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All Microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore, transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200nm, noticeably larger than fixed-sectioned, or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

[Extract] Microsporidia are intracellular pathogens of animals andhumans. Although not hyphal, a recent study suggests theyare fungi descended from an ancestral zygomycete. Theyhave a unique method for invading susceptible tissue: an internal coiled tube everts rapidly and sporoplasm moves into the cell as if through a hypodermic needle. In the human eye, microsporidial infection presents as keratoconjunctivitis or stromal keratitis. The former has been reported in HIV+ patients because of Encephalitozoon and in immunocompetent patients because of several genera. Stromal infection is seen in immunocompetent patients and is caused mostly by Vittaforma corneae. We report a case in which keratoconjunctivitis preceded deep stromal infection in a patient with a low neutrophil count.

Light and electron microscopy studies of a myxosporean, parasitizing the gill filaments of the freshwater fish Brycon hilarii (Valenciennes, 1850) (Characidae) collected in the Paraguay River (18°49'S, 57°39'W) (Pantanal), Brazil, is described. This parasite produces spherical to ellipsoidal polysporic histozoic plasmodia (Pmd) (up to ∼180 μm in diameter) delimited by a double membrane and with several pinocytic channels. The plasmodial cyst contained the youngest developmental stages at the cortical periphery and immature and mature spores more internally. The Pmd developed near the cartilaginous structure of the gill filament, forming a prominent deformation where the gill lamellae disappear. Pyriform spores measured 6.9±0.6 (range 6.5-7.2) μm long, 4.2±0.5 (range 3.9-4.8) μm wide, and 2.5±0.7 (range 1.9-2.8) μm thick. The spores composed of two equal shell valves (∼70 nm thick), adhering together along the straight suture line, surrounded two equal symmetric and elongated to pyriform polar capsules (PC) 4.2±0.6 (range 3.8-4.7) × 1.9±0.6 (1.7-2.5) μm; each PC contained a coiled polar filament with eight or nine (rarely 10) turns and a binucleated sporoplasm cell. Dense irregular masses were observed among the polar filaments coils. An intercapsular appendix was not observed. The sporoplasm contained several globular sporoplasmosomes randomly distributed among an extensive rough endoplasmic reticulum system with numerous vesicles and cisternae. Based on the morphological and ultrastructural differences and specificity of the host, we establish the new species, Myxobolus brycon n. sp.

Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 μm were localized in Bowman's space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0-8.5 × 5.0-6.5 μm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0-4.0 × 2.5-3.0 μm and a single sporoplasm. Each valve possesses 14-24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6-10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU-rich inserts. In all SSU and LSU rDNA-based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.

A myxosporean parasitizing the gill filaments of the freshwater teleost fish Centromochlus heckelii collected in the Tocantins River (Lower Amazonian Region, Brazil) is described using light and electron microscopy. This parasite produces spherical to ellipsoidal cyst-like plasmodia up to 250 mum in diameter, with a thick wall strengthened by several stratified juxtaposed crossed collagen layers, whose thickness varies according to the number of the layers. Several compressed fibroblasts are observed among the collagen fibrils. Deposits of spherical dense material are scattered at the internal periphery of the cysts. Plasmodia and different developmental stages, including immature and mature spores, filled the central region of the cysts. The spore body is ellipsoidal in valvar view and biconvex in sutural view. It is formed by two equal-sized and symmetric valves measuring 12.7 microm long (12.2-13.1) (n=50), 6.6 microm wide (6.3-6.9) (n=25), and 4.0 microm (3.7-4.4) (n=20) thick. A thin layer formed by fine and anastomosed microfibrils is observed at the spore surface. Two equal, elongated pyriform polar capsules measure 2.9 microm (2.7-3.3) x 1.7 microm (1.4-2.0) (n=25), each containing four or five oblique polar filament coils. The binucleated sporoplasm contains numerous spherical sporoplasmosomes, glycogen particles, and a large vacuole with fine granular matrix. Based on the morphological and ultrastructural differences and specificity of the host, we describe this isolate as a new myxosporidian, Myxobolus heckelii n. sp. (Myxozoa, Myxosporea).

