Minchinia mercenariae n. sp. (Haplosporidia) in the Hard Clam Mercenaria mercenaria: Implications of a Rare Parasite in a Commercially Important Host

ABSTRACT. During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria , from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae . No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.     

Molecular analysis of a haplosporidian parasite from cultured New Zealand abalone Haliotis iris

In the Austral summer and autumn of 2000 and 2001, mortalities of black-footed abalone Haliotis iris (Martyn, 1784) occurred in a commercial facility in New Zealand. Histological analyses suggested that infection by a haplosporidian parasite was responsible. To confirm identification as a haplosporidian and to help determine if this parasite represented a new, undescribed species, DNA was extracted from infected host tissues scored as positive for infection by histological examination. Small-subunit rRNA (SSU rRNA) gene sequences from both the host abalone and a parasitic organism were amplified by PCR and characterized. Although the sequence for this parasite was novel, not matching any known SSU rRNA gene sequences, phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. Parsimony analyses placed the parasite at the base of the phylum Haplosporidia, ancestral to Urosporidium crescens and the Haplosporidium, Bonamia, and Minchinia species. Sequencing of multiple parasite DNA clones revealed a single polymorphic site in the haplosporidian SSU rRNA gene sequence.     

Hemolymph melanization and alterations in hemocyte numbers in Lymantria dispar larvae following infections with different entomopathogenic microsporidia

Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae can be infected in the laboratory with a variety of entomopathogenic microsporidia. In many cases, however, L. dispar is only a semi‐permissive host for such infections. In this study, we analyzed changes in the melanization of hemolymph and hemocyte numbers in L. dispar larvae after inoculation with various entomopathogenic microsporidia. We compared the infections produced by microsporidia isolated from L. dispar and infections produced by isolates from other Lepidoptera to which L. dispar is only a semi‐permissive host. Microsporidiosis induced a significant activation of the prophenoloxidase system leading to melanization; activation was highest when the pathogen caused heavy infections of the fat body, which was the case with two microsporidia originally isolated from L. dispar. Infection of only the silk glands or light infection of the fat body by two Vairimorpha spp. from other lepidopteran hosts elicited a lower response. Very light infections caused by a microsporidium isolated from Malacosoma americanum were not accompanied by elevated hemolymph melanization activity. Heavy infections by Endoreticulatus spec. that remained restricted to the gut tissue likewise did not elicit melanization. One Vairimorpha spec. from L. dispar induced a significant increase in total hemocyte numbers; the other infections led to temporarily decreased numbers. Microscopic examinations showed that parts of infected tissue were encapsulated by hemocytes. We conclude that measured alterations in hemolymph melanization and hemocyte numbers were likely to be induced by the damaging effects of heavy infections. Observed defense responses did not prevent the progression of infections.     

Bitter crab syndrome in two species of king crabs from the Sea of Okhotsk

The disease caused by parasitic dinoflagellates of the genus Hematodinium was found in the red Paralithodes camtschaticus and blue P. platypus king crabs from the Sea of Okhotsk. The hemolymph of diseased crabs was cream colored, opaque, and dense. Numerous dinokaryotic trophonts and multinucleate plasmodia of the parasite were revealed in the hemolymph and internals of diseased animals. The parasitic infection was recorded in females and juvenile males from August to mid-October.     

Observations on the pathogenesis of the imperfect fungus,Fusarium solani,in the California brown shrimp,Penaeus californiensis

The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate.     

Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae)

The fine structure of maturing spores of a haplosporidian parasite found in the gill, mantle and foot tissues of Ruditapes decussatus L. (Mollusca, Bivalvia), a species of commercial importance in Portugal, is described. When observed free in suspension, immature spores exhibit one or two epispore cytoplasmic extensions (ECE) which constitute a projection of a portion of the exosporoplasm, sometimes without ultrastructural organisation, surrounded by the plasmalemma. Free spores observed by light microscopy (LM) after 3-5 days of incubation in filtered sea-water exhibit no ECE attached to the spore wall. The mature spore is ovoid to ellipsoid, operculate, uninucleate and measures c. 4.8 microm long and c. 3.9 microm wide. The spore shape and size and the identity of the host living in the same geographical region suggest that this species is the same as previously described using LM observations as Haplosporidium tapetis Vilela, 1951 and later transferred to Minchinia Labbé, 1896.     

Haplosporidiosis of the Pacific oyster, Crassostrea gigas.

