Reorganization Band Research Articles

A comparative fine structural and phylogenetic analysis of resting cysts in oligotrich and hypotrich Spirotrichea (Ciliophora)

So far, neither morphology nor gene sequences have provided a reliable classification of halteriid and hypotrichid spirotrichs. Thus, we performed a comparative study on the fine structure of the resting cysts in some representative species, viz., the oligotrichs Halteria grandinella and Pelagostrombidium fallax and the oxytrichid hypotrichs Laurentiella strenua, Steinia sphagnicola, and Oxytricha granulifera. Main results include: (i) there are three different, very likely non-homologous cyst surface ornamentations, viz., spines (generated by the ectocyst), thorns (generated by the mesocyst), and lepidosomes (produced in the cytoplasm); (ii) Halteria has a perilemma; (iii) Halteria, Meseres and Pelagostrombidium have fibrous lepidosomes, while those of Oxytricha are tubular; (iv) the cyst wall structure of Pelagostrombidium and Strombidium is distinctly different from that of halteriids and oxytrichids, which are rather similar in this respect; (v) cyst ornamentation does not provide a reliable phylogenetic signal in oxytrichid hypotrichs because ectocyst spines occur in both flexible and rigid genera. The new observations and literature data were used to investigate the phylogeny of the core Spirotrichea. The Hennigian argumentation scheme and computer algorithms showed that the spirotrichs are bound together by the macronuclear reorganization band, the subepiplasmic microtubule basket, and the apokinetal stomatogenesis. The Hypotrichida and Oligotrichida are united by a very strong synapomorphy, viz., the perilemma, not found in any other member of the phylum. Halteriid and oligotrichid spirotrichs form a sister group supported by as many as 13 apomorphies. Thus, the molecular data, which classify the halteriids within the core hypotrichs, need to be reconsidered.

Macronuclear chromatin structure in Euplotes

Electron microscopy after section staining with heavy metals shows that in Euplotes the macronuclear chromatin is organized into a large number of distinct, electron dense granules. Estimations of the total number of granules and determinations of the ratio macronuclear to micronuclear DNA (microspectrophotometry) suggest that the number of chromatin granules is greater than what could be expected if each granule was the equivalent of a micronuclear chromosome. Mathematical analysis of granule diameters suggests the presence of many different size groups ranging from 0.1–1.2 μ, smaller granules being more frequent than larger granules. The granules probably consist of accumulations of fibrils 30–40 Å in diameter. Some of the fibrils seem to run in pairs for fairly long distances, two 30 Å fibrils being separated by a 40 Å interspace. Fibrils project from the surface of the granules into nucleoplasm, nucleoli and adjacent chromatin granules. During reorganization (DNA-replication) the chromatin granules break up into fibrils. At the distal part of the reorganization band, fibrils reaggregate to form granules, the average volume of which is 6–8 times greater than that of the pre-reorganization granules. Since DNA and dry mass only double in the macronucleus as a whole and since there is no apparent difference in the packing of fibrils in the granules, the results indicate that material from more than one pre-reorganization granule is incorporated into one post-reorganization granule or that fusion of granules takes place. Post-reorganization granules must decrease in size during the cell life cycle, presumably by division. Macronuclei were labelled with 3H-thymidine for one full cell cycle and segregation of label was studied on individual chromatin granules from squashed G1 cells up to the third “cold” cycle after labelling. Autoradiography on acridine orange stained squash preparations showed dilution but no segregation of label among those granules which were large enough to be detected by fluorescence microscopy. The results suggest that the individual chromatin granules cannot be the equivalents of diploid subnuclei or of micronuclear chromosomes.

Electron Microscopy of the Hypotrich Ciliate Oxytricha platystoma, with Consideration of its Macronuclear Organization

SYNOPSIS. The body of Oxytricha platystoma is covered with 2 membranes, the outer or pellicular membrane and the inner or cytoplasmic membrane. Each cirrus consists of a bundle of 25‐50 cilia. There are 2 macronuclei and 2 micronuclei. The micronucleus is a very small body containing dense Feulgen‐positive material. The macronucleus is an elongated body surrounded by a double membrane. The dense Feulgen‐positive material is embedded in a less dense nucleoplasm. Some larger bodies found inside the nucleus may be nucleoli. There is a single reorganization band in each macronucleus. It consists of a solution plane and a reconstruction plane, and has a total thickness of 1.5 μ. The macronucleus divides amitotically, whereas the micronucleus divides mitotically.