Elongate plasmodia with myxosporean spores belonging to the genus Unicapsula, Davis, 1924 were found in the skeletal muscle of the striped seabream, Lithognathus mormyrus (L.), a candidate for the mediterranean aquaculture. The only species of Unicapsula described from the Mediterranean is Unicapsula pflugfelderi Schubert et al. 1975, which occurs in the picarel, Spicara smaris (L.). For morphological and molecular comparison of U. pflugfelderi from S. smaris with Unicapsula sp. from L. mormyrus measurements of plasmodia and spores, ultrastructural details and 18S and 28S rDNA sequences were analysed. Whereas plasmodia were 2-3 times larger in S. smaris than in L. mormyrus (length 2.47-0.81 mm; width 0.22-0.09 mm; P = 0.000), spore morphology showed minor differences and both 18S and 28S rDNA sequences were 100% identical identifying the myxozoan as U. pflugfelderi. Scanning electron microscopy of the spores revealed a different shell valve distribution than the one used for the diagnosis of the genus Unicapsula. This resulted in a review of the genus Unicapsula dividing it into two morphological groups of different spore valve arrangement. TEM revealed the presence of a yet undescribed crystalline structure in the sporoplasm of the spores.

A new species of myxozoan, Kudoa trifolia sp. n., was found in various organs of the golden grey mullet, Liza aurata (Risso), and the thinlip mullet, L. ramada (Risso), from the western Mediterranean. Spores developed in subspherical plasmodia of 0.28-1 mm diameter within connective tissue, predominantly in the spleen, the outer wall of the gall bladder and the gut, the mesenteries and occasionally also in the gills. The spores of K. trifolia differ from the commonly known shape of Kudoa by considerable enlargement of one of the four valve cells, thus forming a 'spore body', which contains the major part of the binucleate sporoplasm. Scanning electron microscopy of the spores revealed the presence of grape-like appendages, which occur in bundles terminally on the valve cells. Phylogenetic analysis based on the 18S rDNA sequence of K. trifolia showed that this species is deeply embedded in the genus Kudoa despite its aberrant morphology and host tissue location. This suggests important amendments to the morphological diagnosis of the genus Kudoa.

Henneguya rhamdia n. sp. is described in the gill filaments of the teleost fish Rhamdia quelen, collected from the Peixe Boi River, State of Pará, Brazil. This myxosporean produced spherical to ellipsoidal plasmodia, up to 300 microm in diameter, which contained developmental stages, including spores. Several dense bodies up to 2 microm in diameter were observed among the spores. The spore body was ellipsoidal (13.1 microm in length, 5.2 microm in width, and 2.5 microm in thickness) and each of the two valves presented a tapering tail (36.9 microm in length). These valves surrounded the binucleated sporoplasm cell and two equal ellipsoidal polar capsules (4.7 x 1.1 microm), which contained 10-11 (rarely 12) polar filament coils. The sporoplasm contained sporoplasmosomes with a laterally eccentric dense structure with a half-crescent section. Based on the data obtained by electron microscopy and on the host specificity, the spores differed from previously described Henneguya species, mainly in their shape and size, number and arrangement of the polar filament coils, and sporoplasmosome morphology.

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.

The initial site of infection in the fish host for Tetracapsula bryosalmonae, causative agent of proliferative kidney disease (PKD) is poorly understood. Following the recent recognition that freshwater bryozoans harbour the infective stages to salmonid fish, experimental transmission studies were undertaken to investigate (1) the route of entry of the parasite into the fish host and (2) the minimum exposure time required to induce clinical signs of PKD. In-situ hybridization (ISH) studies were carried out on naive rainbow trout exposed to the naturally infected bryozoan Fredericella sultana for up to 90 min. The sporoplasm of T. bryosalmonae was detected entering the fish via mucous cells in the skin epithelium within the first minute of exposure. In addition, T. bryosalmonae cells were infrequently detected in the skeletal musculature of exposed experimental fish up to 72 h post-exposure. The route of migration through the fish to the kidney and spleen was not determined. All fish exposed to infected, disrupted bryozoans for 10, 30 and 90 min and maintained for up to 8 weeks developed clinical PKD.

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.

