Analysis of possible mechanisms of orthonectid emergence from their hosts

In the present study authors claim that the adult orthonectids can not move through host tissues by themselves. In various species of these enigmatic parasites there are at least two different mechanisms of emission of males and females from the host body. Intoshia linei, the orthonectid from Lineus ruber (Heteronemertini), and Intoshia variabili, the parasite of a flatworm Macrorhynchus crocea, realize the first way of emission. The plasmodium of these species forms tube-like outgrowths, which pierce the host tissues reaching the host body surface. The cytoplasm structure of these outgrowths differs from the cytoplasm of the central mass of plasmodium. Small mitochondria with electron dense matrix, lipid granules and vesicular bodies being common in the central part are absent in these outgrowths. Plasmodial outgrowths reach the host body surface and adult orthonectids move inside them using their cilia and stopping from time to time. The plasmodial outgrowths penetrate the ciliated epithelium, then males and females leave the host. Duration of emission may vary in different species from 6 to 13 days. The second mechanisms of emission is common for the orthonectid parasites of mollusks. Our observations of Rhopalura philinae from the gastropod Philine scabra lead to the conclusion that males and females leave their host practically simultaneously. When the plasmodium attains the terminal stage of its development most of the host entrails are already displaced by plasmodial mass. It causes breaks in host body walls and hence to emission of sexual individuals. During this process, which lasts about 24 hours, the mollusk dies. The same mechanism was observed in Rhopalura littoralis--parasite of the gastropod Onoba aculeus. Our investigations of emission ways reveal that the plasmodium of orthonectids has a potency of directing growth and can form certain structures. The process of forming the plasmodial outgrowths is coordinated in time and space. These outgrowths have certain directions inside the host body and the maturation of sexual individuals is clear related with the development of plasmodium outgrowth system. Our results suggest that forming of plasmodial outgrowths is an element of development of the united and highly integrated system. It is necessary to emphasize the capability of plasmodium to accomplish such morphogenetic transformations. This fact argues that plasmodium is a part of parasite organism and not host cells modified, like some experts supposed.     

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.     

Comparative study of ultrastructure of interlamellar and intralamellar types of Henneguya exilis Kudo from channel catfish

The inter- and intralamellar types of Henneguya exilis Kudo (Myxosporida) infections from channel catfish are similar in spore structure and sporogenesis, but differ in the structure of their plasmodium wall and surface coat and in their relationship with the host cells. The 2 clinical types differ also in the sites of development and growth patterns of plasmodia within a gill filament. Interlamellar plasmodia are limited by 2 outer unit membranes which give rise to both single-and double-membraned pincytic canals. Intralamellar plasmodia are limited by a single outer unit membrane which gives rise to single-membraned pinocytic canals. Interlamellar plasmodia are covered by a fine granular coat of highly variable thicknesses; in some regions there is direct contact between the parasite and cells of the host. There is some evidence that host cell cytoplasm as well as interstitial material are taken in by interlamellar plasmodia. In contrast, intralamellar plasmodia are covered by a fine granular coat of almost uniform thickness, which prevents direct contact between the parasite and cells of the host; probably only interstitial material is taken by these plasmodia.     

The Structure and Origin of the Plasmodium of Rhopalura ophiocomae(Phylum Orthonectida)

Abstract Adult orthonectids develop from germinal cells within a cytoplasmic matrix called a plasmodium. This is generally assumed to be formed by the parasite. In the case of Rhopalura ophiocomae, which lives in the brittle star Amphipholis squamata, the plasmodia occupying the perivisceral coelom are closely associated with the walls of the genital bursae or the gut, and they are covered by peritoneum. They have been reported to contain scattered small nuclei distinct from those within germinal cells, embryos, and adults, but the results of the present study indicate that such nuclei probably do not exist. Furthermore, electron micrographs show that some plasmodia are in continuity with the cytoplasm of contractile cells that lie beneath the peritoneum of a genital bursa or the gut of the host. The matrix of a plasmodium of R. ophiocomaeappears, therefore, to consist of cytoplasm of a contractile cell. It is proposed that after a contractile cell has been entered by an infective cell of the parasite, it hypertrophies, bulging progressively farther into the perivisceral coelom and lifting up the peritoneum, which remains in intimate contact with it.     

