Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

Cell size and dorsal cilia: are they useful features for the identification of Stylonychia mytilus (Ciliophora, Spirotrichea) and its subpopulations?

It was investigated whether (1) the number of cilia of the dorsal kineties 3 and 4 and (2) the cell length are species-specific characters which can be used to distinguish the sibling species S. mytilus and S. lemnae. The number of cilia of the dorsal kineties 3 and 4 is a relatively constant, reliable species-specific character in all investigated strains of both species and rather independent from the origin and the nutritional condition of the cells. The cell length is also a reliable species character, if strains of both species from Germany are compared (under identical nutritional conditions). However, all S. mytilus strains from China, Australia and Peru are significantly smaller forming one (or some) "small" subpopulations or subspecies, compared with a "big" subpopulation from Germany. The small ones cannot always be distinguished by size alone from S. lemnae cells. Thus the cell size in S. mytilus is not in all geographic regions a species character, but can be used to characterize subspecies.     

Effects of Cadmium in Stylonychia lemnae, Stylonychia notophora and Oxytricha granulifera: Isolation of a Cadmium-Binding Protein

ABSTRACT The effects of cadmium on three ciliates are reported here. Cultures of Stylonychia lemnae, Stylonychia notophora and Oxytricha granulifera were treated with different doses of Cd according to tolerance. The two species of Stylonychia are very sensitive to the metal, white O. granulifera tolerates higher doses. Adding 50 μM of Cd to the medium did not damage cells. The accumulated metal is almost totally present in the particulate fraction after day 3. Two Cd-Zn linking fractions were separated from the soluble fraction of culture treated on day 1. The first protein linking 17 μg Cd/mg showed an ultraviolet absorption spectrum similar to that of Cd-thioneins. Preliminary amino acid analyses indicated that it contained 13% cysteine. The second protein, linking 60 μg Cd/mg, was a glycoprotein. Its ultraviolet absorption spectrum and amino acid analysis showed that this binding protein was far from being a metallothionein: its cysteine content was very low and aromatic and cyclic residues were present. This Cd-linking compound seems to be unique, since it was very different both from metallothioneins and chelatins isolated by other protozoa. The protective role of these chelating proteins is discussed.     

多节核棘尾虫的发现及其与邻近属种的形态比较

黑龙江省帽儿山镇沼泽地生存着一种在体形和皮层形态上酷似贻贝棘尾虫的腹毛类纤毛虫,其大核呈多个结节相连的索状,这与传统的棘毛虫属的特征有别,但S期大核复制带两个仍在两端出现,这一点全似棘尾虫属,又鉴于腹毛类的其它属中也有大核结数目不同的种（Borror,1983),故单纯大核结节数目不同不应被视为属间判别,因此本文将此虫定为棘尾虫属一新种,名之为多节核棘尾虫,据此,棘尾虫属的传统鉴别特征在核形上也予以修正。     

Effect of heat shock on growth and division of Stylonychia mytilus

Stylonychia mytilus cells grown at 23 degrees C exhibit an immediate arrest at G1 and S stages in the cell cycle when subjected to a heat shock of 1 h at 35 degrees C. The duration of arrest was seen to be dependent on the stage at which heat shock was given. It varied from 3 to 7 h and was synchronously accompanied by the delay in the completion of cell cycle. G2 and the early dividing stage D1 were found to be even more sensitive to heat shock than G1 and S phases. Cells divide normally when heat shock was given at the late dividing stage D2. However, the G1 stage of progeny cells was prolonged to 30 h from normal 5.5 h. These observations have been compiled from the cytological studies of normal and heat-shocked Stylonychia mytilus cells at different stages of cell cycle.     

Identification of an amplification promoting DNA sequence from the hypotrichous ciliate Stylonychia lemnae.

The macronucleus of the hypotrichous ciliate Stylonychia lemnae contains a 1218 bp long DNA molecule which becomes highly amplified during vegetative growth due to a continuous overreplication over a long time range. The region which is located upstream the open reading frame of the overamplified 1.2kbp Stylonychia DNA molecule enabled plasmids containing an inefficiently transcribed thymidine kinase gene to persist and amplify upon transfection into mouse L fibroblasts under selective conditions. This region contains long AT-rich stretches. The AT-rich sequences interact with a previously characterized HMG-I like protein from mouse Ehrlich ascites tumour cells. A binding activity for AT-rich stretches could also be identified in macronuclear extracts from Stylonychia lemnae. We suggest a common mechanism for overamplification in Stylonychia macronuclei during vegetative growth and amplification of plasmid DNA in heterologous mouse cells under the influence of a common element.     

