Urosporidium sp. hyperparasite of the turbellarian Paravortex cardii in the cockle Cerastoderma edule

Cysts with spores showing different degree of maturity and a single plasmodium were observed in the connective tissue of the turbellarian Paravortex cardii located in the digestive lumen of the cockle Cerastoderma edule. The study of spore morphology by transmission electron microscopy revealed that they correspond to an haplosporidian belonging to the genus Urosporidium. Spore ornaments were similar to those described from Urosporidium spisuli, infecting a nematode parasite of the Atlantic surf clam, Spisula solidissima.     

Scanning electron microscopy and molecular characterization of a new Haplosporidium species (Haplosporidia), a parasite of the marine gastropod Siphonaria pectinata (Mollusca: Gastropoda: Siphonariidae) in the Gulf of Mexico

Based on scanning electron microscopy and the small subunit ribosomal RNA (SSU rRNA), Haplosporidium tuxtlensis n. sp. (Haplosporidia), a parasite found in the visceral tissues of the false limpet Siphonaria pectinata (Linnaeus, 1758), is described. The spores are ellipsoidal (3.61 ± 0.15 μm × 2.69 ± 0.19 μm), with a circular lid (2.94 ± 0.5 μm) representing the operculum. The spore wall bears filaments occurring singly, or in clusters, of 2 to 8, fusing distally. Phylogenetic relationships of H. tuxtlensis n. sp. were assessed with other described species using the SSU rRNA sequence. Haplosporidium tuxtlensis n. sp. is sister taxon to Haplosporidium pickfordi Barrow, 1961. The morphological characteristics (spore wall structure, shape, size, and filament structure) and the unique host identity corroborate it as a new species. Additionally, this is the first record of Haplosporidia infecting striped false limpets in the Gulf of Mexico.     

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.     

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.     

Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters (Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude -20.4'S longitude 115.53'E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite's rRNA gene. An oligonucleotide probe was constructed from the parasite's SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite's rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123-13], in archived rock oyster tissues from the same coastline.     

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.     

Spore ornamentation of Haplosporidium pickfordi Barrow, 1961 (Haplosporidia), a parasite of freshwater snails in Michigan, USA

Spore ornamentation is increasingly recognized as a key character for species differentiation and genus assignment in the phylum Haplosporidia. Unfortunately, spore ornamentation is known for only a small number of described species so it is difficult to assign most species to genera with any confidence. Scanning and transmission electron microscopy were used to determine the presence and morphology of spore ornamentation of Haplosporidium pickfordi collected from the digestive gland of the snail Physella parkeri in Douglas Lake, Michigan. Spores possess filaments that are derived from the spore wall and originate from two separate areas at the posterior end of the spore. When spores are first isolated from host tissue, filaments are fused into a sheet that wraps around the spore, passing under the opercular lid. These filaments gradually unravel when spores are held in water and after about 14 d most filaments project freely from the posterior end of the spore. The number of filaments could not be determined with certainty, but appears to be approximately nine. Filaments are 100 nm in diam. and up to 50 microm in length. The presence of spore wall-derived filaments confirms the placement of the parasite in the genus Haplosporidium.     

Six new species of microsporidia of the genus Amblyospora (Microspora: Amblyosporidae) from blood sucking mosquitoes (Diptera: Culicidae) from the west Siberia

