Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 m sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 m sorbitol layer. Regeneration occurred over a period of 10–24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater. Received: 26 September 1997 / Revision received: 17 January 1998 / Accepted: 7 February 1998     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

Development and ultrastructure of a microsporidian parasite in midgut cells of the larch sawfly, Pristiphora erichsonii (Hymenoptera: Tenthredinidae)

The developmental stages of a microsporidium from larvae of Pristiphora erichsonii were investigated. Meronts appeared to be the only stage containing a diplokaryon. Nuclei of sporonts in a parasitophorous vesicle underwent at least three divisions and uninucleate sporoblasts developed from these multinucleate sporonts. As many as 38 spores were observed with a vesicle. A thin pansporoblastic membrane limited the vesicle and was derived from rough endoplasmic reticulum of the host cell. The microsporidium was tentatively identified as Pleistophora sp. Infection levels of the microsporidium in natural populations of P. erichsonii reached 25%.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

Ultrastructural characteristics and small subunit ribosomal DNA sequence of Vairimorpha cheracis sp. nov., (Microspora: Burenellidae), a parasite of the Australian yabby, Cherax destructor (Decapoda: Parastacidae)

This is the first record of a species of Vairimorpha infecting a crustacean host. Vairimorpha cheracis sp. nov. was found in a highland population of the Australian freshwater crayfish, Cherax destructor. The majority of spores and earlier developmental stages of V. cheracis sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, each of which gave rise to four sporoblasts in a rosette-shaped plasmodium, so that eight uninucleate spores were produced within the persistent ovoid SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and averaged 3.4x1.9 microm in dimensions. The anterior polaroplast was lamellar in structure, and the posterior polaroplast vesicular. The polar filament was coiled 10-12 times, lateral to the posterior vacuole. The small subunit ribosomal DNA (SSU rDNA) of V. cheracis sp. nov. was sequenced and compared with other microsporidia. V. cheracis sp. nov. showed over 97% sequence identity with Vairimorpha imperfecta and five species of Nosema, and only 86% sequence identity with V. necatrix. The need for a taxonomic revision of the Nosema/Vairimorpha group of species is discussed.     

Occurrence of a New Microsporidan: Enterocytozoon bieneusi n. g., n. sp., in the Enterocytes of a Human Patient with AIDS1

A new microsporidium is reported infesting the enterocytes of a Haitian patient with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 m?m × 0.5 m?. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.     

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.     

Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS

A new microsporidium is reported infesting the enterocytes of a Haitian patients with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 micron X 0.5 micron. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.     

Asexual Reproduction and Segmental Regeneration, but not Morphallaxis, are Inhibited by Boric Acid in Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae)

Body fragmentation, in some animal groups, is a mechanism for survival and asexual reproduction. Lumbriculus variegatus (Müller, 1774), an aquatic oligochaete worm, is capable of regenerating into complete individuals from small body fragments following injury and reproduces primarily by asexual reproduction. Few studies have determined the cellular mechanisms that underlie fragmentation, either regenerative or asexual. We utilized boric acid treatment, which blocks regeneration of segments in amputated fragments and blocks architomic fission during asexual reproduction, to investigate mechanistic relationships and differences between these two modes of development. Neural morphallaxis, involving changes in sensory fields and giant fiber conduction, was detected in amputated fragments in the absence of segmental regeneration. Furthermore, neural morphallactic changes occurred as a result of developmental mechanisms of asexual reproduction, even when architomy was prevented. These results show that fragmentation in L. variegatus, during injury or asexual reproduction, employs developmental and morphallactic processes that can be mechanistically dissociated by boric acid exposure. In regeneration following injury, compensatory morphallaxis occurred in response to fragmentation. In contrast, anticipatory morphallaxis was induced in preparation for fragmentation during asexual reproduction. Thus, various forms of regeneration in this lumbriculid worm can be activated independently and in different developmental contexts.     

浒苔(Enteromorpha prolifera)藻体发育的显微观察

通过显微镜观察以及石蜡切片法观察了浒苔属浒苔藻体的发育过程,阐述了浒苔从孢子释放到形成成熟藻体这一发育过程的显微特征。结果表明:浒苔孢子由成熟体细胞分化发育而成,每个成熟体细胞可形成10～30个孢子;孢子首先分裂为2细胞结构,2细胞具有明显的极性,基细胞发育成为假根,顶端细胞不断进行横分裂形成丝状体;丝状体顶端以下细胞通过纵分裂形成管状叶状体,顶端细胞始终保持横分裂;纵分裂分为切向分裂与径向分裂,切向分裂增加管状叶状体管周细胞数从而使管体增大,径向分裂形成管外壁突起细胞,最终发育为分枝;管状叶状体管腔内部有绒毛状的突起及糖蛋白成分的网架结构;当管状叶状体管周细胞达到30～50h,管腔内部突起消失,网架结构收缩拉紧,管壁细胞贴近形成片状叶状体;整个浒苔藻体始终保持极性发育。     

Cryptosporidia: epicellular parasites embraced by the host cell membrane

The ultrastructure of two gastric cryptosporidia, Cryptosporidium muris from experimentally infected rodents (Mastomys natalensis) and Cryptosporidium sp. 'toad' from naturally infected toads (Duttaphrynus melanostictus), was studied using electron microscopy. Observations presented herein allowed us to map ultrastructural aspects of the cryptosporidian invasion process and the origin of a parasitophorous sac. Invading parasites attach to the host cell, followed by gradual envelopment, with the host's cell membrane folds, eventually forming the parasitophorous sac. Cryptosporidian developmental stages remain epicellular during the entire life cycle. The parasite development is illustrated in detail using high resolution field emission scanning electron microscopy. This provides a new insight into the ultrastructural detail of host-parasite interactions and species-specific differences manifested in frequency of detachment of the parasitophorous sac, radial folds of the parasitophorous sac and stem-formation of the parasitised host cell.     

