FINE STRUCTURE OF MEMBRANOUS AND MICROFIBRILLAR SYSTEMS IN THE CORTEX OF PARAMECIUM CAUDATUM

An electron microscope study of the cortex of Paramecium caudatum has revealed new details pertinent to several unresolved problems. The lateral boundaries of the alveoli do not regularly follow the crests of the polygonal ridges and thus their staining with silver cannot account for the external lattice seen by light microscopists. A granulo-fibrillar material is present, however, within the peaks of the ridges, which would account for the external lattice if so stained. Perforations are present between adjacent alveoli which make the whole mosaic of alveolar sacs within the cell's cortex continuous—both the membranes and the lumen. A microfibrillar system exhibiting a cross-striated pattern lies in the superficial cortex. These bands are inserted at their ends in the epiplasm and have a fine structure and arrangement suggesting a muscular function. The infraciliary lattice is a branching system of fibers with electron-opaque posts at the center of each branching locus. This system is distinct from the striated bands in morphology and in space. The epiplasm is discontinuous along the crests of the ridges, which may account for the pellicles' disposition to tear along these lines. A three-dimensional drawing is presented to show the interrelationships between the above membranous and microfibrillar systems.     

Basal Body-Associated Nucleation Center for the Centrin-Based Cortical Cytoskeletal Network in Paramecium

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.     

Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.     

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea)

This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens.     

Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

Ultrastructure of the Parabasalid Protist Holomastigotoides

ABSTRACT. The ultrastructure of two species of Holomastigotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber, axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.     

Rumen ciliates from Tanzanian short horn zebu cattle,Bos taurus indicus,and the infraciliature of Entodinium palmare n.sp. and Enoploplastron stokyi ()

Rumen ciliates of ten Tanzanian short horn zebu cattle were examined. A total of 15 genera and 46 species were identified, including a new Entodinium species. The ciliate density was 22.2×10⁴ ml^?1. The number of species per host and the diversity index showed high values, 33.8 and 2.80, respectively. Rumen ciliates had a low percentage composition of the genus Entodinium (7.0–25.0%) and a slightly higher percentage composition of the genera, Eudiplodinium (19.3%), Diplodinium (14.1%), and Ostracodinium (13.1%). Entodinium palmare n.sp., Eudiplodinium kenyensis, and Enoploplastron stokyi were found in all cattle examined. The former two species have been found only in African zebu cattle. Entodinium palmare n. sp. has a characteristic right surface of the body like the “palm of a hand” because of the concave part on the postero-dorsal part of the body, and has the same pattern of infraciliary bands as in other Entodinium species. Enoploplastron stokyi has a characteristic pattern of infraciliary bands analogous to those of Epidinium ecaudatum and Ostracodinium mammosum; with the right side of the adoral polybrachykinety gradually tapering and a slender short vestibular polybrachykinety.     

Electrokaryotypes of macronuclei of several Paramecium species

A comparative study of macronuclear DNA molecules from the following Paramecium species: the P. aurelia complex, P. caudatum, P. bursaria, P. putrinum and P. multimicronucleatum was performed using pulsed-field gel electrophoresis. The electrophoretic pattern was constant and unique for each species, and is referred to herein as its electrokaryotype. Large differences were observed between Paramecium species according to the range and major size of macronuclear DNA fragments, while different strains of the same species, even belonging to different syngens, were characterized by the same electrokaryotype. In this respect sibling species from the P. aurelia complex are as similar as syngens in other Paramecium species, but are unlike conventional species. The principles and value of electrokaryotype analysis for application to ciliates are discussed.     

The architecture of the flagellar apparatus ofPrymnesium patellifera (Prymnesiophyta)

The flagellar apparatus of the small prymnesiophytePrymnesium patellifera has been analysed and a reconstruction is presented. Externally, the cell carries two sub-equal flagella and a short non-coiling haptonema. Within the cell, there are four microtubular roots and a number of fibrous bands, the latter interconnecting the two basal bodies and the haptonema base. One of the roots (r1) consists of a sheet of up to 25 microtubules originating close to the proximal extremity of the haptonema base, but the other three roots are composed of between 1 and 4 microtubules only. Distally, a large striated fibrous auxiliary connecting root extends across the anterior part of the cell linking root r1 and a mitochondrial profile on the opposite side of the cell. The arrangement of the components of the flagellar apparatus ofP. patellifera is commensurate with the general pattern found in many prymnesiophytes other than members of the Pavlovales, but there are a number of differences in detail from the other species described hitherto.     

