The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida)

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.     

Pterospora schizosoma,a New Species of Aseptate Gregarine from the Coelom of Axiothella rubrocincta (Polychaeta,Maldanidae)1

ABSTRACT. An undescribed species of aseptate gregarine, for which the name Pterospora schizosoma is proposed, occurs in the coelom of the maldanid polychaete Axiothella rubrocincta (Johnson). The body of the mature gamont is divided into two cylindrical trunks, which are joined anteriorly. Posteriorly, each trunk bifurcates twice, thereby producing four terminal branches. The length of the entire organism varies from 230–600 μm. The organism actively pumps endoplasm from one trunk to the other, and the nucleus (average = 56.2 × 25.9 μm) moves with the flowing endoplasm. In all four of the previously described species, the gamont's main trunks arise from a substantial body mass whereas in P. schizosoma the trunks make up most of the body mass. Pterospora schizosoma's oocysts possess a distinct outer and inner capsule, which contains eight tightly appressed sporozoites.     

Phylogeny of marine Gregarines (Apicomplexa)--Pterospora, Lithocystis and Lankesteria--and the origin(s) of coelomic parasitism

Gregarines constitute a large group of apicomplexans with diverse modes of nutrition and locomotion that are associated with different host compartments (e.g. intestinal lumena and coelomic cavities). A broad molecular phylogenetic framework for gregarines is needed to infer the early evolutionary history of apicomplexans as a whole and the evolutionary relationships between the diverse ultrastructural and behavioral characteristics found in intestinal and coelomic gregarines. To this end, we sequenced the SSU rRNA gene from (1) Lankesteria abbotti from the intestines of two Pacific appendicularians, (2) Pterospora schizosoma from the coelom of a Pacific maldanid polychaete, (3) Pterospora floridiensis from the coelom of a Gulf Atlantic maldanid polychaete and (4) Lithocystis sp. from the coelom of a Pacific heart urchin. Molecular phylogenetic analyses including the new sequences demonstrated that several environmental and misattributed sequences are derived from gregarines. The analyses also demonstrated a clade of environmental sequences that was affiliated with gregarines, but as yet none of the constituent organisms have been described at the ultrastructural level (apicomplexan clade I). Lankesteria spp. (intestinal parasites of appendicularians) grouped closely with other marine intestinal eugregarines, particularly Lecudina tuzetae, from polychaetes. The sequences from all three coelomic gregarines branched within a larger clade of intestinal eugregarines and were similarly highly divergent. A close relationship between Pterospora schizosoma (Pacific) and Pterospora floridiensis (Gulf Atlantic) was strongly supported by the data. Lithocystis sp. was more closely related to a clade of marine intestinal gregarines consisting of Lankesteria spp. and Lecudina spp. than it was to the Pterospora clade. These data suggested that coelomic parasitism evolved more than once from different marine intestinal eugregarines, although a larger taxon sample is needed to further explore this inference.     

Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp

Many aseptate gregarines from marine invertebrate hosts are thought to have retained several plesiomorphic characteristics and are instrumental in understanding the early evolution of intracellular parasitism in apicomplexans and the phylogenetic position of cryptosporidians. We sequenced the small-subunit (SSU) ribosomal RNA genes from 2 archigregarines, Selenidium terebellae and Selenidium vivax, and 2 morphotypes of the marine eugregarine Lecudina polymorpha. We also used scanning electron microscopy to investigate the surface morphology of trophozoites from Lecudina tuzetae, Monocystis agilis, the 2 species of Selenidium, and the 2 morphotypes of L. polymorpha. The SSU ribosomal DNA sequences from S. vivax and L. polymorpha had long branch lengths characteristic of other gregarine sequences. However, the sequence from S. terebellae was not exceptionally divergent and consistently emerged as 1 of the earliest 'true' gregarines in phylogenetic analyses. Statistical support for the sister relationship between Cryptosporidium spp. and gregarines was significantly bolstered in analyses including the sequence from S. terebellae but excluding the longest branches in the alignment. Eugregarines formed a monophyletic group with the neogregarine Ophryocystis, suggesting that trophozoites with elaborate cortex folds and gliding motility evolved only once. The trophozoites from the 2 species of Selenidium shared novel transverse striations but differed from one another in overall cell morphologies and writhing behavior.     