Histology, ultrastructure and prevalence of Henneguya piaractus (Myxosporea) infecting the gills of Piaractus mesopotamicus (Characidae) cultivated in Brazil

The histopathological and ultrastructural characteristics of Henneguya piaractus, a parasite of the gill lamellae of Piaractus mesopotamicus, are reported here. Histological analysis showed that the plasmodia were of the intralamellar type. The development of the plasmodia resulted in marked dilatation of the infected lamellae, with the neighbouring lamellae being displaced laterally. Discreet epithelial hyperplasia was observed, but there was no inflammatory reaction. Ultrastructural analysis showed that the plasmodium had a single thin wall that was in direct contact with the host cells. Pinocytic canals and points of phagocytosis were observed in the wall. The prevalence of the parasite varied according to host size, with the lowest prevalence occurring in hosts up to 10 cm long.     

The sequence of regulatory events in the sporulation control network of Physarum polycephalum analysed by time-resolved somatic complementation of mutants

The developmental decision for sporulation of Physarum polycephalum plasmodia is under sensory control by environmental factors like visible light or heat shock and endogenous signals like glucose starvation. Several hours after perceiving an inductive stimulus, plasmodia become committed to sporulation; thereby, they lose their unlimited replicative potential and execute a developmental program that involves differentiation into various cell types required to form a mature fruiting body. Plasmodia are multinuclear single cells which spontaneously fuse upon physical contact. Fusion of mutant plasmodia and cytoplasmic mixing allows complementation studies to be performed at the functional level. Mutant cells altered in their ability to sporulate in response to phytochrome activation by far-red light were cured by fusion with wild-type or other mutant plasmodia. Phytochrome activation in one plasmodium and subsequent fusion with a non-induced plasmodium revealed that complementation of the two mutations depended on (i) which of two genetically distinct plasmodial cells was stimulated; and (ii) on the delay time elapsed between stimulation and cytoplasmic mixing. Such experiments allow us to determine the kinetics and the causal sequence of the regulatory events tagged by mutation.     

The Sequence of Regulatory Events in the Sporulation Control Network of Physarum polycephalum Analysed by Time-resolved Somatic Complementation of Mutants

The developmental decision for sporulation of Physarum polycephalum plasmodia is under sensory control by environmental factors like visible light or heat shock and endogenous signals like glucose starvation. Several hours after perceiving an inductive stimulus, plasmodia become committed to sporulation; thereby, they lose their unlimited replicative potential and execute a developmental program that involves differentiation into various cell types required to form a mature fruiting body. Plasmodia are multinuclear single cells which spontaneously fuse upon physical contact. Fusion of mutant plasmodia and cytoplasmic mixing allows complementation studies to be performed at the functional level. Mutant cells altered in their ability to sporulate in response to phytochrome activation by far-red light were cured by fusion with wild-type or other mutant plasmodia. Phytochrome activation in one plasmodium and subsequent fusion with a non-induced plasmodium revealed that complementation of the two mutations depended on (i) which of two genetically distinct plasmodial cells was stimulated; and (ii) on the delay time elapsed between stimulation and cytoplasmic mixing. Such experiments allow us to determine the kinetics and the causal sequence of the regulatory events tagged by mutation.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure

The characteristics of Myxobolus cuneus n. sp. and its relationship to the host Piaractus mesopotamicus are described based on light and electron microscopy and histological observations. Polysporic plasmodia measuring 20 microm to 2.1 mm in size were found in 63.3 % of the P. mesopotamicus examined. The parasite was found in the gall bladder, urinary bladder, gills, spleen, fins, head surface, liver and heart. Generative cells and disporoblastic pansporoblasts occurred along the periphery of the plasmodia, and mature spores were found in the internal region. The mature spores had a pear shaped body in frontal view, with a total length of 10.0 +/- 0.6 microm and a width of 5.1 +/- 0.3 microm (mean +/- SD). The spore wall was smooth with sutural folds. The polar capsules were elongated, were pear shaped, and equal in size (length 5.7 +/- 03 microm; width 1.7 +/- 0.2 microm), with the anterior ends close to each other. The polar filaments were tightly coiled in 8-9 turns perpendicular to the axis of the capsule. The plasmodia were always found in connective tissue (wall of the arterioles of the gill filaments, serous capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). The parasite caused important damage in the gills, where development occurred in the wall of gill filament arterioles; a mild macrophage infiltrate was also observed. In advanced developmental stages, the plasmodia caused deformation of the arteriole structure, with a reduction and, in some cases, obstruction of the lumen. The parasite was found throughout the period studied and its prevalence was unaffected by host size, season or water properties.     