Infraciliature of eight Triplumaria species (Ciliophora, Entodiniomorphida) from Asian elephants with the description of six new species

Intestinal ciliates excreted in the feces of Asian elephants were surveyed. Fourteen species in the order Entodiniomorphida were detected. Nine Triplumaria species in the family Cycloposthiidae were found. Using the silver impregnation, two known species, T. antis and T. dvoinosi, were redescribed and six new species, T. sukuna n. sp., T. zuze n. sp., T. solea n. sp., T. suwako n. sp., T. fulgora n. sp., and T. harpagonis n. sp., were described. T. sukuna, T. zuze, T. solea, and T. suwako have the perivestibular polybrachykinety along the vestibular opening. The buccal infraciliary bands of T. suwako are similar to those of T. selenica found from elephants and the buccal infraciliary bands of T. sukuna, T. zuze, and T. solea are similar to those of T. grypoclunis described from rhinoceroses. T. antis, T. dvoinosi, T. fulgora, and T. harpagonis have the vestibular polybrachykinety extending down inside the vestibulum as found in rumen ciliates in the family Ophryoscolecidae. The caudalial ciliary zones of T. dvoinosi and T. fulgora were retractable as found in rumen ophryoscolecids. Raabena bella and Pseudoentodinium elephantis showed high composition values over 30%. Ciliate densities in the three fecal samples were 0.15, 1.09, and 2.07×10(4)/ml.     

THE FLAGELLAR APPARATUS OF CHRYSOCHROMULINA SP. (PRYMNESIOPHYCEAE)1

The flagellar apparatus of an undescribed species of Chrysochromulina Lackey that bears “eyelash” scales is reconstructed. The transitional region consists of two transitional plates each with an axosome, with no stellate pattern between them. Fine osmiophilic rings lie between the flagellar membrane and the outer doublets in the transitional region. The two jagella and the haptonema are inserted in a subapical depression that is lined ventrally by a spine-like projection formed by one of the parietal chloroplasts. The angles of insertion are similar to those of some other Chrysochromulina species in that both the haptonema and the right basal body lie at an extreme angle to the left basal body. The connectives of the apparatus consist of a striated distal band with a dorsal extension to the R1 and a ventral extension overlying the R2, a striated distal accessory band, an auxiliary connective from the right basal body to the adjacent ventral chloroplast, a well-developed intermediate band, two striated proximal bands, and a striated proximal accessory band. Of the microtubular roots in this Chrysochromulina species, three are associated with the left side of the cell (an R1 of 8+3; a small crystalline compound root, the R1C, associated with the R1; an R2 of three micro-tubules), and two are associated with the right basal body (an R3 of 2/2 microtubules with which the single-stranded R4 converges to form a 2/2+1 and then a 2/3 tiered arrangement). Comparisons are drawn with other species in the genus and related genera, particularly Prymne-sium.     

Paramyosin in invertebrate muscles. I. Identification and localization

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.     

C-banding analysis of six species of lung flukes, Paragonimus spp. (Trematoda: Platyhelminthes), from Japan and Korea