Morphology and phylogenetic position of two novel marine gregarines (Apicomplexa,Eugregarinorida) from the intestines of North‐eastern Pacific ascidians

Eugregarine apicomplexans parasitize marine, freshwater and terrestrial invertebrates, and have lifecycles involving trophozoites (feeding stages) with complex morphologies and behaviour. The genus Lankesteria refers to marine aseptate eugregarines that parasitize ascidians. We described the surface ultrastructure of two new gregarine species, L. chelyosomae sp. n. and L. cystodytae sp. n. that inhabit the intestines of Chelyosoma columbianum and Cystodytes lobatus, respectively, collected from the North‐eastern Pacific Ocean. Apart from inhabiting different hosts and major differences in the cell size of L. chelyosomae sp. n. (mean length 182 µm) and L. cystodytae sp. n. (mean length 70 µm), the morphology of both gregarine species was quite similar. The trophozoites ranged from elliptoid to obdeltoid in shape and were brownish in colour. The nucleus was situated at the anterior end of the cell just behind a pointed mucron. A dense array of epicytic knobs was present over the entire surface of trophozoites in both species, and longitudinal epicytical folds were only weakly developed. We also sequenced the small subunit rDNA from the gregarines collected from both hosts, which supported the establishment of two new Lankesteria species. Phylogenetic analyses of the new DNA sequences and those derived from other alveolates, demonstrated that both new species clustered in a strongly supported clade consisting of other Lankesteria species, Lecudina species, and some environmental sequences. These morphological and molecular phylogenetic data suggested that improved knowledge of gregarine diversity could lead to the recognition of more than one distinct clade (genus) of gregarines within ascidian hosts.     

Scanning Electron Microscopy of Gregarines (Protozoa, Sporozoa) and Its Contribution to the Theory of Gregarine Movement

SYNOPSIS. Scanning electron microscopy was used to reveal detailed surface structure of 4 septate ( Gregarina cuneata, G. steini, G. rhyparobiae, Pileocephalus blaberae ) and one aseptate species ( Nematocystis elmassiani ) of eugregarines. The epicyte of all these gregarines is differentiated into a system of regular longitudinal folds. In the septate species these folds undulate so that these organisms glide along. The undulatory pattern is absent from Nematocystis , which does not glide. The theories and the mechanism of gregarine gliding are discussed.     

The fine structure of the developing oocyst of Pterospora floridiensis (Apicomplexa,Urosporidae)

Developing oocysts of the gregarine Pterospora floridiensis Landers 2001 were examined by transmission electron microscopy. Each oocyst had an outer capsule and an inner capsule that contained 8 sporozoites. In early stages of development the inner capsular wall was separated from the developing sporozoites and residual mass, and was not appressed to the sporozoites. Early stage sporozoites were connected to a residual mass and were filled with endoplasmic reticulum, golgi and numerous developing secretory vesicles. In late stages of oocyst and sporozoite development, the inner capsular wall was closely appressed to the sporozoite surface. The inner capsular wall was ～60-100 nm thick and the outer capsular wall was ～160-320 nm thick. There were no extensions on the outer wall for which the genus was named. Late stage sporozoites had no residual mass connection, were more electron dense, and contained three distinct types of dense secretory structures: 1) small oval/spherical dense vesicles, 2) large (350-400 nm) vesicles near the anterior end, and 3) elongated dense tubular bodies that converged at the apex. Few ultrastructural reports exist of developing gregarine oocysts and sporozoites, and as more studies are completed these morphological characteristics may be important in interpreting molecular phylogenetic analyses.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae (Pterospora andromedea and Sarcodes sanguinea) found in the Greater Yellowstone Ecosystem

