The Fine Structure of the Phoront of the Apostomatous Ciliate,Hyalophysa chattoni*

The phoront of the apostomatous ciliate, Hyalophysa chattoni, is an encysted stage that is carried on the exoskeleton of its crustacean host until the ecdysis of the host. At molting the phoront rapidly metamorphoses to the feeding stage, excysts, and immediately begins to feed on exuvial fluid trapped in the cast-off exoskeleton. The fine structure of the resting phoront resembles that of the preceding migratory stage, the tomite. A prominent ventral tuft of cilia, the ogival field, has vanished, and the trichocysts that paralleled the kinetics have disappeared. The dense inclusion bodies that were concentrated around the mouth and falciform fields have dispersed and greatly decreased in number. The cytoplasm and its membranous organelles do not appear visibly condensed or altered from the preceding stage in the life cycle. The phoront is merely quiescent instead of dormant. Unlike the few ciliate cysts previously examined by electron microscopy, the phoront's cyst is not divisible into separable layers. It resembles the loricae of certain suctoria in being formed principally of a fibrous substance, the outer surface of which has a paracrystalline pattern. The peduncle attaching the cyst to the crab's gill is a continuation of the cyst wall although its structure is somewhat modified. The most conspicuous innovation in the phoront's fine structure is the massive tracts of microtubules that run longitudinally through the macronucleus. The microtubules are in intimate contact with Feulgen-positive chromatin masses which are crowded toward the periphery of the macronucleus.     

The Fine Structure of the Mature Tomite of Hyalophysa chattoni

SYNOPSIS. The fine structure of the tomite stage of Hyalophysa chattoni was examined with particular attention to its kinetal apparatus. The pellicle, thick and dense compared with that of other ciliates, is formed of three layers. The inner layer is composed of short fibrils oriented perpendicular to the surface. The cytoplasm around the oral passage and beneath falciform field 8 is crowded with dense inclusion bodies of unknown function. Dorsal to the oral passage is the rosette, a disc-shaped organelle subdivided by septa in the form of incomplete radii about a central chamber containing a tuft of cilia. The septa are composed of 3 membranes enclosing a fine layer of cytoplasm. At their inner ends 20 mμ fibers run dorsally and ventrally. Dense clumps of fibrous material line the luminal surface of the septa. Rows of fusiform trichocysts parallel the kineties. The trichocysts are composed of a finely periodic, moderately electron-dense material surrounded by 20 mμ fibrils oriented along the long axis of the trichocyst. Between and below the kinetosomes and the rows of trichocysts are electron-dense vesicles 300 mμ in diameter and bounded by a loose membrane. The large “trichocysts,” the “gros trichocystes” of Chatton and Lwoff, whose appearance heralds the beginnings of trichocystogenesis, prove to be canaliculi opening to the surface. Four separate ciliary membrane systems—the oral ciliature (XYZ), falciform field 8, falciform field 9, and the ogival field—are located on the ventral surface of the tomite. Each differs from the others and from the somatic kineties in the fibrillar organization around its kinetosomes. In the somatic kineties the kinetodesmos is a dense, periodic fiber which is formed of stacks of up to 18 subfibers, each arising from the base of a kinetosome. The kinetosomes are short (300 mμ) and contain dense central granules. In some kineties, alternating between the kinetosomes, are elliptical kinetosome-like structures which do not bear cilia and perhaps provide a reservoir of kinetosomes for future growth of the kinety.     

Salinity tolerance in Hyalophysa chattoni (Ciliophora, Apostomatida), a symbiont of the estuarine grass shrimp Palaemonetes pugio

The apostome ciliate Hyalophysa chattoni, a symbiont of the estuarine grass shrimp Palaemonetes pugio, was tested for its growth and reproductive ability in a wide range of salinities from 0.1 to 55 ppt. Shrimp, with their attached ciliates, were slowly acclimated to different salinities in order to assess protozoan cell size and division. The trophont and tomont stages of the ciliate life cycle were analyzed. In both stages, cell size increased with salinity from 0.1 to 20 ppt. Cell size leveled in the 20-35 ppt range, and decreased at higher salinities. The number of daughter cells produced per tomont cyst correlated with increased cell size, and also correlated with increased salinity. Additionally, increased salinity correlated with an increase in the percentage of cells able to divide and excyst as tomite stages. These results indicate that H. chattoni is able to grow and divide more effectively at salinities closer to seawater than in the estuarine environment from which they were collected. Though able to survive salinities from 0.1 to 55 ppt, the species is better adapted for an existence in the higher salt concentrations.     

