Fifteen new species from the myxomycete genus Lycogala.

Based on a study of 255 collections from four continents and four floristic kingdoms, we describe 15 new species of the genus Lycogala. The new species, all morphologically close to L. epidendrum, L. exiguum, and L. confusum, differ from each other by the structure of the peridium and, in some cases, also by the color of the fresh spore mass and the ornamentation of the capillitium and spores. Species delimitation is confirmed by two independently inherited molecular markers, as well as previously performed tests of reproductive isolation and genetic distances. We studied authentic material of L. exiguum and L. confusum and found fresh specimens of these species, which allowed us to obtain molecular barcodes and substantiate the separation of new species from these taxa. We propose to retain the name L. epidendrum for the globally most abundant species, for which we provide a more precise description and a neotypification. Two formerly described species, L. leiosporum and L. fuscoviolaceum, we consider to be dubious. We do not recognize the species L. terrestre.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Ultrastructure of vegetative cells and resting cysts, and live observations of the encystation and excystation processes in Diophrys oligothrix Borror, 1965 (Protista, Ciliophora).

Ciliated protists can form cysts to resist unfavorable environmental conditions and then excyst when environmental conditions become favorable. This study used electron and light microscopy to investigate the structure of vegetative cells and resting cysts, as well as the encysting and excysting processes of Diophrys oligothrix. For the first time, ampules were revealed beneath the pellicle in the genus Diophrys, and their extrusome types differed between Diophrys species. Membrane-packed discs of diverse shapes were found in the cytoplasm just beneath the pellicle around the cytopharynx and were separated by rows of microtubule units. Beneath the discs, some double-layer microtubule structures were detected as well. During encystment, the ventral ciliature was folded in a ventral cavity of the cell, and the caudal cirri were retracted directly into the cyst in a separate cavity on the dorsal side. In the resting cysts, high autophagic activity occurred, possibly including digestion of membrane-packed discs and ampules. Two macronuclear nodules kept their basic shape, although the chromatin aggregation and fusion region were observed in ultrathin sections. The cyst wall contained two layers, namely, the ectocyst, and endocyst. In mature cysts, basal bodies and ciliary shafts were observed, demonstrating that D. oligothrix forms non-kinetosome-resorbing cysts. The process of excystment occurred in two modes, either with or without participation of a contractile vacuole.

Islandinium minutum subsp. barbatum subsp. nov. (Dinoflagellata), a New Organic-Walled Dinoflagellate Cyst from the Western Arctic: Morphology, Phylogenetic Position Based on SSU rDNA and LSU rDNA, and Distribution.

A study of modern sediment from the Western Arctic has revealed the presence of a distinctive brown-colored cyst with a spherical central body bearing unbranched processes that are usually solid with a small basal pericoel. Distinctive barbs project from some processes, and process tips are usually minutely expanded into conjoined barbs. The archeopyle is apical and saphopylic. This cyst corresponds to Islandinium? cezare morphotype 2 of Head et al. (2001, J. Quat. Sci., 16:621). Phylogenetic analyses based on the small and large subunit rRNA genes infer close relationship with Islandinium minutum, the type of which is that of the genus. Re-examination of specimens of I. minutum reveals the presence of minute barbs on its processes, but differences with Islandinium? cezare morphotype 2 remain based on size, process distribution, and barb development. Furthermore, the internal transcribed spacer shows I. minutum to be distinct from this morphotype. On the basis of these small but discrete differences, we propose the new subspecies Islandinium minutum subsp. barbatum subsp. nov. Molecular sequencing of other cysts encountered, namely Echinidinium karaense, an unidentified flattened cyst, and "Polykrikos quadratus", places them in the Monovela clade, the latter showing greater morphological variability than previously thought.

Morphological and molecular characterization of a new succulenticolous Physarum (Myxomycetes, Amoebozoa) with unique polygonal spores linked in chains.