Pterospora andromedea, a mycoheterotroph, has been shown to form obligate symbioses with only three species of Rhizopogon in section Amylopogon: R. salebrosus, R. arctostaphyli and an undescribed molecular taxon. Sarcodes sanguinea, another my coheterotroph in Ericaceae, and sister taxon to Pterospora andromedea, has been found to form symbioses with two species of Rhizopogon in section Amylopogon: R. ellenae and R. subpurpurascens. To date no overlap has been recorded between these two achlorophyllous plants and their associated mycobionts. Tissue from Pterospora andromedea rootballs and Rhizopogon spp. basidiocarps were collected from the Greater Yellowstone Ecosystem. The mycobionts were identified using sequence analysis of the ITS locus and compared with sequences of Rhizopogon spp. section Amylopogon from GenBank. Sequences of two additional loci, ATP6 and RPB2 were also generated and analyzed. In addition to Rhizopogon salebrosus, Pterospora andromedea was found for the first time in association with a fourth mycobiont, Rhizopogon ellenae, a known associate of Sarcodes sanguinea. The discovery of a new symbiont may provide evidence for an undiscovered lineage of Pterospora andromedea inhabiting the Greater Yellowstone Ecosystem. In addition, overlap in obligate mycobionts between closely related mycoheterotrophs provides interesting new information on the phylogenetic history and coevolution of the mycoheterotrophs in the Monotropoideae (Ericaceae).     

Description of Ganymedes yurii sp. n. (Ganymedidae), a New Gregarine Species from the Antarctic Amphipod Gondogeneia sp. (Crustacea)

A novel species of aseptate eugregarine, Ganymedes yurii sp. n., is described using microscopic and molecular approaches. It inhabits the intestine of Gondogeneia sp., a benthic amphipod found along the shore of James Ross Island, Weddell Sea, Antarctica. The prevalence of the infection was very low and only a few caudo‐frontal syzygies were found. Morphologically, the new species is close to a previously described amphipod gregarine, Ganymedes themistos, albeit with several dissimilarities in the structure of the contact zone between syzygy partners, as well as other characteristics. Phylogenetic analysis of the 18S rDNA from G. yurii supported a close relationship between these species. These two species were grouped with other gregarines isolated from crustaceans hosts (Cephaloidophoroidea); however, statistical support throughout the clade of Cephaloidophoroidea gregarines was minimal using the available dataset.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Is rarity of pinedrops (Pterospora andromedea) in eastern North America linked to rarity of its unique fungal symbiont?

Like other myco-heterotrophic plants, Pterospora andromedea (pinedrops) is dependent upon its specific fungal symbionts for survival. The rarity of pinedrops fungal symbiont was investigated in the eastern United States where pinedrops are rare. Wild populations of eastern pinedrops were sampled, and the plant haplotypes and fungal symbionts were characterized with molecular techniques; these data were compared to those from the West with phylogenetic analyses. The frequency of the fungal symbiont in eastern white pine forests was assessed using a laboratory soil bioassay and in situ pinedrops seed baiting. Only one plant haplotype and fungal symbiont was detected. The plant haplotype was not unique to the East. The fungal symbiont appears to be a new species within the genus Rhizopogon, closely related to the western symbionts. This fungal species was not frequent in soils with or without pinedrops, but was less frequent in the latter and in comparison to the fungal symbionts in western forests. Seed baiting resulted in few germinants, suggesting that mycelial networks produced by the eastern fungal symbiont were rare. Results suggest that eastern pinedrops rarity is influenced by the distribution and rarity of its fungal symbiont.     