Microsporidia of the genus Amblyospora parasiting the adipose body of mosquito larvae of the genus Aedes and Culex has been studied with both light and electron microscopy. Six new species of microsporidia are described based on ultrastructural characteristics of spores and sporogony stages. Amblyospora flavescens sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 165 nm. Exospore is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, large vesicular, lamellar. The anisofilar polar filament with 10--11 coils (3 1/2 + 2 1/2 + 4-5). Fixed spores are 6.3 +/- 0.1 x 4.24 +/- 0.1 microm. Amblyospora kolarovi sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 265-315 nm. Exospore shapes tucks on the surface of spore. It is two-membranous. Subexospore is quagge, structural. Endospore is electron-translucent. Polaroplast consists of two parts: lamellar and large vesicular. The anisofilar polar filament with 11-13 coils (3 + 8-10). Fixed spores are 5.4-5.6 x 3.5-4.2 microm. Amblyospora orbiculata sp. n. Mature spores are widely egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 155 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, vesicular, lamellar. Polar filament is anisofilar, with 11 1/2 coils (4 1/2 + 1 + 6). Fixed spores are 6.3 +/- 0.1 x x 4.0 +/- 0.1 microm. Amblyospora rugosa sp. n. Mature spores are egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 225 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 17 1/2 coils (3 1/2 + 1 + 13). Fixed spores are 5.3 +/- 0.1 x 3.7 +/- 0.1 microm. Amblyospora undata sp. n. Mature spores are egg-shaped. The spore wall is three-layered, about 220 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. The anisofilar polar filament with 8 coils (3 + 5). Fixed spores are 5.0 +/- 0.1 x 3.0 +/- 0.1 microm. Amblyospora urski sp. n. Mature spores have widely oval form. The back pole is concave. The spore wall with three layers, about 280 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 6 coils (2 + 4). Fixed spores are 4.4 +/- 0.1 x 2.9 +/- 0.1 microm.     

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

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

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

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

Ultrastructural studies of Henneguya rhamdia n. sp. (Myxozoa) a parasite from the Amazon teleost fish, Rhamdia quelen (Pimelodidae)

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.     

Ultrastructure of the microsporidian Inodosporus octospora (Thelohaniidae), a parasite of the shrimp Palaemon serratus (Crustacea, Decapoda)

A parasite of the muscle of the shrimp Palaemon serratus has been examined by light and electron microscopy. Development occurs among myofibrils and induces ultrastructural alterations of the muscle fibers causing white discoloration. This microsporidian is characterized by uninucleate, later diplokaryotic and di-diplokaryotic meronts. The mother cell develops by rosette-like budding into 8 uninucleate sporoblasts, each containing 3 tape-like filaments attached to the wall that is enclosed in a persistent sporophorous vacuole. Each sporoblast gives rise to a uninucleate spore that possesses 3 elongated tape-like filaments attached to the spore wall, like spore tails. The morphological characters of the spores, redescribed in the present study, suggested that the spores belonged to Inodosporous octospora. The possibility that in the future members of Inodosporus sp. may be considered a new parasite group is discussed.     

Myosporidium merluccius n. g., n. sp. infecting muscle of commercial hake (Merluccius sp.) from fisheries near Namibia

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.     

First report of Pandora neoaphidis resting spore formation in vivo in aphid hosts

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores.     

Ultrastructure of developing basidiospores in <Emphasis Type="Italic">Rhizopogon roseolus</Emphasis> (= <Emphasis Type="Italic">R. rubescens</Emphasis>)

The ultrastructure of developing basidiospores in Rhizopogon roseolus is described. When viewed in the fruiting body chamber using scanning electron microscopy, basidiospores appear narrowly ellipsoid and have smooth walls. Eight basidiospores are usually produced on the apex of each sterigma on the basidium. Transmission electron micrographs showed that basidiospores formed by movement of cytoplasm (including the nuclei) via the sterigmata, and then each basidiospore eventually became separated from its sterigma by an electron-lucent septum. The sterigma and basidium subsequently collapsed, resulting in spore release. Freshly released spores retained the sterigmal appendage connected to the collapsed basidium. After spore release, the major ultrastructural changes in the spore concerned the lipid bodies and the spore wall. During maturation, lipid bodies formed and then expanded. Before release, the spore wall was homogeneous and electronlucent, but after release the spore wall comprised two distinct layers with electron-dense depositions at the inner wall, and the dense depositions formed an electron-dense third layer. The mature spore wall complex comprised at least four distinct layers: the outer electron-lucent thin double layers, the mottled electron-dense third layer, and the electron-lucent fourth layer in which electron-lucent granular substances were dispersed.     

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).     

Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.