A new plasmodiocarpic and sporocarpic species of myxomycete in the genus Physarum is described and illustrated. This new species appeared on decayed leaves and remains of succulent plants (Agave, Opuntia, Yucca) growing in arid zones. It differs from all other species in the genus in having polyhedral spores linked in chains like a string of beads, a unique feature within all known myxomycetes. Apart from detailed morphological data, partial sequences of both the small-subunit ribosomal RNA and elongation factor 1-alpha genes, generated from four isolates collected in two distant regions, i.e., Mexico and Canary Islands, are also provided in this study. Combined evidence supports the identity of the specimens under study as a new species.

A New Heterolobosean Amoeboflagellate, Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea, Korea.

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5-28.0 µm and 5.4-12.6 µm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 µm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo- and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1-5.8S rRNA gene-ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.

Myxobolus chushi n. sp. (Myxozoa:Myxosporea) parasitizing Schizothorax niger (Heckel), a native cyprinid fish from Wullar Lake in Kashmir Himalayas.

In the study, a new species, Myxobolus chushi n. sp. infecting gills of wild specimens of Schizothorax niger (Heckel) inhabiting Wullar Lake in Kashmir Himalayas, (J&K) India has been described based on morphology of the myxospore and using partial 18S rDNA sequencing. Pathological changes in the gills have been studied with the help of histological sections stained with Luna's method. Twenty fish specimens were examined, out of which four had oval, white plasmodia in gills measuring 2.0×0.5mm. The myxospores were spherical to ovoidal in shape with slightly attenuated posterior end, measuring 11.17±0.23 (10.60-11.40)µm in length and 9.14±0.06 (8.80-9.20)µm in width, having a prominent pore at the anterior end. The polar capsules were pyriform in shape, measuring 4.25±0.15 (4.00-4.40)µm in length and 2.38±0.27 (2.00-2.65)µm in width having polar filaments forming coils up to 5 in number. Parietal folds 9 in number present on the posterior part of the shell. The intensity of infection was recorded to be moderate as indicated by gill plasmodial index (GPI=2). The plasmodium was located in the vascular network occupying whole of the gill lamella therefore typed as intralamellar vascular type, LV3. Analysis of 18S small subunit (SSU) rDNA sequence of the isolate demonstrated 90% homogeneity with M. sp. KLT-2014 infecting scales of Labeo rohita from Myanmar and 89% with M. dermiscalis infecting scales of Labeo rohita from India.

Description of a new Neoactinomyxum type actinosporean from the oligochaete Branchiura sowerbyi Beddard.

Actinosporean infection of oligochaetes living in the mud of a commercial gibel carp pond with myxosporean disease was studied. Six actinospore types were detected exclusively from the oligochaete Branchiura sowerbyi Beddard with very high prevalence (18%). Five out of the six types were identified as the same actinosporeans described in previous reports, the sixth actinosporean was identified as a new Neoactinomyxum type and described here based on morphological and molecular characterisation. Spore body of the actinospore was globular, much smaller than caudal processes. Three caudal processes were disc-like in apical view, hemispherical in side view, closer together and encircling the spore body. The number of sporoplasm cells was detected as eight in one specimen. The new actinosporean markedly differed from other Neoactinomyxum types in literature having much bigger caudal processes. DNA sequence analyses further confirmed the morphological identification, and revealed the actinosporean described here (KU641392) possessed less than 94% sequence similarity with myxozoans available in the GenBank database.

Didymium azorellae, a new myxomycete from cushion plants of cold arid areas of South America.

A new species of Didymium (Myxomycetes), D. azorellae, isolated from plant debris in a moist chamber, collected during studies of cold arid areas of Argentina and Peru, is described. It can be distinguished by its small size, the tightly packed layer of lime crystals on the peridium, the very scant, or absent, capillitium, and the unique spore ornamentation, especially by scanning electron microscopy. The species developed on dead leaves of cushion plants growing in the extremely harsh environments of the central Andean puna at almost 5000 m elevation and the Andino-Patagonian steppe. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. In order to confirm the identity of the new species described in this paper, a molecular study was conducted based on partial sequences of both the 18S rRNA and the elongation factor 1-alpha gene. Phylogenetic analysis including two specimens from different countries of the newly described species, Didymium azorellae, strongly supports the grouping of these specimens as a separate clade from the rest of the analyzed species.