The development of Myxobolus dispar Thelohan, 1895, a myxosporean parasite of the gills of common carp (Cyprinus carpio L.) was studied in experimentally infected oligochaetes Tubifex tubifex Muller. After infection of uninfected tubificids with mature spores of M. dispar, development of actinosporean stages was first observed light microscopically 21 days after initial exposure. In histological sections, early pansporocysts were located in the gut epithelium of experimental oligochaetes, while advanced stages occupied mostly the outer layers of the gut and the coelozoic space. Mature pansporocysts, each containing 8 raabeia spores, appeared 199 days after initial exposure. Following damage of the intestinal wall and rupture of the pansporocysts, free actinosporean stages were found in the gut lumen of the oligochaetes. Actinospores of M. dispar emerged from the worms after 217 days of intra-oligochaete development. They were floating in the water and showed a unique raabeia form. Each raabeia spore had three pyriform polar capsules and a cylindrical-shaped sporoplasm with approximately 32 secondary cells. The spore body joined the three caudal projections without a style. Caudal projections were bifurcated at the end and the two main branches had further small bifurcations. The total length of the raabeia spore was approximately 158 µm. The prevalence of infection in 240 experimentally infected Tubifex specimens was 99.2%. No infection was found in the control oligochaetes.

This is the first ultrastructural study of the development of a marine actinosporean and of a species belonging to the genus Spkaeractinnmyxon Caullery & Mesnil, 1904. S. ersei n. sp. is described from a limnodriloidine oligochaete, Doliodrilus diver- ticulatus ErsCus, 1985, from Moreton Bay, Queensland, Australia. Development is asynchronous, there being all stages from two-celled pansporoblasts through to mature spores present simultaneously within a host. Spores develop in groups of eight within pansporoblasts in the coelom and when mature are located also in the intestinal lumen. The primordial spore envelope and sporoplasm develop separately in the pansporoblast until the polar filament is formed within the polar capsule and the capsulogenic cell cytoplasm has begun to degrade. The sporoplasm then enters the spore through a separated valve junction. Mature spores are triradially symmetrical with three centrally located polar capsules and a single binucleate sporoplasm with about 46 germ cells. Swellings or projections of the epispore do not occur when spores exit the host and contact sea water. Supplementary key words. Australia, electron microscopy, marine oligochaete, taxonomy. RESHWATER actinosporeans infecting oligochaetes have re- F cently been shown to be alternate stages in the life cycles of freshwater fish myxosporeans (25). However, it is not known whether marine Actinosporea and Myxosporea are involved in similar cycles. Kent et al. (I I) postulated that a non-oligochaete host may be required or that direct fish-to-fish transmission may occur in marine environments. Myxosporea are important pathogens in marine fishes. Infections by Ceratomyxa spp., Sphaerospora spp. and Kudoa spp. are common in wild marine teleosts and are frequently reported in mariculture (4, 11. Infec- tions detract from the marketability of fish products as the result of cosmetic blemishes or myoliquefaction. The mode of trans- mission of infections is unknown but may include actinospo- rean stages. Several ultrastructural studies have examined de- velopmental stages of actinosporeans. Comprehensive studies describing spore development have been conducted on only three species; namely, Aurantiactinomyxon eiseniellae by Mar- ques & Ormihes ( 171, Neoactinomyxon eiseniellae by Marques (1984. Contribution 2 la connaissance des Actinomyxidies: ul- trastructure, cycle biologique, systkmatique. Dissertation. Univ- ersitk des Sciences et Techniques du Languedoc, Montpellier, France) (16) and Triactinomyxon legeri by Lom & Dykovg

Penetration of triactinomyxon–sporoplasms of Myxobolus cerebralis through skin, fins, gills and buccal cavity have been demonstrated experimentally in rainbow trout. Furthermore the multiplication–stages of penetrated triactinomyxon–sporoplasms reach the cartilage via peripheral nerves and the central nervous system (CNS). This is in contrast to the assumption that the agent reaches the cartilage via blood, lymph, and/or coelomic fluid. During the first hour following penetration, the sporoplasm migrates between the epidermal cells. Then, it enters the epithelia and multiplies intracellularly. These stages migrate deeper into the subcutis, then through the peripheral nerves and CNS. After about 21 days the parasites reach the head cartilages. During their migration they also multiply to increase parasite numbers. The ultrastructure of the proliferative phase (presporogonic development) and the sporogonic phase of the life cycle are demonstrated and discussed.