五味子属种子形态及其分类学意义

用光学显微镜及扫描电镜对五味子属（Schisandra)10种96号样品的种子宏观形态和种皮的微观形态特征进行观察,首次对该属的种皮微观形态特征进行系统报道,并编排有分种检索表。研究结果表明：该属的种子宏观形态呈现平滑、细皱纹或瘤状突起,并认为五味子属种子形态可有从平滑到有细皱纹再到有瘤状突起的演化趋势;该属种皮表面微观形态可分为疣状突起型和网纹型。结合其它性状,本文认为网纹型所代表的两种植物应与本属中其它植物建立并列的分类单元。五味子属植物在种子形态特征上存在一定的差异,可为属以下、种以上的分类提供依据,并为探讨该属的系统演化提供有价值的参考资料。     

Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeus albidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium

A selenid gregarine Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae), harboring the gut lumen of the oligochaete Enchytraeus albidus, was studied by light and electron microscopy. The trophozoite of Ditrypanocystis sp. is attached to the gut wall with its apical end to be anchored eventually between enterocytes in the crypts. Simultaneously, between the surfaces of the parasite and the host cell a peculiar contact is formed made of membranous channels and vesicles of unknown origin, the host cell surface in the contact area lacking cilia. The trophozoite becomes progressively enclosed within a parasitophorous vacuole made of layers of fused ciliar membranes of enterocytes. The fused cilia may be a source of membranes lining channels and vesicles of the contact area. Such a mode of parasitophorous arrangements has never been described before for gregarines, however, it bears a some likeness with that of the coccidian genus Cryptosporidium (similarity and differences being discussed). With regard to some molecular phylogeny constructions, claiming the "sister" relationship between gregarines and the coccidian genus Cryptosporidium (Carreno et al., 1999; Leander et al., 2003), this common feature in host-parasite relationships enabled us to put forward an idea of a possible evolutionary route from extracellularity of gregarines to intracellularity of coccidia, as exemplified by species of Cryptosporidium.     

Lotharella reticulosa sp. nov.: a highly reticulated network forming chlorarachniophyte from the Mediterranean Sea

A new chlorarachniophyte Lotharella reticulosa sp. nov. is described from a culture isolated from the Mediterranean Sea. This strain is maintained as strain RCC375 at the Roscoff Culture Collection, France. This species presents a multiphasic life cycle: vegetative cells of this species were observed to be coccoid, but amoeboid cells with filopodia and globular suspended cells were also present in the life cycle, both of which were not dominant phases. Flagellate cells were also observed but remained very rare in culture. The vegetative cells were 9-16 μm in diameter and highly vacuolated, containing several green chloroplasts with a projecting pyrenoid, mitochondria, and a nucleus. The chloroplast was surrounded by four membranes possessing a nucleomorph in the periplastidial compartment near the pyrenoid base. According to ultrastructural observations of the pyrenoid and nucleomorph, the present species belongs to the genus Lotharella in the phylum Chlorarachniophyta. This taxonomic placement is consistent with the molecular phylogenetic trees of the 18S rRNA gene and ITS sequences. This species showed a unique colonization pattern. Clusters of cells extended cytoplasmic strands radially. Then, amoeboid cells being born proximately moved distally along the cytoplasmic strand like on a "railway track". Subsequently the amoeboid cell became coccoid near the strand. In this way, daughter cells were dispersed evenly on the substratum. We also observed that the present species regularly formed a structure of filopodial nodes in mid-stage and later-stage cultures, which is a novel phenotype in chlorarachniophytes. The unique colonization pattern and other unique features demonstrate that RCC375 is a new chlorarachniophyte belonging to genus Lotharella, which we describe as Lotharella reticulosa sp. nov.     

Molecular phylogeny of Pacific Archigregarines (Apicomplexa), including descriptions of Veloxidium leptosynaptae n. gen., n. sp., from the sea cucumber Leptosynapta clarki (Echinodermata), and two new species of Selenidium

