New contributions to the taxonomy of urostylid ciliates (Ciliophora, Hypotrichia), with establishment of a new genus and new species.

Hypotrichia, one of the most complex and highly differentiated groups in Ciliophora, has been the object of extensive studies, especially in recent years. Nevertheless, methodological difficulties and insufficient faunistic studies have limited our understanding of their biodiversity and phylogeny. In this study, one novel urostylid ciliate, Pseudoholosticha zhaoi nov. gen., nov. spec. and two populations of Anteholosticha monilata (Kahl, 1928) Berger, 2003, type species of the latter genus, are studied using an integrative approach (live observation, protargol impregnation, scanning electron microscopy, and phylogenetic analysis) to provide further insights into the diversity, classification, and phylogeny of this group of ciliates. Pseudoholosticha nov. gen. can be separated from other morphologically similar genera mainly by the absence of buccal and caudal cirri. A key to 12 morphologically similar genera and illustrations of their cirral patterns are provided. The validation of the new genus and new species is supported by both morphological and phylogenetic analyses. The first 18S rRNA gene sequence of A. monilata, with detailed morphological data, provided a reliable clarification of A. monilata populations and corroborated the phylogenetic position of the type species of the polyphyletic genus Anteholosticha.

Morphology and ontogenesis of two new Hemiholosticha species (Ciliophora, Hypotrichia, Hemiholostichidae nov. fam.).

The morphology and ontogenesis of two new hypotrich ciliates, Hemiholosticha solitaria and Hemiholosticha germanica, were studied using live observation, protargol impregnation, and scanning electron microscopy. Both species share a medium-sized, almost globular body with a short anterior projection; two macronuclear nodules with a single micronucleus in between; a central contractile vacuole; three or four ventral, one postoral, one right and one left marginal cirral row; and three dorsal kineties extending along ribs. However, H. germanica is distinguished from congeners by a higher number of cirri in ventral rows R1 and R2 (3-6 vs. 2 cirri in each row). Hemiholosticha solitaria differs from congeners by having four (vs. three) ventral cirral rows and by the lack (vs. presence) of intracellular green algae. The ontogenesis of H. solitaria follows the H. pantanalensis mode in that (i) the oral primordium develops in a deep pouch and generates the first two cirral streaks in addition to adoral membranelles and undulating membranes, (ii) the undulating membrane anlage does not produce any cirri, and (iii) the longitudinal ventral cirral row R3 originates from two anlagen. The ontogenetic peculiarities along with the 18S rRNA gene phylogenies suggest classification of Hemiholosticha, Psilotrichides, and Urospinula into a new family, Hemiholostichidae.

Two Urosoma species (Ciliophora, Hypotrichia): A multidisciplinary approach provides new insights into their ultrastructure and systematics.

The general morphology and ultrastructure of two soil hypotrichous ciliates, Urosoma emarginata and U. salmastra, were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. Phylogenetic analyses, based on the newly sequenced small subunit ribosomal (SSU) rRNA genes, were conducted on three U. emarginata populations and one U. salmastra population. Our findings support for the validity of Perilemmaphora Berger, 2008, a rankless taxon comprising spirotrich ciliates having a perilemma. The cortical granules of both species are extrusomes representing a new type of mucocyst in U. emarginata and possibly a new type of pigmentocyst in U. salmastra. Additionally, the lithosomes were revealed as subglobose structures composed of a low electron-dense, homogeneous inner part and an electron-dense outer part. The ultrastructural features of the cortical granules, together with ontogenetic and molecular phylogenetic data, suggest that the genus Urosoma might need to be divided. It is posited that ultrastructural features of hypotrichous ciliates in general may have important taxonomic value warranting further investigation.

Morphology and Morphogenesis of a Novel Saline Soil Hypotrichous Ciliate, Gonostomum sinicum nov. spec. (Ciliophora, Hypotrichia, Gonostomatidae), Including a Report on the Small Subunit rDNA Sequence.