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7 × 1.1 μm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10 nm) and an electron-dense exospore (2 nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848 bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.     

Henneguya adiposa Minchew (Myxosporida) in the Channel Catfish: Ultrastructure of the Plasmodium Wall and Sporogenesis*

Wall ultrastructure and sporogenesis were studied in plasmodia of Henneguya adiposa Minchew which infects the channel catfish, Ictalurus punctatus (Rafinesque). Plasmodia were located among connective tissue bands of the adipose fin and were always separated from host fibrocytes by collagen fibers. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite's ectoplasm. The membrane was highly convoluted, producing an irregular parasite surface, and was covered by a fine granular coat of almost uniform thickness. Early sporogenic stages were located in a zone of cytoplasm rich in mitochondria, just interior to the zone of pinocytic canals. Later sporogenic stages, including mature spores, were concentrated in the center of the plasmodia. Sporogenesis began with the envelopment of one generative cell, the sporont, by a 2nd, nondividing, cell—the enveloping cell. The sporont and its progeny proceeded through a series of divisions until 10 cells were present within the enveloping cell. Once divisions were completed, the 10 cells became arranged into 2 identical spore-producing units, each consisting of one binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Later stages of spore development indicated that capsulogenesis, valvogenesis and sporoplasm maturation occurred concomitantly.     

Comparative Study of Ultrastructure of Interlamellar and Intralamellar Types of Henneguya exilis Kudo from Channel Catfish*†

SYNOPSIS. The inter- and intralamellar types of Henneguya exilis Kudo (Myxosporida) infections from channel catfish are similar in spore structure and sporogenesis, but differ in the structure of their plasmodium wall and surface coat and in their relationship with the host cells. The 2 clinical types differ also in the sites of development and growth patterns of plasmodia within a gill filament. Interlamellar plasmodia are limited by 2 outer unit membranes which give rise to both single-and double-membraned pinocytic canals. Intralamellar plasmodia are limited by a single outer unit membrane which gives rise to single-membraned pinocytic canals. Interlamellar plasmodia are covered by a fine granular coat of highly variable thicknesses; in some regions there is direct contact between the parasite and cells of the host. There is some evidence that host cell cytoplasm as well as interstitial material are taken in by interlamellar plasmodia. In contrast, intralamellar plasmodia are covered by a fine granular coat of almost uniform thickness, which prevents direct contact between the parasite and cells of the host; probably only interstitial material is taken by these plasmodia.     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.     

Cytological and molecular description of Hamiltosporidium tvaerminnensis gen. et sp. nov., a microsporidian parasite of Daphnia magna, and establishment of Hamiltosporidium magnivora comb. nov