Haplosporidan parasites were observed in 10/100 spat and 1/171 adult Pacific oysters, Crassostrea gigas, reared in Matsushima Bay, Japan. Eight of the infected spat contained mild to severe plasmodial infections. The multinucleated plasmodia were 6-12 microm x 7-15 microm and were associated with an infiltration of hemocytes that occurred throughout the vesicular connective tissues of all infected oysters. Two oysters, one adult and one spat, contained advanced sporogonic infections. These were characterized by the presence of sporocysts and immature and mature operculated spores that measured 5.6-6.0 microm x 6.0-8.0 microm and were found exclusively within the digestive tubule epithelium. Electron microscopic examination revealed that mature spores contained a hinge operculum, striated and layered wall, spherule, single nucleus, and haplosporosome formative regions. Parasite morphology and infection pattern closely resemble that of Haplosporidium nelsoni, a pathogen of American oysters (C. virginica).     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration.

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

A new pathogenic virus in the Caribbean spiny lobster Panulirus argus from the Florida Keys

A pathogenic virus was diagnosed from juvenile Caribbean spiny lobsters Panulirus argus from the Florida Keys. Moribund lobsters had characteristically milky hemolymph that did not clot. Altered hyalinocytes and semigranulocytes, but not granulocytes, were observed with light microscopy. Infected hemocytes had emarginated, condensed chromatin, hypertrophied nuclei and faint eosinophilic Cowdry-type-A inclusions. In some cases, infected cells were observed in soft connective tissues. With electron microscopy, unenveloped, nonoccluded, icosahedral virions (182 +/- 9 nm SD) were diffusely spread around the inner periphery of the nuclear envelope. Virions also occurred in loose aggregates in the cytoplasm or were free in the hemolymph. Assembly of the nucleocapsid occurred entirely within the nucleus of the infected cells. Within the virogenic stroma, blunt rod-like structures or whorls of electron-dense granular material were apparently associated with viral assembly. The prevalence of overt infections, defined as lethargic animals with milky hemolymph, ranged from 6 to 8% with certain foci reaching prevalences of 37%. The disease was transmissible to uninfected lobsters using inoculations of raw hemolymph from infected animals. Inoculated animals became moribund 5 to 7 d before dying and they began dying after 30 to 80 d post-exposure. The new virus is apparently widespread, infectious, and lethal to the Caribbean spiny lobster. Given the pathogenic nature of the virus, further characterization of the disease agent is warranted.     

Severe apicomplexan infection in the oyster Ostrea chilensis: a possible predisposing factor in bonamiosis

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed >85% contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1% of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2% of moderately infected oysters and 9% of heavily infected oysters. Of oysters with empty gonad follicles, 75% were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8% of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.     

Ecological and physiological effects of PaV1 infection on the Caribbean spiny lobster (Panulirus argus Latreille)

Pathogens can profoundly impact the ecology of the organisms they infect through changes in host behavior that influence demographic processes. For example, juvenile Caribbean spiny lobsters (Panulirus argus Latreille) infected with the PaV1 virus (Panulirus argus Virus 1) are avoided by their normally social conspecifics, which alters local spatial distributions and presumably rates of disease transmission. PaV1 infections are nearly always lethal, but prior to succumbing to the disease, infection may impact other host dynamics (e.g., movement, growth, or survival) that effect transmission of the virus. We used mark-recapture surveys and laboratory studies to determine the impact of PaV1 infection on lobster movement and physiological condition, and in turn, the effect of condition on susceptibility to infection. Significantly more healthy lobsters were recaptured than heavily diseased lobsters in 5-d mark-recapture surveys, indicating either greater emigration or greater mortality of infected lobsters. Results of a laboratory bioassay suggest that lobsters with early-stage infections moved at an equivalent rate to healthy lobsters, however, as infection progressed, lobsters inoculated with PaV1 moved less than healthy lobsters ultimately remaining sedentary. Infected lobsters captured in nature also had significantly lower hemolymph (blood) serum protein values, indicating poor physiological condition. However, poor condition did not predispose lobsters to greater risk of infection because prevalence was not different between starved lobsters and healthy lobsters challenged with PaV1. Although lobsters infected with PaV1 eventually become sedentary and of poor nutritional condition, during the early stages of infection they remain active and are thus capable of dispersing the virus throughout the population.     