Ultrastructural examination of Myxobolus nuevoleonensis in cysts on the bones at the bases of the anal, dorsal, and caudal fins of shortfin mollies Poecilia mexicana and guppies P. reticulate showed that sporogenesis and morphology of the mature spores were generally similar to those of the other myxosporeans. The sporoplasm, with one or two spherical nuclei, contained several ellipsoid sporoplasmosomes 70–120 nut in diameter. The polar capsule wall consists of three recognizable zones.

The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin-based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.

Dero digitata infected with Aurantiactinomyxon sp. were obtained from a pond with channel catfish Ictalurus punctatus during an outbreak of proliferative gill disease. These worms were maintained until actinosporeans were released. Aurantiactinomyxon sp. was exposed to channel catfish gills then observed microscopically. After exposure to gills, the sporoplasm of the actinosporean began to change and a motile stage was observed. During the motile stage, pseudopodia-like structures retracted and protruded. In the final stage, all movement ceased, and the sporoplasm rounded up and detached from the valves. This motile stage is a possible link to the penetration of Aurantiactinomyxon sp. into channel catfish gills.

A new myxosporean, Sphaerospora dicentrarchi n. sp., was found in numerous organs of wild and cultured sea bass (Dicentrarchus labrax L.). It is distinguished from all previously reported Sphaerospora spp. by the shape and small size of the spores, location in the host and its geographical distribution. Prevalence of infection was 100% and 83.5% in wild and cultured fish, respectively. Outstanding ultrastructural features are the presence of a binucleate sporoplasm, valvogenic spheroidal structures and capsulogenic lipid inclusions. Other data concerning ultrastructure and sporogenesis are presented.

Spores of the microsporidium Nosema algerae were stimulated to germinate in vitro while observed with video-enhanced contrast microscopy. Field-by-field playback of tape-recorded sequences yielded the first serial illustrations and kinematic analysis of the explosive discharge of the polar filament and the sporoplasm. The filament emerges from the anterior pole of the spore in a regularly pitched helicoidal course along a nearly straight axis, with a mean maximum instant velocity of 105 μm/s. Just before elongation is completed the filament tip follows a tortuous path that often results in a curved or spiralling terminal configuration. Then elongation stops and, after a lag that may vary from less than 15 to over 500 ms, the sporoplasm pours out at the filament tip forming a globule that quickly grows up to a size larger than its original volume within the spore. Concomitantly, the helical filament becomes straightened and frequently the spore body is pulled forward. Thereafter a relaxed filament, usually 5–10% shorter than when maximally extended, remains connecting the empty spore case and the sporoplasmic droplet. Experiments with hyperosmolar media produced a considerable slowdown of filament extrusion and often precluded sporoplasm discharge. The present results are fully consistent with the hypothesis of a hydrostatic pressure-triggered mechanism of spore germination, and revealed that the process is composed of two discrete phases separated by a variable lag: (1) complete eversion of the polar filament, and (2) passage of the main sporoplasm mass along the tube. The data provide a preliminary basis toward the conception of a quantitative physical model of microsporidian spore germination. © 1992 Wiley-Liss, Inc.

The first ultrastructural study of the actinosporean genus Triactinomyxon was carried out on Triactinomyxon legeri from the intestinal epithelium of Tubifex tubifex. The developmental cycle starts with bi- and uninucleate cells. We propose that these cells may be an early proliferative phase of the cycle and may unite to give rise to the four-cell stage, initiating pansporoblast formation. Valvogenic cells transform in the long stylus and anchor-like projections of the spore. In the capsulogenic cells, the primordium of the polar capsules originates as a simple, dense, club-shaped structure not observed in other actinosporeans. In all other respects, actinosporean ultrastructure follows more or less similar patterns. Comparison of actinosporean and myxosporean species gives evidence of considerable structural similarity, exemplified in both classes by the occurrence of cell junctions in their multicellular spores, identical polar capsules and their morphogenesis, cell-in-cell condition, pansporoblast formation, and presence of dense bodies (sporoplasmosomes) primarily in the sporoplasm. This unity of patterns speaks in favor of the postulated actinosporean-myxosporean transformation, which warrants further study.

Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.