The Fine Structure of the Cytostome of the Apostomatous Ciliate Hyalophysa chattoni*

SYNOPSIS. The fine structure of the organelles concerned with the ingestion of exuvial fluid by the trophont of the apostome ciliate, Hyalophysa chattoni, has been examined. One of the taxonomic characteristics of the order Apostomatida is that cytostomes of ciliates within the taxon are reduced and evolving toward astomy. When examined by electron microscopy the cytostome of H. chattoni appears as a small region of active pinocytosis which is continuous with a very large cortical area, the extended cytostome. The fine structure of the extended cytostome resembles that of the cytostomes of ciliates from other orders in that it is covered by a single membrane underlain with microtubular ribs. Beneath the extended cytostome are accumulations of peculiar organelles that may represent stored membrane for recycling during food vacuole formation. Associated with the site of pinocytosis is a complex fiber that may be contractile.     

Hyalophysa lynni n. sp. (Ciliophora,Apostomatida), a new pathogenic ciliate and causative agent of shrimp black gill in penaeid shrimp

The parasitic ciliate causing shrimp black gill (sBG) infections in penaeid shrimp has been identified. The sBG ciliate has a unique life cycle that includes an encysted divisional stage on the host’s gills. The ciliature of the encysted trophont stage has been determined and is quite similar to that of the closely related apostomes Hyalophysa bradburyae and H. chattoni. Hyalophysa bradburyae is a commensal ciliate associated with freshwater caridean shrimp and crayfish, while H. chattoni is a common commensal found on North American marine decapods. Based on 18S rRNA gene sequence comparisons, the sBG ciliate is more closely related to the marine species H. chattoni than to the freshwater species H. bradburyae. The unique life cycle, morphology, 18S rRNA gene sequence, hosts, location, and pathology of the sBG ciliate distinguish this organism as a new species, Hyalophysa lynni n. sp.     

Growth, Fine Structure and Cyst Formation of a Microbial Mat Ciliate: Pseudocohnilembus pusillus (Ciliophora, Scuticociliatida)

ABSTRACT Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.     

Hyalophysa Iwoffi sp.n. from the Fresh-water Shrimp Palaemonetes paludosus and Revision of the Genus Hyalophysa

SYNOPSIS. Hyalophysa Iwoffi sp. n. is described from the shrimp, Palaemonetes paludosus . Its life cycle is like that of other exuviotrophic apostomes. Hyalophysa Iwoffi differs from the type species, H. chattoni , in the details of its infraciliature and the shape of its macronucleus during the trophont stage. Emended diagnoses of the genus, Hyalophysa , and the species, H. chattoni , also are given.     

The Fine Structure of the Cyst Wall of the Ciliated Protozoon Didinium nasutum1

SYNOPSIS. Electron microscope observations of the complex cyst wall of Didinium nasutum are reported. The cyst wall is composed of 3 major coats. The outermost coat, the ectocyst, consists of short strands of filamentous material which forms a diffuse, amorphous layer approximately 8–9 μ thick. Culture debris, bacteria and unidentified inclusions have been observed adhering to the outer coat. The mesocyst, approximately 2.5 μ thick, has 2 distinct regions. The outer region is differentiated into several slightly thicker layers which in face view have a honeycomb appearance. The deeper region of the mesocyst consists of compact lamellae lacking the obvious honeycomb appearance of the layers of the outer region. Finally, the endocyst (0.3 μ thick), which arises in the mature cyst in the space that develops between the pellicle and the mesocyst, consists of delicate fibrils in a compact matrix. Both mesocyst and endocyst may be undulant and folded. The structure, origin and possible relationships of the various coats composing the cyst wall are discussed. The present study also contributes information on the role and fate of mucocysts and other cytoplasmic structures during the formation of the cyst wall.     