Morphology, cirral pattern, and morphogenesis of the new saline soil hypotrich, Gonostomum sinicum nov. spec. collected from Longfeng Wetland in Daqing, north China, were studied, using detailed live observations and protargol-stained specimens. The new species is characterized as follows: (i) a size in vivo of 100-125 × 30-40 µm, (ii) colorless cortical granules, 0.5 µm across, arranged in short rows, (iii) an adoral zone composed of 28-33 membranelles, (iv) three or four frontoventral rows, one of which extends onto the postoral area, (v) left and right marginal rows composed of 18-27 and 21-35, cirri, respectively, and (vi) usually two transverse cirri. Morphogenesis is as usual for the genus Gonostomum, i.e. the cirral primordia II-VI are primary primordia which split into two sets for proter and opisthe in division middle stages, except for anlage I which develops independently. However, the number of frontoventral transverse anlagen is either five or six not only in different individuals but even in proter and opisthe of the same divider. The phylogenetic analyses based on SSU rDNA sequences showed that the genus Gonostomum is nonmonophyletic, indicating that the patterns of cirri and dorsal kineties are homoplasious characters. The new species G. sinicum nov. spec. is perhaps closely related to Cotterillia bromelicola and two congeners.

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora, Hypotrichia), with preliminary notes on encystation and various types of excystation.

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.

Morphology, ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera.

The morphology, ontogeny and molecular phylogeny of Apobakuella fusca gen. n., sp. n. and Parabistichella variabilis gen. n., sp. n., from south China were investigated. Apobakuella fusca, brown colored, demonstrates bakuellid-like infraciliature, and a similar ontogenesis as the genus Bakuella. It is argued, however, that this species represents a novel genus, Apobakuella, which is characterized by two or more marginal rows on the right, several buccal and parabuccal cirri, and lack of frontoterminal and caudal cirri. Phylogenetic analysis based on SSU rRNA gene sequences supports the close relationship of Apobakuella with Neobakuella and Diaxonella within the core Urostylida. By contrast, Parabistichella variabilis has a dominant frontoventral row, few midventral pairs, a long midventral row, and one marginal row on each side. Its morphogenesis exhibits: (1) partial reorganization of the parental adoral membranelles; (2) over six frontoventral-transverse cirri anlagen; (3) intrakinetal development of the midventral row; and (4) very likely, formation of the frontoventral row from the midventral row anlage. Both the morphological characteristics and the SSU rRNA gene sequences suggest that it is incertae sedis among the basal hypotrichs. Further investigation of key taxa with additional molecular markers is required to reveal a better understanding on the phylogeny of Parabistichella.

Morphological, ultrastructural, and molecular characterization of Euplotidium rosati n. sp. (Ciliophora, Euplotida) from Guam.

We combined morphological (i.e. live, stained, scanning, and transmission electron microscopy) with morphometric and molecular analysis to describe a ciliate species collected from shallow reefs in Guam, grown, and maintained in our laboratory. The species was recognized as a member of Euplotidium, and compared with established species of the genus: Euplotidium itoi Ito 1958; Euplotidium psammophilus (Vacelet 1961) Borror 1972; Euplotidium arenarium Magagnini and Nobili 1964; Euplotidium helgae Hartwig 1980; Euplotidium prosaltans Tuffrau 1985, and Euplotidium smalli Lei, Choi and Xu, 2002. To obtain more elements to compare the species, new morphometric data and additional SSU rRNA gene sequences of E. itoi and of E. arenarium are reported. On the basis of this comparison, we established the new species Euplotidium rosati that has a cirral pattern composed of 12 frontoventral and six transverse cirri, and lacks the left marginal cirrus. Euplotidium rosati harbors on its dorsal surface epixenosomes, the peculiar extrusive symbionts described in other Euplotidium species. The whole body of our observations together with the analysis of the data available in the literature leads us to propose a redefinition of the genus. The results may also be useful to clarify the tangled relationship between Euplotidium and Gastrocirrhus.

The ultrastructure of the extrusomes in Pseudourostyla cristata, 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.

Some ultrastructural observations of the vegetative, resting and excysting ciliate, urostyla grandis (urostylidae, hypotrichida).

In order to reveal the differentiation characteristics of organelles of ciliates under different physiological status, the cellular ultrastructure of Urostyla grandis was studied by transmission electron microscopy. In the resting cells most ciliary shafts, kinetosomes and sub-pellicle microtubules were resorbed, and the endoplasmic reticulum (ER) disappeared with the autophagocytosis taking place within the cytoplasm. As well, the nuclear matrix of the macronucleus was extruded into the cytoplasm, forming pseudopodia-like structures with large quantities of heterochromatin (CH) attached to the inner nuclear membrane. During excystment, membraneous structures developed and gradually increased in number to form the ER. Autophagic vacuoles (AVs) appeared containing mitochondria, paraglycogen particles (PGP), membranous structures, etc. Moreover, the number of nucleoli decreased with the chromatin, condensing in parallel with the process of recombination. Based on these observations, it could be concluded that the de-differentiation of microtubular organelles and the changes occurring in macronuclei in the resting Urostyla grandis, as well as the differentiation of cytoplasmic organelles, digestion by AVs, and the recombination of chromatin during excystment, are not similar to events that occur in non-kinetosome-resorbing cysts (NKR).