We examined C-banded karyotypes of six species of lung flukes from Japan and Korea; diploid and triploid Paragonimus westermani, P. miyazakii, P. ohirai, P. iloktsuenensis and P. sadoensis, with special reference to their karyotypic diversification. C-band analysis between the diploid and the triploid westermani revealed that two of three homologues of the triploid resembled those of the diploid in C-band pattern, while the remaining chromosome showed a different pattern from any species examined here. This karyological evidence indicates that the triploid is allotriploid probably induced by interspecific hybridization between the diploid westermani and an unknown species; we, therefore, suggest that the triploid westermani is an independent species and synonymous with P. pulmonalis (Miyazaki 1978). As the morphologically similar three species, ohirai, iloktsuenensis and sadoensis, had the same C-band polymorphism in chromosome No. 4, these species are classified as the local races of P. ohirai. Paragonimus miyazakii has one common C-band (5q) with the diploid westermani, but other bands (1q, 4q, 6q, 7p and 7q) are different. From these observations, the six species examined are phylogenetically divided into three groups: (1) westermani group containing diploid and triploid (= pulmonalis) species, (2) miyazakii and (3) ohirai including two geographic races, iloktsuenensis and sadoensis.     

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross- bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick- freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.     

A new Chinese species of Psathyrostachys (Poaceae) with notes on its karyotype

The new species Psathyrostachys stoloniformis native to China is described. The karyotype was investigated by C-, N-, and Ag-banding. The new species has 2n = 2x = 14, including 8 metacentric and 6 SAT-chromosomes with minute satellites, distributed either on 3 or on 4 chromosome pairs. The C-banding pattern comprises from one to six conspicuous and from zero to three very small bands per chromosome. Often two bands placed close together appear as one large band. The karyotype suggests that P. stoloniformis is closely related to P. juncea. N-banding produced very weak or no bands. Meiotic analyses of interspecific hybrids revealed a high chiasma frequency indicating that P. stoloniformis has the same basic genome (N) as P. huashanica , and P. fragilis.     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: essential role of a centrin-rich filamentous cortical network, the infraciliary lattice

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.     

Pneumocystis carinii: immunoblotting and immunofluorescent analyses of serum antibodies during experimental rat infection and recovery

Serum antibodies to Pneumocystis carinii were measured in rats by the indirect fluorescent antibody and immunoblotting techniques. Serum IgG and IgM antibodies developed with environmental exposure to P. carinii, were low or absent during immunosuppression to induce P. carinii pneumonia, and rose when immunosuppression was withdrawn. The IgG and IgM antibodies formed at the same time, but the titers of each antibody varied in individual rats. Serum IgG antibodies by immunoblotting recognized bands of 45, 50, and 116 kDa as the major reactive moieties of P. carinii. The bands were detected with sera from all rat groups in a temporal pattern which closely paralleled antibody formation by indirect immunofluorescence. The pattern of immunoblotting reactivity varied among individual rats, particularly with immunosuppression. Additional bands were detected with prolonged exposure to P. carinii. Thus, the rat makes both IgG and IgM antibodies to P. carinii, and specific P. carinii antigens identified in this immune response might be targeted for future serologic studies.     

COMPARATIVE ULTRASTRUCTURE OF VEGETATIVE AND SEXUAL THECAE OF PERIDINIUM LIMBATUM AND PERIDINIUM CINCTUM (DINOPHYCEAE)

Peridinium limbatum (Stokes) Lemmerman reproduces sexually in nitrogen deficient medium. Sexual reproduction is homothallic and similar to that reported for other Peridinium species. Thecate planozygotes of P. limbatum and P. cinctum enlarge greatly. Suture bands connecting thecal plates enlarge unequally in P. cinctum producing a “warty” zygote and equally in P. limbatum maintaining the regular but enlarged appearance of the cell. Zygotes can be recognized by greatly enlarged striated suture bands.     

The evolutionary significance of ultrastructural variations in the micronuclear spindle apparatus in the genus Paramecium.

Ultrastructural variations of micronuclear mitosis in three species of Paramecium have been reviewed for the purpose of hypothesizing possible evolutionary relationships. P. bursaria is considered to be the most primitive species because it has complex kinetochore-equivalents termed microlamellae which attach to microfilaments. P. multimicronucleatum is considered more advanced since it has typical kinetochores which intercalate between the kinetochores and polar microfilaments. P. aurelia is considered the most advanced member of the genus, and has typical diffuse or well defined kinetochores and chromosomal microtubules while it lacks obvious meshworks of microfilaments. A hypothetical model of chromosome movement in the genus involves the interaction of microtubules and microfilaments.