A new species in the genus Cyrtostrombidium (Ciliophora,Oligotrichia, Oligotrichida): its morphology,seasonal cycle and resting stage

Cysts of an oligotrich ciliate were collected from natural sediment samples in Onagawa Bay, northeastern coast of Japan, and examined for their excystment capability. A high excystment ratio was obtained at lower temperatures of 10 or 15 degrees C; no excystment occurred at 20 degrees C. Excysted vegetative cells were observed after protargol staining and were identified as a new species, Cyrtostrombidium boreale n. sp. The seasonal changes in the vegetative population and sedimentation of newly formed cysts were also investigated in situ. Planktonic vegetative cells were abundant during the cold season from February to May, when the water temperature was lower than 10 degrees C. Mass encystment occurred abruptly just after the seasonal peak of the vegetative population in April. These results indicate that C. boreale is a cold-water species and aestivates during the longer, warm period from late spring to fall.     

The fine structure of secretion in Hyalophysa chattoni: formation of the attachment peduncle and the chitinous phoretic cyst wall.

The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electrondense dense secretory bodies from the cell's ventral surface.     

Fine Structure of the Cyst and Some Sporulation Stages of Ichthyosporidium (Microsporida)*

SYNOPSIS. Ichthyosporidium sp. Schwartz, 1963, apparently identical with the type species, I. giganteum (Thélohan, 1895) Swarczewsky, 1914, was studied with the electron microscope. Only late stages, a mature cyst containing sporulation stages and a cyst in the terminal (necrotic) stage were observed. The cyst, originating from host tissue, is a highly organized structure that is integrated with the surrounding connective tissue by means of numerous conspicuous processes. It is interpreted as essentially a manifestation of a defensive reaction of the host that is elicited by the parasite and then used to its advantage. Eventually the cyst dies and disintegrates. This type of cyst, peculiar among those associated with microsporidia, may be regarded as a distinctive character of the poorly defined genus Ichthyosporidium. Other observations let to an hypothesis which reconciles several different views regarding the identity of the Golgi complex. According to this new interpretation, these different views concern different aspects af the total complex. When all such views are integrated, a “classical Golgi” can be recognized in the presporoblastic stages and the “primitive Golgi” concept disappears. This “classical Golgi” then becomes highly modified during spore morphogenesis, giving rise to many of the internal organelles that are peculiar to the spore.     

The Formation and Structure of the Cell Wall in Fibres and Tracheids

A study has been made of the changes in organization of theprimary wall of xylem fibres and tracheids during the phaseof surface growth using both direct electronmicroscopic observationsand replica techniques. It has been demonstrated that the transversemicrofibril orientation on the inner surface of the cell walldiffers from that on the outer surface. It has been concludedthat the observations made are consistent with some form ofmulti-net mechanism of growth in these cells. Autoradiographicstudies were consistent with this conclusion. Measurements of the variation in path difference in fibres andtracheids undergoing secondary wall formation showed a maximumnear the centre of the cells decreasing towards the cell tips.From these observations and parallel autoradaographic studiesit has been concluded that secondary thickening begins nearthe centre of the cells and progresses towards the cell tips.Since all layers of the secondary wall are lamellated theseobservations imply that more lamellae are present near the centreof the cells than at their tips, so that as secondary thickening proceeds the concentric lamellae of microfibrils grow progressivelytowards the ends of the cells. The lamellae of the secondarywall appear to develop in close association with the limitingcytoplasmic membrane (the plasmalemma). No evidence was obtainedof the inclusion of cytoplasmic components in the developingsecondary wall. At the conclusion of secondary thickening thecytoplasmic membranes retract on to the inner surface of thesecondary wall, forming the so-called ‘terminal lamella’which shows some fibrillar texture. It has been concluded thatthis appearance reflects some intrinsic texture of the lamellaitself, rather than the presence of cellulose microfibrils.     

Fine Structure of Gametogony and Oocyst Formation in Sarcocystis sp. in Cell Culture