Sphaerospores were found in the kidneys of alevin channel catfish (Ictalurus punctatus) from a farm in Central California. Multicellular developmental stages, similar to C-blood protozoans described for Sphaerospora spp. from cyprinid fishes, were observed in circulating blood and numerous tissues. Upon a 2nd examination of the same population of fish 10 days later, sporogonic stages were seen developing into mature sphaerospores in the lumina of the kidney tubules. Sporogenesis was asynchronous with simple unicellular stages adjacent to more complex forms with developing polar capsules and valves. Only one elliptical spore (5.6 microns in width, 6.5 microns in thickness by 5.8 microns in length) developed within the surrounding pseudoplasmodium. Thin valves surrounded two sporoplasm cells and two subspherical polar capsules (1.7 x 1.9 microns) which contained a polar filament with four to five turns. The blood stages of the Sphaerospora sp. described here are similar to the trophozoites seen in channel catfish with proliferative gill disease (PGD). Early stages of PGD also observed in the same population of channel catfish containing developmental and sporogonic stages of this newly recognized Sphaerospora sp. may suggest a causal relationship between this new myxosporean and the gill disease.

Microsporidia are important parasitic eukaryotic organisms that can infect invertebrates, insects, fish and mammals. While the majority of reported cases of human microsporidiosis involve diarrhea, the spectrum of diseases caused by these organisms includes keratoconjunctivitis, disseminated disease, hepatitis, myositis, sinusitis, nephritis, prostatitis, and cholangitis [8,11]. There are over 144 genera and 1200 described species of microsporidia. Recent molecular phylogeny suggests that the Microsporidia are related to fungi [5,10]. All of the microsporidia contain a unique invasion organelle consisting of a single polar tube, polaroplast and anchoring disc [11]. Upon appropriate stimulation the polar tube is rapidly discharged out of the spore forming a hollow tube through which the sporoplasm passes. The mechanism of the interaction(s) of the polar tube with the host cell membrane remains to be determined. Polar tubes are resistant to dissociation in detergents and acids, but thiol-reducing agents will dissociate polar tubes into their component proteins [11]. We have previously isolated and characterized polar tube proteins (PTPs) using these solubility characteristics [6]. The major polar tube protein (PTP1) of Encephalitozoon hellem was purified to homogeneity from solubilized PTPs using HPLC [6]. This protein was cloned and found to be a proline-rich protein with a predicted mass of 43 kDa [6]. On SDS-PAGE, however, EhPTP1 appears as a broad 55-kDa band [6]. Several potential N- and Olinked glycosylation sites are predicted based on the primary amino acid analysis. Post-translational glycosylation of this protein may be responsible for its aberrant SDS-PAGE migration. Such post-translational modifications may be important for the function of PTP1. We, therefore, undertook a study of the lectin binding of purified polar tube proteins.

SYNOPSIS. By fine-structural studies of speculation stages in the European flat oyster pathogen Marteilia refringens this organism is shown to be a member of the protozoan class Haplosporea. This conclusion is based on observations that haplosporosomes are found in plasmodia and spores and that sporoplasm delimitation occurs by internal cleavage within plasmodia. During sporulation plasmodia become sporangiosori by enlarging, cleaving internally into ∼ 8 sporangia, each of which then cleaves internally into 3 or 4 spore primordia. The primordia divide into 3 uninucleate sporoplasms of graded sizes, the smallest lying in an eccentrically located vacuole of the medium-sized one, and the latter situated within an eccentric vacuole of the largest. Haplosporosomes are found only in the cytoplasm of the largest sporoplasm. Mature, living spores measure 3.9(3.5–4.5)μm diameter and are enclosed in a continuous wall without a mantle of epispore cytoplasm as in other Haplosporea. An amended genus and species description of Marteilia refringens is presented, based on light and electron microscope observations.

