熟后射出胞器向表膜迁移,结构由不同电子密度片层的体部、结晶状的中心轴杆部和多层膜的帽部组成。受外界刺激时胞器冲破皮层射出,形态呈"蘑菇"状。据上述观察结果推测:该射出胞器具有防御作用,它可能起源于高尔基体活动产生的小泡;在亲缘关系较近的纤毛虫中,其射出胞器可能具有相似的分化特征。     

Extrusomes in ciliates: diversification, distribution, and phylogenetic implications

Exocytosis is, in all likelihood, an important communication method among microbes. Ciliates are highly differentiated and specialized micro-organisms for which versatile and/or sophisticated exocytotic organelles may represent important adaptive tools. Thus, in ciliates, we find a broad range of different extrusomes, i.e ejectable membrane-bound organelles. Structurally simple extrusomes, like mucocysts and cortical granules, are widespread in different taxa within the phylum. They play the roles in each case required for the ecological needs of the organisms. Then, we find a number of more elaborate extrusomes, whose distribution within the phylum is more limited, and in some way related to phylogenetic affinities. Herein we provide a survey of literature and our data on selected extrusomes in ciliates. Their morphology, distribution, and possible function are discussed. The possible phylogenetic implications of their diversity are considered.     

Trichocyst Ribbons of a Cryptomonads Are Constituted of Homologs of R-Body Proteins Produced by the Intracellular Parasitic Bacterium of Paramecium

Trichocysts are ejectile organelles found in cryptomonads, dinoflagellates, and peniculine ciliates. The fine structure of trichocysts differs considerably among lineages, and their evolutionary relationships are unclear. The biochemical makeup of the trichocyst constituents has been studied in the ciliate Paramecium, but there have been no investigations of cryptomonads and dinoflagellates. Furthermore, morphological similarity between the contents of cryptomonad trichocysts and the R-bodies of the endosymbiotic bacteria of Paramecium has been reported. In this study, we identified the proteins of the trichocyst constituents in a red cryptomonad, Pyrenomonas helgolandii, and found their closest relationships to be with rebB that comprises the R-bodies of Caedibacter taeniospiralis (gammaproteobacteria), which is an endosymbiont of Paramecium. In addition, the biochemical makeups of the trichocysts are entirely different between cryptomonads and peniculine ciliates, and therefore, cryptomonad trichocysts have an evolutionary origin independent from the peniculine ciliate trichocysts.     

Structure,function, and formation of extrusive organelles — microtoxicysts in the rhizopodPenardia cometa

Summary A study of the structure, function, and development of extrusive organelles (microtoxicysts) in the unusual bacteriovorous rhizopodPenardia cometa is performed. Microtoxicysts are located in the cortical cytoplasm of the cell body and in special thickenings on reticulopodia. Similar types of extrusomes have been observed in some cercomonads. The microtoxicysts are organized as membrane-bound vesicles of a complex form. An oviform-conical axial element lies inside each vesicle and is directed with its narrower end towards the plasma membrane. An internal cylindrical tube occupies the central part of the axial element; it is turned out as the organelle is shot out. The extrusomes ofP. cometa originate from and develop in derivates of the endoplasmic reticulum, the initial diameter of proextrusome vesicles is twice the diameter of the mature organelles. At late stages of maturation the microtoxicysts adopt their characteristic form and orientation. The mode of construction, ejection and development is compared with that in some other carnivorous and bacterivorous protists.     

The structure of extrusive organelles in alveolate and other heterotrophic flagellates

The structure of ejective organelles (extrusomes) in 12 species of heterotrophic flagellates belonging to 8 taxonomic groups is considered. Trichocysts of various structures have been found in colpodellids, Spumella and Metromonas. Kinetocysts of amoeboid flagellates, thaumatomonads, contain a cylindrical element. These kinetocysts are attached to bacteria after discharge. When being discharged, trichocysts of colpodellids produce filaments which have transversal striation. Discobolocysts have been found in excavate flagellate Reclinomonas, and ejectisomes--in Goniomonas. Toxicysts have been marked in carnivorous alveolate flagellate Colponema. The most of studied extrusomes lie inside the cytoplasm, while exstrusomes in chrysomonad Spumella are also found inside the nucleus. Trichocysts of colpodellids and Metromonas as well as ejectisomes of Goniomonas are located near the branches of Golgi apparatus. The comparison of the data obtained confirms the hypothesis about the correlation of taxonomic position of flagellates and the structure of their extrusomes that allows in some cases using these features as phylogenetic markers.     