Fine structure of gametocytes and oocyst formation of Sarcocystis sp. from Quiscalus quiscula Linnaeus grown in cultured embryonic bovine kidney cells was studied. Microgametocytes measured up to ～5 μm diameter. During nuclear division of the microgametocyte, dense plaques were found adjacent to the nucleus just beneath the pellicle; occasionally microtubules were present within these plaques. These microtubules subsequently formed 2 basal bodies with a bundle of 4 microtubules between them. Microgametocytes also contained numerous mitochondria, micropores, granules, vacuoles, and free ribosomes. Each microgamete was covered by a single membrane and consisted of 2 basal bodies, 2 flagella, a bundle of 4 microtubules, a perforatorium, a mitochondrion, and a long dense nucleus which extended anteriorly and posteriorly beyond the mitochondrion. The bundle of 4 microtubules is thought to be the rudiment of a 3rd flagellum. Macrogametes were covered by a double membrane pellicle, and contained a large nucleus (～2.5 μm), vacuoles, and a dilated nuclear envelope connected with the rough endoplasmic reticulum (ER). In young macrogametes (～4 μm), the ER was arranged in concentric rows in the cortical region, and several sizes of dense granules were found in the cytoplasm. However, in later stages (～8 μm) the ER was irregularly arranged and was dilated with numerous cisternae; only large dark granules remained and a few scattered polysaccharide granules were found. No Golgi apparatus or micropores were observed. After the disappearance of dark granules 5 concentric membranes appeared. Four of these fused to form an oocyst wall composed of a dense outer layer (～66 nm thick) and a thin inner layer (～7 nm). The 5th or innermost membrane surrounded the cytoplasmic mass which was covered by a 2-layered pellicle and contained a nucleus, small amounts of ER, large vacuoles, and mitochondria. The sexual stages described greatly resemble those of Eimeria and Toxoplasma.     

The Fine Structure of the Cinctum of Ellobiophrya conviva (Ciliophora: Peritricha)

Ellobiophrya conviva clasps tentacles of the bryozoan Bugula neritina with a ring-like structure formed from aboral extensions of its body that taper into two slender arms. The tips of the arms overlap and join to form a unique organelle, the bouton. Each arm contains a massive myoneme that splays out at the bouton. The bouton consists of the cupped tips of the arms and a cavity, which is filled with dense homogeneous material. Long digitations containing longitudinal microtubules at their periphery project from the inner surface of the tip of each arm into the cavity. Deep folds of pellicle with pores opening into their depths line the wall of the cavity. Conventional kinetosomes are not visible in the bouton, but circular or elliptical arrays of microtubules are found at the bases of digitations. The nonfunctional scopula of the adult is in a depression enclosed by pellicular folds. The bouton is distant from the scopula, but its fine structure somewhat resembles it, supporting Chatton and Lwoff's hypothesis that the cinctal arms carry parts of the scopula at their tips. The fine structure of the cinctum supports their suggestion that the cinctal arms are homologous to the spasmonemes of vorticellid peritrichs.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

Notes on the Distribution of Hyalophysa and Gymnodinioides on Crustacean Hosts in Coastal North Carolina and a Description of Hyalophysa trageri sp. n.

SYNOPSIS. Hyalophysa chattoni Bradbury, Hyalophysa Iwoffi Bradbury & Clamp, and Gymnodinioides inkystans Minkiewicz are found on species of palaemonids, astacurans, grapsid crabs, ocypodids, anomurans, and portunids in coastal North Carolina. Hyalophysa trageri sp. n. is described from species of Uca and Sesarma. Its life cycle is like that of other exuviotrophic apostomes. Hyalophysa trageri differs from other species in the genus in the pattern of its infraciliature and the shape of the macronucleus during the trophont stage.
In coastal North Carolina, H. chattoni is euryhaline and infests a great number of species. Hyalophysa Iwoffi is found in many locations on the freshwater shrimp Palaemonetes paludosus (Gibbes), and in one location on the crayfish Cambarus sp. Hyalophysa trageri is the most limited in range, being found only on semiterrestrial crabs. On some host species G. inkystans is found alone, but is sympatric with Hyalophysa on others.
Possible factors restricting the distribution of these apostomes are discussed. These include the structure of their encysted stages, salinity, and the behavior of their hosts.     

Observations on the Fine Structure of Plant Cell Walls III. The Sieve Tube Wall in Cucurbita

The sieve tube wall in Cucurbita was examined in ultra-thinsections of petioles treated in different ways for the removalof non-cellulosic wall components. The sections were stainedwith permanganate. The microfibrillar components of the wallare arranged in concentric lamellae. The earliest (outermost)part of the wall is similar to that of ordinary parenchyma inhaving its lamellae composed of thinly-distributed microfibrilsreadily separated from one another by certain treatments suchas pectinase extraction. In the characteristically-thickenedinner (nacreous) layer the microfibrils are very densely packedand the lamellae do not separate readily. The microfibrils inthis layer of the wall are very close to transverse and the‘crossed fibrillar’ orientation is not easily discernible.