RESUME L'etude du caractere planctonique de differentes spores d'Actinomyxidies montre une complexite croissante dans leur adaptation au milieu aquatique. Au contact de l'eau, les trois cellules epeisporales de chaque spore se transforment en flotteurs de forme differente suivant les especes. Ces flotteurs peuvent s'unir entre eux en un style equivelent a un quatrieme flotteur ou associer diversement les huit spores issues d'un měme pansporocyste. C'est le cas dans le genre Synactinomyxon dont la diagnose est modifiee pour inclure une deuxieme espece S. Iongicauda n. sp. Un type nouveau est decrit chez lequel la preeence d'ancres a l'extremite des cellules episporales permet de maintenir efficacement reunies plusieurs dizaines de spores emises simultanement. Nous avons observe dans les genres Aurantiactinomyxon, Synactinomyxon, Echinactinomyxon l'emission du sporoplasme. II est libere en entier et capable de se deplacer dans l'eau pendant plus d'une heure grǎce a des mouvements amoeboides. Chez Aurantiactinomyxon eiseniellae les etudes ultrastructurales montrent que l'enveloppe du pansporocyste, d'une part, les epispores et les capsules polaires d'autre part sont realisees a partir de cellules distinctes et profondement modifiees. Quant au sporoplasme, autrefois decrit comme un plasmode avec de nombreuses paires de noyaux, il contient, en fait, des ensembles identiques dont chacun est constitue de l'union d'un noyau satellite et d'une cellule uninucleee. ABSTRACT The study of the planktonic character of different Actinomyxidia spores reveals increasingly complex adaptations to an aquatic environment. On contact with water, the three episporal cells of each spore transform into floats, the forms of which differ according to species. These floats can join together so that a fourth type of float is formed, or they can unite in various ways the eight spores originating from the same pansporocyst. This is the case in the genus Synactinomyxon whose diagnosis is modified to include a second species S. Iongicauda n. sp. A new type is described in which the presence of anchors at the extremities of the episporal cells permits several dozen spores that have been emitted simultaneously to be kept together. We have observed the emission of the sporoplasm in the genera Aurantiactinomyxon, Synactinomyxon, and Echinactinomyxon. It is freed completely and for more than an hour is capable of changing its position in the water by amoeboid movements. In the case of Aurantiactinomyxon eiseniellae, ultrastructural studies show that the pansporocyst envelope on the one hand, and the epispores and polar capsules on the other hand, are formed from separate but profoundly modified cells. The sporoplasm, however, sometimes described as a plasmodium with numerous pairs of nuclei, contains, in fact, identical complexes, each consisting of a uninucleate cell united with a satellite nucleus.

SYNOPSIS 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.

A new myxosporean, Myxobolus jiroveci sp. nov. has been found to infect the brain of bullhead, Cottus gobio L., in several localities in Czechoslovakia and England. Trophozoites, up to about 200 μm in size, were encountered in various regions of the brain where they occupied the space within the distended myelin sheath of the axons. Trophozoites provoke local compression and atrophy of the neighbouring nervous tissue. Host reaction — mostly glial cell proliferation, less often perivascular lymphocytic infiltration — can be associated with heavy infections. The sporoplasmosomes, spherical, double membrane-bound dense bodies in the sporoplasm of mature spores, are described and compared with similar bodies of other myxosporeans and with the haplosporosomes found in representatives of the phylum Haplosporea.

A new histozoic species of myxosporidian, Myxosoma microspora n.sp., infecting the gill filaments of Mugil cephalus is described. Cysts measuring 0.5–1.0 mm in diameter were found attached to the gill filaments. Spherical or slightly oval, spores 4.8–5.2 μm in diameter, were present and possessed a thin outer mucous envelope which appeared as small conical protuberances at the ends of the equatorial axis. Polar capsules were pyriform in shape, equal in size and measured 1.6–2.0 × 1.0–1.2 μm; the polar filaments were 22–28 μm in length. There was a bean-shaped sporoplasm measuring 3.5 × 1.5 μm. No iodine vacuole was detected when the parasite was stained with Lugol's iodine.

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.

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.

Guidelines for the preparation of a species description of myxosporean species are proposed. The establishment of a new myxosporean species has to be based on the morphology of both spores and vegetative stages as observed in a fresh state. All important diagnostic features are listed and diagrams for size measurements are presented.

In this second study of the myxozoan Zschokkella russelli Tripathi from Wales, infection occurred in 71-0% of five-bearded rocklings, Ciliata mustela L.; all infected fish were mature and immature fish were uninfected. Histologieal studies showed ehanges to gallbladder and liver that mostly conformed with earher findings, but sinusoidal dilation, which had not been seen previously, was present in three of 22 infected livers. Thickening around hepatic ducts was shown by transmission electron microscopy (TEM) to comprise fibroblasts and associated collagen bundles, while the attenuated epithelium of hepatic ducts bore few mierovilli. Zschokkella russelli plasmodia were seen by TEM to have fine tubular structures, particularly adjacent to developing sporonts. Spores showed long extensions to the sporoplasm, while spore valves had up to 18 surface ridges each and depressions between ridges were supported by microtubules. Otherwise, in its general ultrastructure and sporogenesis Z. russelli resembled other myxosporida.