The crystal lattice of Paramecium trichocysts before and after exocytosis by X-ray diffraction and freeze-fracture electron microscopy

Paramecium trichocysts are unusual secretory organelles in that: (a) their crystalline contents are built up from a family of low molecular mass acidic proteins; (b) they have a precise, genetically determined shape; and (c) the crystalline trichocyst contents expand rapidly upon exocytosis to give a second, extracellular form which is also an ordered array. We report here the first step of our study of trichocyst structure. We have used a combination of x-ray powder diffraction, freeze-etching, and freeze-fracture electron microscopy of isolated, untreated trichocysts, and density measurements to show that trichocyst contents are indeed protein crystals and to determine the elementary unit cell of both the compact intracellular and the extended extracellular form.     

Interstitial Ciliates: Benthic Microaerophiles or Planktonic Anaerobes?

We observed marine benthic interstitial ciliates Geleia sp. and Tracheloraphis sp. inhabiting the water column of a chemically stratified salt pond. This habitat is uncharacteristic for interstitial ciliates, yet they displayed active and abundant planktonic populations (up to 800 and 250 cells/liter, respectively) and a well-defined pattern of vertical distribution. Completely absent from the oxygenated epilimnion, they first appeared at the oxic/anoxic interface and were present throughout the anoxic hypolimnion. The data could not be explained by a passive removal (e.g. by currents) of these ciliates from their conventional habitat (soft sediments) to water column. The results suggest that 1) these ciliates favored an anoxic environment, and 2) they switched to a planktonic lifestyle as appropriate conditions (seasonal anoxia) developed in the water column. This sharply contrasts the classic view of these ciliates as specifically benthic and aerobic (albeit microaerophilic) organisms. We hypothesize that Geleia sp. and Tracheloraphis sp. can readily grow in either water column or benthos, but are typically found in sediments simply because they contain their preferred (anoxic) niche.     

The ultrastructure of the extrusomes in <Emphasis Type="Italic">Pseudourostyla cristata</Emphasis>, a hypotrichous ciliated protozoan

By using scanning and transmission electron microscopy, the present study demonstrates a great number of trichocyst-like extrusomes distributed in the cortical cytoplasm of the protozoan Pseudourostyla cristata, a hypotrichous ciliate. Of these, the mature organelles are rod-shaped with a cap consisting of tubular structures, a tip located at the apex of the cap, a body consisting of strateform structures of uneven electron density and an elongated shaft located along the longitudinal central axis of the body. The electron microscopic observations suggest that the extrusive organelles in P. cristata might undergo a morphogenetic process including the following sequential events: the occurrence of the vesicles in the cytoplasm, the condensation of the fibrous substances within the vesicles, the appearance of the electron-dense shaft, and the formation of the cap. In contrast with a large quantity of extrusomes in trophozoit P. cristata, there are no such extrusive organelles in the encysted cells of the ciliate. The phenomena that P. cristata ciliates can readily enter physiological reorganization or encysting phases and discharge a great number of their extrusomes when prepared for SEM and TEM observation suggest that the extrusive process of the extrusomes in P. cristata might have an important influence on the life activity of the ciliate and could be one of the causes leading to the physiological reorganization and the encysting of the ciliate. These reactions of P. cristata might be a protective or defensive response to the environmental changes.     

Ultrastructure,biogenesis, and functions of extrusive organelles in selected non-ciliate protists

Summary The ultrastructural features, biogenesis and functions of several selected protist extrusive organelles are discussed. Most of the review focuses on some common extrusive organelles that were not considered by Hausmann and several types which have been described since that review of 16 years ago. For convenience, extrusomes are categorized as projectile or mucocyst extrusomes. The projectile extrusomes are further subdivided into non-penetrating and cell penetrating extrusomes. This review is restricted to projectile extrusomes such as ejectisomes, the microsporidian invasion apparatus, and the gun cell of oomycetes. Mucocysts include the apicomplexan rhoptries, the K₂ bodies of oomycetes, and the spermatial vesicles and adhesive vesicles of red algae. The possible phylogenetic importance of some extrusive organelles is briefly considered.     

Defence function of pigmentocysts in the karyorelictid ciliate Loxodes striatus

The karyorelictid ciliate Loxodes striatus has pigment granules which are similar in size, structure and distribution to the pigmentocysts in the heterotrich ciliates, Blepharisma japonicum and Stentor coeruleus, which are known to be extrusomes for chemical defence against predators. We examined whether the pigment granules of L. striatus are also defensive organelles. We showed that: (1) pigment granules of L. striatus are extrusive organelles; (2) bleached cells of L. striatus produced by inducing a massive discharge of pigment granules are more vulnerable than normally pigmented cells to the raptorial ciliate Dileptus margaritifer and the turbellarian Stenostomum sphagnetorum, while they are indistinguishable from intact cells in external morphology and the capacity to grow; (3) the cell-free fluid (CFF) which contains the pigment discharged from pigment granules of L. striatus induced in D. margaritifer behavioural and pathological reactions which are essentially the same as those observed in the interaction with L. striatus, and this effect of the CFF disappeared when the pigment was bleached by light. We conclude that pigment granules of L. striatus are extrusomes for chemical defence against predators, and that the defence is based on the toxic pigment contained in these organelles.     