We describe the new microsporidium Hamiltosporidium tvaerminnensis gen. et sp. nov. with an emphasis on its ultrastructural characteristics and phylogenetic position as inferred from the sequence data of SSU rDNA, alpha- and beta-tubulin. This parasite was previously identified as Octosporea bayeri Jírovec, 1936 and has become a model system to study the ecology, epidemiology, evolution and genomics of microsporidia - host interactions. Here, we present evidence that shows its differences from O. bayeri. Hamiltosporidium tvaerminnensis exclusively infects the adipose tissue, the ovaries and the hypodermis of Daphnia magna and is found only in host populations located in coastal rock pool populations in Finland and Sweden. Merogonial stages of H. tvaerminnensis have isolated nuclei; merozoites are formed by binary fission or by the cleaving of a plasmodium with a small number of nuclei. A sporogonial plasmodium with isolated nuclei yields 8 sporoblasts. Elongated spores are generated by the most finger-like plasmodia. The mature spores are polymorphic in shape and size. Most spores are pyriform (4·9-5·6×2·2-2·3 μm) and have their polar filament arranged in 12-13 coils. A second, elongated spore type (6·8-12·0×1·6-2·1 μm) is rod-shaped with blunt ends and measures 6·8-12·0×1·6-2·1 μm. The envelope of the sporophorous vesicle is thin and fragile, formed at the beginning of the sporogony. Cytological and molecular comparisons with Flabelliforma magnivora, a parasite infecting the same tissues in the same host species, reveal that these two species are very closely related, yet distinct. Moreover, both cytological and molecular data indicate that these species are quite distant from F. montana, the type species of the genus Flabelliforma. We therefore propose that F. magnivora also be placed in Hamiltosporidium gen. nov.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Nuclei in the plasmodium of Intoshia variabili (Orthonectida) as revealed by DAPI staining

DAPI staining of wholeamounts was used to reveal the parasitic plasmodium of the orthonectid Intoshia variabili in its host, the turbellarian Macrorhynchus crocea. The nuclei of the parasite differ drastically from those of the host in size, morphology, and the estimated DNA content. Our findings indirectly support the idea that the orthonectid plasmodium is a distinct parasitic organism, rather than modified host cells.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an Intracellular Parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other Filamentous Brown Algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Nuclear sieving ofDidymium iridis plasmodia

The normal diploid plasmodium of the slime moldDidymium iridis can pass through a membrane filter having a uniform pore size of 3 μm and form a new reticulum on the other side; tetraploid plasmodia will not pass through this filter. In similar experiments using senescent plasmodia (which contain both diploid and large polyploid nuclei) only diploid nuclei and cytoplasm pass through the filter. The resultant “filtered” plasmodia live longer than untreated subline plasmodia. Studies involving the transfilter contact of two plasmodia across 1-μm-pore-size Nucleopore filters indicate that heterokyaron incompatibility does not involve a cytoplasmic toxin.     

Henneguya adiposa Minchew (Myxosporida) in the channel catfish: ultrastructure of the plasmodium wall and sporogenesis.

Wall ultrastructure and sporogenesis were studied in plasmodia of Henneguya adiposa Minchew which infects the channel catfish, Ictalurus punctatus (Rafinesque). Plasmodia were located among connective tissue bands of the adipose fin and were always separated from host fibrocytes by collagen fibers. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite's ectoplasm. The membrane was highly convoluted, producing an irregular parasite surface, and was covered by a fine granular coat of almost uniform thickness. Early sporogenic stages were located in a zone of cytoplasm rich in mitochondria, just interior to the zone of pinocytic canals. Later sporogenic stages, including mature spores, were concentrated in the center of the plasmodia. Sporogenesis began with the envelopment of one generative cell, the sporont, by a 2nd, nondividing, cell--the enveloping cell. The sporont and its progeny proceeded through a series of divisions until 10 cells were present within the enveloping cell. Once divisions were completed, the 10 cells became arranged into 2 indentical spore-producing units, each consisting of one binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Later stages of spore development indicated that capsulogenesis, valvogenesis and sporoplasm maturation occurred concimitantly.     

Histochemistry and ultrastructure of the metacercarial cyst of Bolbogonotylus corkumi (Trematoda: Cryptogonimidae).

Histochemical and ultrastructural studies were conducted on the metacercarial cyst of the cryptogonimid trematode Bolbogonotylus corkumi from the muscle tissue of fantail darters Etheostoma flabellare. The metacercarial cyst consisted of an outer host capsule and an inner parasite cyst. The host capsule was composed of an outer region of fibroblasts, collagen, macrophages, and unidentified cells, and an inner region containing degenerating cells. The parasite cyst was thin, homogenous, and noncellular in nature. The host capsule stained strongly for connective tissue and proteins and moderately for lipids, nucleic acids, nonspecific esterase activity, and acid and alkaline phosphatase activities. The parasite cyst stained intensely for acid mucopolysaccharides and moderately for acid phosphatase activity. A thick glycocalyx occurred between the parasite cyst and metacercarial tegument.