This ultrastructural study examines Sporogenesis of Kudoa paniformis, a monosporous myxosporean that causes a softened muscle texture in cooked Pacific whiting, Merluccius productus. Capsulogenesis and valvogenesis occur in a manner similar to that of other myxosporeans, while some aspects of generative cell interaction and sporoplasm ultrastructure differ from most other myxosporeans that have been examined with electron microscopy. Capsulogenesis of K. paniformis includes the formation of an external tubule which is continuous with the polar capsule, followed by the apparent internalization and modification of the external tubule to form a polar filament winding within the polar capsule. Valvogenesis occurs with the accumulation of electron-dense material on the cytoplasmic side of the outer membrane of each valvogenic cell to form a valve wall, Sporogenesis of K. paniformis differs from other myxosporeans in two aspects: (1) spore development does not appear to begin as a result of one generative cell engulfing another, and (2) the sporoplasm of developing and mature spores contains two morphologically distinct cells, one of which surrounds the other.

The stages of development leading to sporogenesis of Ceratomyxa shasta (Noble) were investigated by light and electron microscopy. Salmonid fishes were infected by exposing them to water containing the infectious stage and intestinal material was fixed at weekly intervals. Signs of intestinal infection were barely detectable by 7 days following exposure where trophozoites and later developmental forms were present, but by 14 days a large number of pansporoblasts could be detected in varying stages of development. By 21 days the majority of caeca were completely occluded and infection had spread throughout the connective tissues attached to the caeca. The early developing trophozoites contained two or more nucleated cells within a mother cell. There was some evidence of multiplication of nuclei by fission. The sporoblasts usually contained twelve nucleated cells that gave rise to two groups of six cells (sporonts) and resulted in the formation of two spores in each mother cell. Each spore was formed by two sets of bilaterally arranged cells consisting of the main germinative cell or sporoplasm, the anteriorly placed capsule cells and the outer envelope or spore valve cell that surrounded the others and formed the spore covering. As the spore matured the two germinative cells interacted with each other by pseudopodial extensions and appeared to fuse to form a diploid cell. The position of the cells laterally and slightly posteriorly to the central suture line formed a bilaterally curved spore. Mature spores when examined with the electron microscope were condensed, dark staining and relatively featureless, with a lateral measurement of 15 μm and an anterior-posterior measurement of 7μm.

Sporogenesis of Sphaerospora angulata occurs in the lumen of renal tubules, and of Sphaerospora carassii sporogenesis occurs in the gill epithelium of carp (Cyprinus carpio) from Hungarian pond farms. In infected fish, the lumen of the renal tubules is filled with disporous pansporoblasts of S. angulata. Spores are formed through the participation of paired valvogenic and capsulogenic cells, and a dikaryotic sporoplasm, all of which lie in the cytoplasm of an envelope cell. The polar filament is formed through the apparent invagination of components of the elongate external tubule into the capsular primordium, coupled with the incorporation of dense material from the lumen of the primordium and external tubule. The cytological events leading to spore formation in monosporous pansporoblasts of S. carassii are similar to those of S. angulata and most other species of Myxosporea thus far. While neither parasite appears to induce severe pathogenesis, they may contribute to morbidity in concurrent infections with other microorganisms. Since spores of S. angulata and S. carassii are not formed in plasmodia and the earliest stage observed in this study was 2-celled, earlier stages of these parasites must occur elsewhere in the carp host.