Ultrastructure of the photosynthetic ciliate Mesodinium rubrum

New cellular structures, bifurcated oral tentacles, were observed in many specimens of the photosynthetic ciliate Mesodinium rubrum from the northern Baltic Sea. Cross-sections of tentacles revealed rings (cylinders) of 14 microtubules with spokes. The number of microtubules per ring decreased from 14 to 12 or 11 inside the cell but no true kinetosomes were detected. These "micro-rings" were often associated with extrusomes and the tentacle tips consisted of extrusomes. A nucleus of a symbiotic alga was present, surrounded by algal cytoplasm containing plastids and delimited from the ciliate cytoplasm by two membranes. Each plastid was bounded by four membranes and was associated with one nucleomorph, suggesting a symbiotic origin as a cryptophyte. The unique symbiotic organization and the organelles of 14 microtubules make Mesodinium rubrum an organism of unusual evolutionary interest.     

Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, <Emphasis Type="Italic">Spirostomum ambiguum</Emphasis>

Several species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence.     

Mutations Affecting the Trichocysts in Paramecium aurelia. I. Morphology and Description of the Mutants*

Six types of genic mutants have been isolated. Their phenotypes range from animals with no trichocysts (trichless), to animals with morphologically abnormal trichocysts (football, stubby, pointless, screwy-cigar), to animals which are incapable of extruding otherwise normal looking trichocysts (nondischarge). The football mutant possesses football-shaped trichocysts, which, unlike wild-type trichocysts, do not attach at the cortex. The stubby mutant possesses shorter trichocysts which have a very highly variable morphology. The screwy-cigar animals have thinner and usually longer trichocysts than those found in wild-type cells. The trichocysts of the pointless mutant have all the components of the wild-type organelles but not in their proper relationship. Electron microscopic studies of the mutants have demonstrated that although the morphology of the various mutant trichocysts may differ, their ultrastructure and early developmental stages are comparable to those of trichocysts found in wild type. The mutations are usually pleiotropic, affecting other systems besides trichocysts. The existence of these mutants, particularly trichless, poses some interesting questions regarding the function of trichocysts, and also gives insight into the development of trichocysts.     

A comparative ultrastructural study on the nanoscale extrusomes of tintinnids (Alveolata,Ciliophora, Spirotricha) and their phylogenetic significance

The capsules, putative extrusomes in tintinnid ciliates, are known since 1971. Based on their ultrastructure, shape, and size, five capsule types were distinguished and suggested to be of phylogenetic significance. However, detailed morphometric data and transmission electron micrographs are lacking to verify former conclusions. In the current study, comprehensive analyses of transmission electron microscopic data were performed, investigating 14 species from 13 genera and more than seven families collected in European coastal waters and in the Northeast Pacific. Our data suggest two main capsule types (large and ampulliform vs small and ellipsoidal/ovoidal) each including two subtypes characterised by their internal structures. Species groupings inferred from the capsule (sub-)types emerge also as closely related in gene trees. Additionally, the ampulliform type unites the Undellidae, Xystonellidae, and Tintinnid clade 2, while the shared possession of the small ellipsoidal type proposes a close relationship of Tintinnid clade 11 with the Rhabdonellidae and Cyttarocylididae. Thus, the capsules provide promising features to shed light on several unresolved evolutionary relationships among tintinnid genera and families; yet, information on capsules is still missing for many monophyletic groupings. Finally, we provide the first ultrastructural clues for the extrusive character of these organelles.     

The Trichocysts of Chilomonas paramecium*

SYNOPSIS Axenic cultures of Chilomonas paramecium were grown in media lacking a C-source, resulting in breakdown in autophagosomal vesicles of large numbers of trichocysts. Return of the starved organisms to complete media was followed by a wave of trichocyst formation. Stages in the degeneration and subsequent reformation of trichocysts are described as well as attempted labeling of the developing organelles with ³H-thymidine. A modification of the method of Anderson et al. (2) was used for isolating quantities of exploded trichocysts from Chilomonas. Attempts at isolation of the trichocyst in its coiled state were unsuccessful. Isolated trichocysts mounted on electron microscope grids were subjected to various types of enzymatic digestions.     

Secretory organelle docking at the cell membrane of Paramecium cells: Dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2–3 h by different means, including cytochalasin B (but not D), high cell density, or Ca²⁺ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40–50 min, with a rate of 20–40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca²⁺ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca²⁺ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Secretory organelle docking at the cell membrane of Paramecium cells: dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2-3 h by different means, including cytochalasin B (but not D), high cell density, or Ca2+ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40-50 min, with a rate of 20-40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca2+ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca2+ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.