Morphological re-description and molecular characterization of Kudoa pagrusi (Myxosporea: Multivalvulida) infecting the heart muscles of the common sea bream fish Pagrus pagrus (Perciformes: Sparidae) from the Red Sea, Egypt.

In the present study, 100 samples of different sizes of the common sea bream fish Pagrus pagrus were collected from the Egyptian water along the Gulf of Suez, Red Sea and examined for the prevalence of myxosporidian parasites in general and Kudoa spp. in particular. Fish samples were thoroughly externally examined. After dissection, all the internal organs were removed and examined. A total of 60 out of 100 fish specimens were found to be infected with Kudoa stages. Parasitic infection was restricted to the heart muscles of the examined fish. None of the other organs was found to be infected. Macroscopic cysts (plasmodia) heavily infested the different parts of the heart muscles. Each plasmodium measured 1.2-2.5 (1.53 ± 0.2) mm × 0.63-0.80 (0.65 ± 0.2) mm. Mature spores are quadratic in shape in the apical view showing four equal valves and four symmetrical polar capsules. Fresh spores were 5.0-7.1 (5.7 ± 0.2) µm long × 5.4-8.5 (6.1 ± 0.3) µm wide. On the basis of spore morphology, the present species was identified as Kudoa pagrusi. Morphometric characterization revealed that the relatively small size of this Kudoa species was the distinctive feature that separates it from all previously described species. Molecular analysis based on small subunit ribosomal DNA (SSU rDNA) sequences revealed that the highest percentage of identity was observed with K. scomberomori and followed by K. shiomitsui, K. hypoepicarclialis, K. amamiensis, and K. kenti. The kudoid spores showed morphometric variations to some extents but had essentially identical nucleotide sequences of the SSU rDNA gene sequences closest to those of K. scomberomori and K. shiomitsui recorded from elasmobranchs in the Indo-Pacific Ocean. The present findings support the identification of an ancestral marine origin of the present Kudoa species.

Synopsis of the species of the genus Sphaeromyxa Thélohan, 1892 (Myxosporea: Bivalvulida: Variisporina: Sphaeromyxidae).

Sphaeromyxa spp. are parasites of marine fishes, infecting the gall-bladders or bile ducts. The spores of these species possess characteristic ribbon-like polar filaments, a unique character among myxozoans. This unique character is also a synapomorphy consistent with estimates of phylogeny for this group which forms a lineage distinct from other myxozoans. There are 49 nominal species of Sphaeromyxa Thélohan, 1892 for which a synopsis is provided, reporting spore dimensions, spore shape, locality, and host species. A line drawing is also provided for each species.

Stress management in cyst-forming free-living protists: programmed cell death and/or encystment.

In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes.

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits.

Free-living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.

Description of Dientamoeba fragilis cyst and precystic forms from human samples.

Dientamoeba fragilis is a common enteropathogen of humans. Recently a cyst stage of the parasite was described in an animal model; however, no cyst stage has been described in detail from clinical samples. We describe both cyst and precystic forms from human clinical samples.

Genetic crosses and complementation reveal essential functions for the Plasmodium stage-specific actin2 in sporogonic development.

Malaria parasites have two actin isoforms, ubiquitous actin1 and specialized actin2. Actin2 is essential for late male gametogenesis, prior to egress from the host erythrocyte. Here, we examined whether the two actins fulfil overlapping functions in Plasmodium berghei. Replacement of actin2 with actin1 resulted in partial complementation of the defects in male gametogenesis and, thus, viable ookinetes were formed, able to invade the midgut epithelium and develop into oocysts. However, these remained small and their DNA was undetectable at day 8 after infection. As a consequence sporogony did not occur, resulting in a complete block of parasite transmission. Furthermore, we show that expression of actin2 is tightly controlled in female stages. The actin2 transcript is translationally repressed in female gametocytes, but translated in female gametes. The protein persists until mature ookinetes; this expression is strictly dependent on the maternally derived expression. Genetic crosses revealed that actin2 functions at an early stage of ookinete formation and that parasites lacking actin2 are unable to undergo sporogony in the mosquito midgut. Our results provide insights into the specialized role of actin2 in Plasmodium development in the mosquito and suggest that the two actin isoforms have distinct biological functions.