Electron microscopy of the nuclear apparatus of Urostyla grandis, a hypotrichous ciliate.

SYNOPSIS. A description is given of the interphase nuclear apparatus of Urostyla grandis, which has many macronuclei and a few micronuclei. The macronucleus is bounded by a typical double envelope. Chromatinic material in the macronucleus forms a large, irregular network composed of threads up to 500 mμ in thickness which are thicker than those in the macronuclei of most ciliates. The nucleoli are round bodies about 1–2 μ in diameter and are embedded in the matrix enclosed in chromatin threads. The micronucleus is bounded by a double, porous envelope similar to that of the macronucleus. Chromatinic material in the micronucleus forms a small network of comparatively thin threads, 60–80 mμ thick. At high magnifications, both the chromatin threads in the macronucleus and in the micronucleus appear to be composed of fine twisted fibrils about 10 mμ thick. The reorganization band which traverses each macronucleus in the late interphase is divided into two zones: the forward zone consists of fine, twisted fibrils 30 mμ thick formed by pairs of parallel fibrils each 10 mμ thick. The rear zone consists of small chromatin bodies and thin threads, both 80 mμ in diameter, consisting of fine fibrils 10 mμ thick. Reorganized chromatin threads appear to be thin spiral threads 80–130 mμ thick in striking contrast to the thick threads of 100–500 mμ diameter composing the large, irregular network observed in the early interphase macronucleus.

Endogenous bud formation in Histriculus vorax, a new asexual reproductive process in the hypotrichida.

SYNOPSIS. Histriculus vorax (Stokes) Corliss, a hypotrichous ciliate, has been separated from activated sludge and cultured monoxenically in the laboratory. The asexual life cycle has been observed and the stages of development photographed. There are large variations in shape and size of cells within clonal cultures. Both large (190–250 μ long) and small (70–140 μ long) cells are capable of normal asexual binary fission but only the large cells are able to grow endogenous buds, which, when mature, are extruded through the body wall of the mother cell. Newly deposited buds can either develop directly into an embryonic form, or, if unfavorable conditions prevail, first encyst. Similar embryonic stages bearing caudal and frontal cirri are produced both by direct development and by excystment of the encysted bud. Embryos develop rapidly into trophic forms which finally grow into small adults capable of asexual binary fission. During binary fission the nuclei behave as described previously for other members of the Oxytrichidae. A reorganization band forms at the outer end of each macronucleus, and these bands move along the macronuclei towards each other, finally disappearing at the inner ends. A fusion nucleus is then formed which splits into 4 pieces, 2 of which pass to the proter and 2 to the opisthe. During bud formation the reorganisation bands form and move as in binary fission. No fusion nucleus is formed, but the macronuclei divide immediately after reorganization, and the anterior macronucleus of each pair so produced migrates to the budding region. The infraciliature of H. vorax resembles that of Opisthotricha monspessulana.‘Erratic’ kinetosomes, which are present in the adult, divide during binary fission and so produce the infraciliature of the proter and opisthe. During bud formation the budding region receives a number of these ‘erratic’ kinetosomes from the mother cell, and these divide to form the infraciliature of the bud.

Conjugation in Stylonychia muscorum Kahl

SYNOPSIS. The process of conjugation in a strain of Stylonychia muscorum Kahl is described. It follows the general pattern reported earlier in other Oxytrichidae, but the following peculiarities can be noted: 1) mating never occurs between individuals possessing only two micronuclei, 2)exchange of small macronuclear lobes can be occasionally observed, and 3) the reorganization of the exconjugants involves the quick, successive passage of a series of reorganization bands.