Ultrastructural study of the microsporidian chytridiopsis typographi (Chytridiopsida: Microspora) infecting the bark beetle,Ips typographus (Scolytidae: Coleoptera), with new data on spore dimorphism

Two types of sporogony of the microsporidian Chytridiopsis typographi in the midgut of adult bark beetle, Ips typographus, have been examined by means of light and electron microscopy. New data are reported on spore dimorphism and on the formation of pansporoblasts in two types of sporogony. Thin-walled spores, larger in size, are formed in a parasitophorous vacuole in the host columnar cells. Thick-walled spores are formed in a minimal vacuole in the host. The ultrastructure of the spore walls and the cyst wall are different from the organization in other microsporidia. Both spore types have identical internal structures and viable spores.     

Encephalitozoon-Like Organisms in Patients with Alveolar Hydatid Disease: Cell Culture, Ultrastructure, Histoimmunochemical Localization and Seroprevalence

ABSTRACT. We found Encephalitozoon -like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cunculi. The spores that developed in parasitophorous vacuoles were 2.0–2.6 × 1.1–1.5 μm: each contained a single nucleus and 4–5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05–1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.     

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Morphology of mitochondrial nucleoids, mitochondria, and nuclei during meiosis and sporulation of the yeast Saccharomycodes ludwigii

The morphology of mitochondrial nucleoids (mt-nucleoids), mitochondria, and nuclei was investigated during meiosis and sporulation of the diploid cells of the ascosporogenic yeast Saccharomycodes ludwigii. The mt-nucleoids appeared as discrete dots uniformly distributed in stationary-phase cells as revealed by 4',6-diamidino-2-phenylindole (DAPI) staining. Throughout first and second meiotic divisions, the mt-nucleoids moved to be located close to the dividing nuclei with the appearance of dots. On the other hand, mitochondria, which had tubular or fragmented forms in stationary-phase cells, increasingly fused with each other to form elongated mitochondria during meiotic prophase as revealed by 3,3' -dihexyloxacarbocyanine iodide [DiOC(6)(3)] staining. Mitochondria assembled to be located close to dividing nuclei during first and second meiotic divisions, and were finally incorporated into spores. During the first meiotic division, nuclear division occurred in any direction parallel, diagonally, or perpendicular to the longitudinal axis of the cell. In contrast, the second meiotic division was exclusively parallel to the longitudinal axis of the cell. The behavior of dividing nuclei explains the formation of a pair of spores with opposite mating types at both ends of cells. In the course of this study, it was also found that ledges between two spores were specifically stained with DiOC(6)(3).     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

Direct Transition of Outgrowing Bacterial Spores to New Sporangia Without Intermediate Cell Division.

Vinter, Vladimir (Syracuse University, Syracuse, N.Y.), and Ralph A. Slepecky. Direct transition of outgrowing bacterial spores to new sporangia without intermediate cell division. J. Bacteriol. 90:803-807. 1965.-A direct transition was observed of the primary cell developed after germination of Bacillus cereus spores into new sporangia without intermediate division stages. Two simple methods were used for replacement of outgrowing spores into diluted medium or saline. Elongated primary cells prevented from division by limitation of nutrients in the suspending medium were able to form new forespores in 8 hr and sporangia in 12 hr. These new sporangia were still marked by attached envelopes of the original spore. Under the same conditions, cells replaced during the first divisions quickly lysed. Spores formed in the elongated primary cell during "microcycle sporogenesis" possessed normal heat resistance and refractility and were later released from sporangia.     

Nadelspora canceri N. G., N. Sp., an Unusual Microsporidian Parasite of the Dungeness Crab, Cancer Magister

ABSTRACT. The microsporidium Nadelspora canceri n. g., n. sp., is described from the striated musculature of the Dungeness crab ( Cancer magister ) in Oregon, USA. The needle-shaped spores were rounded anteriorly, tapered to a posterior point and measured 7.1–11.8 × 0.2–0.3 μm in fixed preparations. The extremely narrow spore diameter prevented observation of morphological details at the light microscopic level and ultrastructural details of mature spores were difficult to resolve. Meronts were not observed and the monokaryotic merozoites and sporonts were not contained within either parasitophorous or sporophorous vesicles. Sporonts were disporoblastic and gave rise to monokaryotic sporoblasts that became narrow and elongate as they developed into immature spores with a developing polar filament. The nucleus was not clearly resolved in mature spores and may have been surrounded by the lamellar polaroplast. The polar filament was of nearly uniform diameter throughout most of its length and ended abruptly about three-fourths of the distance from the anterior end of the spore. Unusual spherical non-membrane bound granules surrounded the polar filament in a spiral arrangement. The new microsporidium resembles members of the family Mrazekiidiae, but differs in lacking a diplokaryon at any stage. It is probably most closely related to Baculea daphniae from which it differs primarily by spore shape and size. The familial relationships of the genus Baculea have not been determined and it is proposed to include it with Nadelspora in the new family Nadelsporidae.