Glugea hertwigi spores were activated to discharge sporoplasms in Medium 199 with 3% gelatin at pH 9.0; the liberated sporoplasms were transferred to a maintenance medium with 6% gelatin (pH 7.0) supplemented with 2 mM ATP and 10% (v/v) fetal calf scrum. The spherical sporoplasms (measuring 3.5-4 mm in diameter) had single nuclei and had a cytoplasm rich in free ribosomes. Each G. hertwigi sporoplasm was initially bounded by an external (0.1-0.2 mm) satellite body adjoining the plasma membrane. The satellites displayed ordered membrane and appeared to merge with the sporoplasm 15-30 min after spore discharge. The external location of the satellite (in reference to the discharged sporoplasm) seems to be part of the normal sequence of events under the in vitro conditions provided. The surface of G. hertwigi sporoplasms does not bear an obvious surface coat; however, our cytochemical observations indicate the plasma membrane of the sporoplasm was somewhat responsive to concanavalin A-peroxidase, colloidal iron, and native ferritin. During the short term incubations of sporoplasms with ferritin, the particles permeated membrane channels extending into the sporoplasm cytoplasm.

SYNOPSIS. An electron microscope study has been made of the spores of a microsporidian, Thelohania californica, parasitic in Culex tarsalis. Fresh spores measure 7–9 × 5–6 μ and contain a polar filament which when extruded may reach a length of 150 μ or more. Thus, this is a typical microsporidian spore. The spore is enveloped in a resistant outer membrane. The polar filament is attached to this membrane at the narrow anterior end, runs inward for a distance and is coiled spirally close to the inner surface of the outer membrane. There are 12–14 spiral turns in the majority of the spores. In the anterior five turns the filament is distinctly larger in diameter than in the seven or more posterior coils. The polar filament appears to be tubular, but the lumen is filled partially or completely with material of high electron density. A laminated polaroplast surrounds the basal portion of the filament and extends into the middle and posterior regions of the intrasporal cavity. The sporoplasm is uninucleate and lies in close contact with the polar filament and the polaroplast. There is no polar capsule.

SYNOPSIS. Haplosporidium sp. Sprague, 1954, originally reported without description and now considered to belong to genus Minchinia, is described. Vegetative forms with two to many nuclei are common but the mode of vegetative multiplication is obscure. Mitotic division, as in related species, occurs without breakdown of the nuclear membrane and shows a conspicuous intradesmose. A complex endosome resembling an amphinucleolus gives rise during prophase to chromosomes, nucleolus and (probably) centrioles. The minute chromosomes are not easily resolved or distinguished from other nuclear elements but their number is of the order of 3 to 6. Mature spores are ovoid and average about 12.1 μ by 8.4 μ when fixed and stained. The spore wall, about 1 μ thick, contains a circular orifice 2 μ in diameter. An operculum arising from the outer gelatinous coat covers the orifice. The single uninucleate sporoplasm is attached anteriorly to the perimeter of the orifice. Cysts are spherical and with variable number of spores, usually too many spores to count but rarely as few as 8 or 16.

SYNOPSIS. Sporocysts of the trematode Bucephalus cuculus McCrady in Maryland oysters, Crassostrea virginica (Gmelin), have been sometimes found to harbor a microsporidian parasite considered to be a new species of Nosema. The ovoid spores are about 3 by 2 u in sectioned material. The spore membrane has a PAS positive mass at the anterior end. The polar filament extends inwardly from this end and coils within a posterior “vacuole.” An anterior “vacuole,” containing no visible structure besides the basal portion of the filament, is also present. The sporoplasm forms a girdle near the middle of the spore. It contains a tiny elongated chromatin mass believed to consist of two nuclei in close proximity.

SYNOPSIS. Myxosoma cartilaginis n. sp. is described from the cartilage of Lepomis macrochirus (bluegill), L. cyanellus (green sunfish) and Micropterus salmoides (largemouth black bass). The development of the parasite is described from naturally infected fish which were held in spore-free water after infection. The sporoplasm invades cartilage, and becomes a multi-nucleate trophozoite which forms pansporoblasts, each of which produces 2 to 4 spores. The first spores appear in 7 weeks. The histopathology in the above fish consists at first of little cellular reaction, but after 4 to 5 months epithelioid granulomas appear around some of the spore masses. Cartilage liquefaction is present around the parasites for at least 5 weeks. Eosinophilic globules are present in cartilage cells adjacent to the lesions. Diffuse infiltration of the spores from the lesions is described. Of 24 chemicals tested for polar filament extrusion, potassium hydroxide gave the best results. An illustrated synopsis of the Myxosoma of North American fishes is given. Included is some additional information and illustrations of M. hoffmani Meglitsch, 1963. Also included is a table showing the hosts, site of infection, geographic location, spore and polar capsule sizes.