Cytochemistry of macronuclear reorganization

The reorganization phenomenon in the macronuclear was related to the cell life cycle of Euplotes. At generation times of 20 hr the G1 period occupied 5 hr, the S period 12.5 hr and the G2 period 2.5 hr. Signs of cytoplasmic division were present during the last third of the G2 period. The S phase consisted of two phases. During the first 80 per cent of the S phase (S1 phase) DNA synthesis was slow. When only 20 per cent of the S phase remained the bands had travelled approximately 45 per cent of the length of the macronucleus. During the last 20 per cent of the S phase (S2 phase) the rate of DNA synthesis was approximately 4 times as rapid as during the first part. As generation time increased, the length of the S phase also increased. In slow-growing organisms some macronuclei exhibited reorganization bands but no incorporation of 3H-thymidine whereas the majority of reorganization bands did incorporate this isotope. Macronuclei from Euplotes were found by cytochemical techniques to undergo quantitative changes in their macromolecular composition during the reorganization phenomenon. The reorganization phenomenon consisted not only of a duplication of the DNA component as previously described by Gall but also of a sudden increase in macronuclear protein. Microinterferometry indicated that in macronuclei isolated from rapidly multiplying organisms a doubling of almost all nuclear macromolecules took place in the reorganization band. The dry mass changes observed could be due only to a small extent to a doubling in DNA content since DNA made up less than 10 per cent of the dry mass material. Autoradiography with 3H-lysine and 3H-arginine showed that zones distal to the reorganization bands became strongly labelled but that protein labelling seemed to occur independently of reorganization throughout the duplicated and unduplicated segments of the macronucleus. The cytochemical characteristics of the forward and rear zones of the reorganization bands are described.

Deoxyribonucleic Acid Synthesis and Distribution During Growth and Amitosis of the Macronucleus of Euplotes

SYNOPSIS. Short sections of deoxyribonucleic acid (DNA) in the elongated macronucleus of Euplotes eurystomus were labeled by means of a short exposure to tritiated (H3‐) thymidine to follow by autoradiography the fate of the labeled DNA during amitotic reorganization of the macronucleus. During amitosis the radioactive DNA that was restricted during interphase to short sections of the nucleus is dispersed and becomes evenly distributed throughout each daughter macronucleus.Although the reorganization bands normally originate only at the ends of the macronucleus, additional bands can start at other places on the macronuclear surface. Bands of the same macronucleus originate with a high degree of synchrony. DNA synthesis is a constant feature of the rear zone of every reorganization band.

Macronuclear duplication in the ciliated protozoan Euplotes.

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.

An electron microscope study of the cytology of the protozoan Euplotes patella.

1. Structurally the "sensory bristles" in Euplotes patella are typical cilia, but no ciliary rootlets connect their bases. 2. The "neuromotor fibrils" are composed of filaments 21 mmicro in diameter. At the point of junction of the filaments with the peripheral ciliary fibrils a granular structure 65 to 90 mmicro in diameter is seen which has dense central and peripheral zones separated by a less dense layer. Information on the interconnection of organelles is expanded. 3. A system of subpellicular fibrils is described. The external fibrillar system described by others could not be found. 4. The motorium is shown to be a mass of intertwining rootlet filaments. 5. The micronucleus is shown to have a spongy, dense material in a less dense material, all of which is surrounded by a double-layered membrane. 6. The double-layered macronuclear membrane contains annuli whose outside diameter is 70 mmicro; the macronuclear bodies are sometimes closely applied to the membrane. In the macronuclear reorganization bands, the solution plane is a fine network, while the reconstruction plane is devoid of structure at the level of resolution observed. 7. The mitochondria are composed of tubules, only occasionally oriented, usually embedded in a surrounding material of lower density. 8. Microbodies whose diameters are 250 to 350 mmicro are frequently observed in close association with mitochondrial surfaces. 9. The food vacuoles, contractile vacuoles, and ciliary vacuoles are bounded by single-layered membranes. In the food vacuoles, the bacteria are surrounded by membranes individually or in small groups. 10. Cytoplasmic rods localized in the oral region, and cytoplasmic granules dispersed at random, are described. No typical ergastoplasm, endoplasmic reticulum, or Golgi material was observed.