Although archigregarines are poorly understood intestinal parasites of marine invertebrates, they are critical for understanding the earliest stages in the evolution of the Apicomplexa. Previous studies suggest that archigregarines are a paraphyletic stem group from which other lineages of gregarines, and possibly all other groups of apicomplexans, evolved. However, substantiating this inference is difficult because molecular phylogenetic data from archigregarines, in particular, and other gregarines, in general, are severely limited. In an attempt to help fill gaps in our knowledge of archigregarine diversity and phylogeny, we set out to discover and characterize novel lineages of archigregarines with high-resolution light and scanning electron microscopy and analyses of small subunit (SSU) rDNA sequences derived from single-cell (SC) PCR techniques. Here, we describe two novel species of Selenidium, namely Selenidium idanthyrsae n. sp. and S. boccardiellae n. sp., and demonstrate the surface morphology and molecular phylogenetic position of the previously reported species S. cf. mesnili. We also describe a novel genus of archigregarine, Veloxidium leptosynaptae n. gen., n. sp., which branches with an environmental sequence and, together, forms the nearest sister lineage to a diverse clade of marine eugregarines (i.e. lecudinids and urosporids). This molecular phylogenetic result is consistent with the hypothesis that archigregarines are deeply paraphyletic within apicomplexans, and suggests that convergent evolution played an important role in shaping the diversity of eugregarine trophozoites.     

Host specificity in Metamera sillasenorum, n. sp., a gregarine parasite of the leech Helobdella triserialis with notes on transmission dynamics

Eugregarines of the suborder Septatorina are apicomplexan parasites that are found mainly in arthropods. Some exceptions are species in the Metameridae that contains the only 5 septate gregarines recorded from annelids. The type genus is Metamera Duke, 1910 with 2 species, Metamera schubergi Duke, 1910, in European Glossiphonia complanata, and Metamera reynoldsi Jones, 1943, from North American G. complanata. Over the summers of 1995-1998, in Keith County, Nebraska, septate gregarines were found in the glossiphoniid leech Helobdella triserialis. The gregarines were determined to be a new species of Metamera, herein named Metamera sillasenorum. Measurements of size and body proportions of over 600 gregarines and 50 oocysts showed differences from measurements of M. schubergi and M. reynoldsi, and secondary septa in the deutomerite were rarely observed. Field observations indicated that M. sillasenorum is probably host specific. In the laboratory, leeches also exhibited strong feeding preferences; e.g., H. triserialis and G. complanata consumed only snails, whereas Helobdella stagnalis consumed only oligochaetes. Infection experiments demonstrated that freshwater snails ingest the oocysts and are required as mechanical vectors. Oocysts were passed unaltered through the snails' intestines. Glossiphonia complanata did not become infected regardless of heavy exposure to oocysts, although only 5 G. complanata were used in the experiments. The results show that host specificity of M. sillasenorum is most likely due to a combination of host-feeding habits and host-parasite compatibility.     

A new species of Everhartia associated with leaf spots of Phoenix hanceana from Hong Kong

A new species of Everhartia, E. phoenicis , is described and illustrated. The fungus was collected on living leaves of Phoenix hanceana in Twisk, Tai Mo Shan. Hong Kong. Everhartia phoenicis is associated with yellow spots on living leaves and rachides of the host, and is the only species in the genus known to be pathogenic. It differs from other species of Everhartia in producing symmetric, hyaline, aseptate, horseshoe-shaped conidia. Everhartia phoenicis is compared with other species in the genus. Everhartia is very similar to Delortia and the differences between these two genera are discussed. Two species of Everhartia, E. aquatica and E. tumidoapuis arc transferred from Delortia .     

The Mammalian Pathogenic Oomycetes

The oomycetes are fungal-like microbes similar to those found within some members of the kingdom Fungi. Although these two groups of microbes share morphological features, there are several contrasting differences: a) phylogenetic analysis placed the oomycetes basal to plants and green algae; b) oomycetes lack ergosterol in their cytoplasmic membrane; c) chitin is not the main compound in the cell wall of oomycetes; and d) asexual reproduction in the oomycetes occurs by the development of sporangia containing numerous biflagellate zoospores. Pythium insidiosum was considered to be the only oomycete pathogenic for mammals. However, in 1999, Grooters reported that several dogs were diagnosed with an unusual oomycete in the genus Lagenidium causing extensive cutaneous and subcutaneous infections. Thereafter, the infection has been also reported in humans and cats, and it could possibly affect other mammalian species as well. This review highlights the epidemiological, clinical and pathological features, as well as the diagnosis and management of the infections caused by this unique group of mammalian pathogenic oomycetes.