Early development and tissue distribution of Pseudoloma neurophilia in the zebrafish, Danio rerio.

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small-subunit ribosomal RNA gene, standard hematoxylin-eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36-48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.

Commitment to cyst formation in Giardia.

Giardia trophozoites differentiate into infectious cysts (encystment) in response to physiological stimuli; encystment is crucial for Giardia's transmission, survival and pathogenesis. In vitro, Giardia encysts when bile sequesters lipids necessary for this lipid auxotroph, and in vivo they encyst to infect new hosts. In this study, we investigated, for the first time, commitment to encystment in Giardia using both molecular and cellular techniques. We show that after 3-6 h in inducing conditions, encysting trophozoites continue to encyst regardless of whether the inducing stimulus remains. We propose that a trophozoite's inability to revert to a growing or dividing trophozoite represents a commitment to encystment. The onset of commitment correlated with the appearance of encystment specific vesicles (ESVs) and encystment specific protein synthesis. These observations suggest the involvement of regulatory pathways with the ability to 'remember' a transient signal long after its removal; a property that enables encysting trophozoites to complete the encystment process should the unfavourable triggering condition(s) change. The ability to form cysts in response to transient signals or, as we have highlighted in this paper, the ability of a small percentage of the population to form cysts without an inducer is vital for the maintenance of infection within populations.

Updated morphology, histopathology and molecular phylogeny of Myxobolus hearti, cardiac myxosporea in gibel carp, Carassius gibelio (Bloch).

The original description of Myxobolus hearti is supplemented with new data on spore morphology, histopathology and molecular phylogeny. Myxobolus hearti are found in the heart ventricle of the gibel carp, Carassius gibelio (Bloch), where they form whitish oval or irregularly shaped plasmodia. Mature spores are oval or shortly ellipsoidal in frontal view, lemon-shaped in sutural view and eye-shaped in apical view. The spores are 14.12 ± 0.35 (13.6-15) µm long (mean ± SD), 11.85 ± 0.34 ± 0.36 (11-12) µm wide and 7.32 ± 0.36 (7-8) µm thick. The two polar capsules are equal in size, 6.11 ± 0.29 (6-7) µm long and 3.89 ± 0.31(3-4) µm wide, and are long pyriform in shape. Polar filaments have six or seven coils situated perpendicular to the longitudinal axis of the polar capsules. Histopathology indicates that the plasmodia are encased by the host connective tissue, and no inflammatory responses are found in the heart ventricles. Phylogenetic analysis based on the 18S small-subunit ribosomal DNA sequences indicates that M. hearti is, genetically, most similar to Henneguya doneci, a gill-infecting species.

Steely enzymes are involved in prestalk and prespore pattern formation.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.

Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species.

The present study consists of a detailed phylogenetic analysis of myxosporeans of the Myxobolus and Henneguya genera, including sequences from 12 Myxobolus/Henneguya species, parasites of South American pimelodids, bryconids and characids. Maximum likelihood and maximum parsimony analyses, based on 18 S rDNA gene sequences, showed that the strongest evolutionary signal is the phylogenetic affinity of the fish hosts, with clustering mainly occurring according to the order and/or family of the host. Of the 12 South American species studied here, six are newly described infecting fish from the Brazilian Pantanal wetland. Henneguya maculosus n. sp. and Myxobolus flavus n. sp. were found infecting both Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum; Myxobolus aureus n. sp. and Myxobolus pantanalis n. sp. were observed parasitizing Salminus brasiliensis and Myxobolus umidus n. sp. and Myxobolus piraputangae n. sp. were detected infecting Brycon hilarii.