Some ultrastructural observations of the vegetative, resting and excysting ciliate, urostyla grandis (urostylidae, hypotrichida)

In order to reveal the differentiation characteristics of organelles of ciliates under different physiological status, the cellular ultrastructure of Urostyla grandis was studied by transmission electron microscopy. In the resting cells most ciliary shafts, kinetosomes and sub-pellicle microtubules were resorbed, and the endoplasmic reticulum (ER) disappeared with the autophagocytosis taking place within the cytoplasm. As well, the nuclear matrix of the macronucleus was extruded into the cytoplasm, forming pseudopodia-like structures with large quantities of heterochromatin (CH) attached to the inner nuclear membrane. During excystment, membraneous structures developed and gradually increased in number to form the ER. Autophagic vacuoles (AVs) appeared containing mitochondria, paraglycogen particles (PGP), membranous structures, etc. Moreover, the number of nucleoli decreased with the chromatin, condensing in parallel with the process of recombination. Based on these observations, it could be concluded that the de-differentiation of microtubular organelles and the changes occurring in macronuclei in the resting Urostyla grandis, as well as the differentiation of cytoplasmic organelles, digestion by AVs, and the recombination of chromatin during excystment, are not similar to events that occur in non-kinetosome-resorbing cysts (NKR).

Morphology and morphogenesis of a new marine ciliate, Apokeronopsis bergeri nov. spec. (Ciliophora, Hypotrichida), from the Yellow Sea, China.

The morphology and morphogenesis of a new marine hypotrich ciliate, Apokeronopsis bergeri nov. spec., collected from mussel-farming waters near Qingdao, China, are described from living and protargol-impregnated specimens. This ciliate has characteristics that place it in the family Pseudokeronopsidae, namely, two long rows of frontal cirri (bicorona), which are continuous with the long midventral rows, and a single row of marginal cirri on each side of the body. It shares with its only congener, Apokeronopsis crassa, the long rows of buccal and transverse cirri and the wide spacing between the midventral rows of cirri. These characters separate the genus Apokeronopsis from Pseudokeronopsis, which has a single buccal cirrus, fewer transverse cirri and midventral rows of cirri arranged in a typical zig-zag pattern. A. bergeri differs from A. crassa in its shape, colour and in the numbers of membranelles and transverse cirri. Although morphogenesis in A. bergeri is similar to that of A. crassa in most respects, the mode of formation of the buccal cirri is slightly different. The close relationship of A. bergeri with A. crassa, and the more distant relationship with three Pseudokeronopsis species, is supported by a comparison of the sequences of their ITS1-5.8S-ITS2 rDNA regions.

A scaffold for basal body patterning revealed by a monoclonal antibody in the hypotrich ciliate Paraurostyla weissei.

In the hypotrich ciliate Paraurostyla weissei, the infraciliature consists of basal bodies on which cilia are anchored together with associated dense material and microtubular rootlets. This is renewed at each morphogenesis, which occurs both in the fully differentiated cell and in a transient dedifferentiated stage, the zygocyst. In both situations, the cell must reconstitute its typical ciliature by properly patterning its basal bodies. Insights into these morphogenetic processes were obtained through an immunocytochemical study using an antipericentriolar material antibody displaying remarkable properties. In Paraurostyla, this antibody decorates the electron-dense material associated with the basal bodies in the interphase cell (as it does with centrosomes on metazoan cells) but, during morphogenesis, the antibody decorates a transient system of tracks which appear prior to basal body patterning and along which basal bodies will later be distributed. In all cases, tracks are initiated close to parental organelles and then elongate to form a system linking the anlagen together during their migration. During zygocyst morphogenesis, they extend along three main cellular meridians. As this antibody decorates an antigen associated with early steps of morphogenesis, it visualizes a proteinaceous system upstream to basal body patterning and provides a structural continuity between parental and newly assembled basal body systems.