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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.

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.

The complete life cycle of Amblyospora campbelli (Kellen and Wills, 1962) (Microsporida: Amblyosporidae) requires a two-host system involving the mosquito host, Culiseta incidens (Thomson), and an obligatory intermediate copepod host. The parasite has dimorphic spore development producing meiospores (haploid condition) and binucleated spores (diploid condition), either as an exclusive infection or simultaneously (within females only). This is the 1st known report of concurrent spore development within an adult mosquito host, and, therefore, shows the Amblyospora campbelli system to be uniquely different from other Amblyospora spp. cycles previously described. The significance of dimorphic spore development is discussed. In females, diplokaryotic meronts may invade oenocytes, causing a benign-type of infection. A blood-meal is required to initiate sporulation of the binucleate spore. The binucleate spore contains the sporoplasm involved in transovarial transmission. A 2nd sporulation sequence, primarily in adipose tissue, may involve both males and females. In this sequence, repeated merogonic division greatly increased the density of diplokaryotic meronts and generally involved most of the body of the host. Production of meiospores, unlike that for the binucleate spore, appeared to be spontaneous (i.e. no obligatory blood meal). Survivorship of male and female larval mosquitoes was nearly equal. Adult females spread the parasite in three ways: transovarial, transovum, and by meiospore deposition.

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.

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.

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.

SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.

SYNOPSIS. Colonies of Bufo bufo kept for pregnancy diagnostic purposes were found infected with a new species of microsporidian, Plistophora myotrophica, causing atrophy of the striated muscles. Lysis of myofibrils by the parasite leads to the formation of fusiform spaces in the fibers packed with microsporidian spores. A cyst wall is not formed. Toads become emaciated and suffer heavy mortality. In experimental infections the sporoplasm hatches in the gut. Fusiform granular bodies, not identifiable as blood cells, are found in the muscle capillaries at 18 days and possibly represent early stages of the parasite. Between 18 and 23 days division of the parasite occurs in the muscle fibers, first by binary fission, then multiple fission and finally by plasmotomy culminating in the formation of sporonts with 16 to 100 nuclei. Separation into sporoblasts precedes spore formation. In old infections phagocytes, which are probably monocytes, infiltrate between the groups of spores and ingest them. Ingested spores which stain differently from their free counterparts, are dead. Muscle regeneration is initiated by the toad, with the formation of long chains of sarcoblasts, sometimes 30 or more in a chain, responsible for the regeneration and redifferentiation of myofibrils. Tadpoles are refractory to infection. As toads normally lead a solitary life the high local incidence in the area from which the toads were collected is explained only if the toads acquire the infection from spore-contaminated ground in the breeding season.