Induced Autogamy in 2 Species of Stylonychia

SYNOPSIS By using the split-pair method autogamy was induced in Stylonychia mytilus Ehrenberg and S. muscorum Kahl, and the process was thoroly studied in the former. The pairs split early in conjugation continued the process as autogamonts and never united again altho they were placed in the same depression slide.
The pattern of nuclear behavior during autogamy closely resembles that reported for conjugation. The fusion of pronuclei at metaphase is usually observed, but it occasionally fails and the stationary pronucleus appears to continue its development as a hemicaryon. The macronuclear anlage and the definitive micronuclei differentiate from particular products of the 3rd postgamic division.     

Chromatin structure in the macronucleus of the ciliate Stylonychia mytilus.

Evidence is presented that macronuclear chromatin in the hypotrichous ciliate Stylonychia mytilus occurs in discrete fragments, each representing at least single genes. The size of these fragments varies between 3 and more than 70 nucleosomes with an average length of about 18 nucleosomes. This observation is discussed with respect to macronuclear structure of hypotrichous ciliates.     

The organization of internal telomeric repeats in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae.

There exist about 1000-1500 internal telomeric sequences per haploid genome in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae. All these telomeric repeats are contained in a very conserved element. This element consists of two 2 kb direct repeats flanking a 2.6 kb sequence. Immediately adjacent to one of the repeats a 18mer C4A4C4A4C2 telomeric sequence is localized. Sequences homologous to macronuclear DNA follow 180 bp downstream of the C4A4-bloc. These macronuclear homologous sequences are flanked by the second direct repeat. The possible origin and function of these telomere containing elements is discussed.     

食物在贻贝棘尾虫(原生动物,纤毛虫)细胞内的消化过程

利用免疫荧光显微术、透射电镜及电镜酶细胞化学技术对食物在腹毛类纤毛虫-- 贻贝棘尾虫细胞内的消化过程进行了追踪观察.食物在进入消化腔隙的初期,外面包被着一层膜结构,但此外层膜很快被消化而消失;尔后食物在贻贝棘尾虫体内的消化过程可分为两种方式:一种方式为直接在消化腔隙内完成,此过程同1982年Kaul et al.和Das s et al.的报道;而另一部分食物的消化过程表现为食物逐步向细胞质内突入,以致最终完全被细胞质包围形成一被膜包裹的圆泡状结构;在此过程中,细胞质边缘突出众多含有酸性磷酸酶等物质的小囊泡结构,小囊泡与食物融合,其内的酸性磷酸酶等水解酶释放出来, 食物的表膜逐渐瓦解,个体逐渐变小,最终消化后的营养物质被释放到细胞的消化腔隙内; 这种食物通过胞口与胞咽达消化腔隙后,再由细胞质将其包裹形成的食物泡,我们称之为" 后成食物泡".这两种消化方式与贻贝棘尾虫的消化胞器为一腔隙结构相适应[动物学报 5 4(3):510-516,2008].     

The two macronuclear histone H4 genes of the hypotrichous ciliate Stylonychia lemnae

Macronuclear DNA of hypotrichous ciliates is organized in short gene-sized molecules, each containing all regulatory sequences for autonomous replication and expression. In these organisms the histone genes are not clustered but dispersed on different molecules of various sizes. Two histone H4 genes containing fragments, one of 1.7 kb and one of 2.8 kb, were found in the macronucleus of Stylonychia lemnae. Restriction and sequence data reveal that the two genes-sized pieces are derived from different micronuclear precursors. Both histone H4 genes code for the same protein of 103 aminoacids but differ greatly in their 5'-and 3'-regions.     

Application of Coriphosphine Staining to the Study of the Macronuclear Anlage of Stylonychia mytilus

SYNOPSIS. Coriphosphine staining of Stylonychia mytilus exconjugants at different times after separation reveals some details of the developing macronucleus. Green fluorescence is seen in both bands and heterochromatic blocks of the polytene chromosomes. No red fluorescence was observed along these chromosomes. Fragments of the old macronucleus and the pycnotic micronuclei have red or orange fluorescence. Red fluorescence is characteristic also of nucleoli in the new macronucleus.