Pathogenicity of Vibrio tapetis,the etiological agent of brown ring disease in clams

Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe. The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell. Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens. This study was designed to evaluate the pathogenicity of V. tapetis, in an attempt to help explain the mechanisms of mortality. We found high mortalities (up to 100%) for clams following the inoculation of V. tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R. philippinarum. Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation. In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption. Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle. In contrast, no mortalities were observed following injection of V. tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD. This clam rapidly eliminated the bacterium from hemolymph and soft tissues. Clam mortality associated with BRD in the field is likely to result from the penetration of V. tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium. Some bacteria also penetrate through the digestive epithelia. In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Infectivity of resting spores of Massospora cicadina (Entomophthorales: Entomophthoraceae), an entomopathogenic fungus of periodical cicadas (Magicicada spp.) (Homoptera: Cicadidae)

Progeny of eastern oyster, Crassostrea virginica, introduced into France in 1992, were reared in IFREMER facilities to test their growth performances. During the summer of 1993, sporadic mass mortalities (80-90%) occurred among C. virginica spat reared in the IFREMER laboratories in La Tremblade (Charente Maritime, France) and Bouin (Vendée, France). Affected oysters presented mantle retraction and deposition of an anomalous conchiolin layer on the inner surface of the shell. The incidence of oysters with gross signs exceeded 80%. No obvious pathogen was identified in soft tissues by histology and transmission electron microscopy (TEM). However, histological examination showed the presence of anomalous basophilic round structures, 0.5-1 microm in diameter, in gill and mantle connective tissues. These extracellular Feulgen-negative structures reacted positively with the von Kossa stain. TEM examination on mantle and gill samples in diseased spat showed that the basophilic bodies consisted of concentric deposits of an amorphous substance interpreted as containing calcium. These observations may indicate that the mineralization process in spat shells was disturbed without exact determination of the cause. Based on the similarities of the gross signs to those reported in juvenile eastern oysters in the United States, we believe that the cause of the mortalities observed in France was probably the Juvenile Oyster Disease. Moreover, we report for the first time the detection of anomalous amorphous structures in gill and mantle connective tissues associated with mortalities and deposition of an anomalous conchioloin layer on the inner shell surface in C. virginica spat.     

A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.     

An ultrastructural and histopathological study of Henneguya pellucida n. sp. (Myxosporea: Myxobolidae) infecting Piaractus mesopotamicus (Characidae) cultivated in Brazil

During a study of myxosporean parasites of cultivated freshwater fish, a new myxosporean species, Henneguya pellucida n. sp., was discovered. Of the 120 Piaractus mesopotamicus sampled, only 10 specimens (8.3%) were infected. Yellow, round plasmodia measuring 0.5-3 mm were found in the serous membrane of the visceral cavity and in the tunica externa of the swim bladder. Sporogenesis was asynchronous, with the earliest developmental stages aligned prevailingly along the endoplasmic periphery and mature spores in the central zone. The mature spores were pear shaped (total length: 33.3 +/- 1.5 microm, mean +/- SD; width: 4.1 +/- 0.4 microm; body length: 11.4 +/- 0.3 microm; caudal process length: 24.1 +/- 1.5 microm). The polar capsules were elongated (length: 4.0 +/- 0.4 microm; width: 1.6 +/- 0.2 microm). The development of the parasite in the swim bladder produced thickening of the tunica externa and a granulomatous reaction. There was no correlation between the prevalence of the parasite and the chemical and physical characteristics of the water. Infection was recorded only in juvenile specimens ranging in size from 9.5 to 20 cm.     

Haplosporidium sp. (Alveolata: Haplosporidia) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia

Haplosporidium sp. is described from rock oysters Saccostrea cuccullata Born, 1778 experiencing epizootics on the northwestern coast of Western Australia. All stages were observed as focal infections in the connective tissue of the gills, or as disseminated infections in the mantle and around digestive diverticulae. Haplosporidium sp. occurred between epithelial cells of the gut, in focal lesions in the gills, but not in the epithelium of the digestive diverticulae, and sporulation was confined to the connective tissue. Plasmodia developed into sporonts and sporocysts in a loose syncytium that gave rise to binucleate and uninucleate sporoblasts from which spores developed. Spores were flask-shaped, 5.6-6.7 x 3.3-4.0 microm, with a characteristic operculum, a few filamentous wrappings and rod-like structures in the posterior sporoplasm. Mature spores had a wall comprising inner (90 nm wide), middle (30 nm wide) and outer (130 nm wide) layers, and a surface coat of microtubules giving them a furry appearance. Oysters with empty gonad follicles were most heavily infected, and oyster condition and mortality appeared to be related to degree of infection.