Lophomonas as a respiratory pathogen-jumping the gun.

Human infections with the protozoan Lophomonas have been increasingly reported in the medical literature over the past three decades. Initial reports were based on microscopic identification of the purported pathogen in respiratory specimens. Later, a polymerase chain reaction (PCR) was developed to detect Lophomonas blattarum, following which there has been a significant increase in reports. In this minireview, we thoroughly examine the published reports of Lophomonas infection to evaluate its potential role as a human pathogen. We examined the published images and videos of purported Lophomonas, compared its morphology and motility characteristics with host bronchial ciliated epithelial cells and true L. blattarum derived from cockroaches, analyzed the published PCR that is being used for its diagnosis, and reviewed the clinical data of patients reported in the English and Chinese literature. From our analysis, we conclude that the images and videos from human specimens do not represent true Lophomonas and are predominantly misidentified ciliated epithelial cells. Additionally, we note that there is insufficient clinical evidence to attribute the cases to Lophomonas infection, as the clinical manifestations are non-specific, possibly caused by other infections and comorbidities, and there is no associated tissue pathology attributable to Lophomonas. Finally, our analysis reveals that the published PCR is not specific to Lophomonas and can amplify DNA from commensal trichomonads. Based on this thorough review, we emphasize the need for rigorous scientific scrutiny before a microorganism is acknowledged as a novel human pathogen and discuss the potential harms of misdiagnoses for patient care and scientific literature.

Rhabdamoeba marina is a heterotrophic relative of chlorarachnid algae.

Rhabdamoeba marina is a unique and poorly reported amoeba with an uncertain phylogenetic position. We successfully cultured R. marina from coastal seawater in Japan and performed a molecular phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) gene sequence. Our phylogenetic analysis showed that R. marina branched as a basal lineage of Chlorarachnea, a group of marine photosynthetic algae belonging to the phylum Cercozoa within the supergroup Rhizaria. By comparing the ecological and morphological characteristics of R. marina with those of photosynthetic chlorarachneans and other cercozoans, we gained insight into the evolution and acquisition of plastids in Chlorarachnida.

Molecular phylogenetics of sessile Dolium sedentarium, a petalomonad euglenid.

The euglenids are a species-rich group of flagellates with varying modes of nutrition that can be found in diverse habitats. Phagotrophic members of this group gave rise to phototrophs and hold the key to understanding the evolution of euglenids as a whole, including the evolution of complex morphological characters like the euglenid pellicle. Yet to understand the evolution of these characters, a comprehensive sampling of molecular data is needed to correlate morphological and molecular data, and to estimate a basic phylogenetic backbone of the group. While the availability of SSU rDNA and, more recently, multigene data from phagotrophic euglenids has improved, several "orphan" taxa remain without any molecular data whatsoever. Dolium sedentarium is one such taxon: It is a rarely-observed phagotrophic euglenid that inhabits tropical benthic environments and is one of few known sessile euglenids. Based on morphological characters, it has been thought of as part of the earliest branch of euglenids, the Petalomonadida. We report the first molecular sequencing data for Dolium using single-cell transcriptomics, adding another small piece in the puzzle of euglenid evolution. Both SSU rDNA and multigene phylogenies confirm it as a solitary branch within Petalomonadida.

Expanding the molecular and morphological diversity of Apusomonadida, a deep-branching group of gliding bacterivorous protists.

Apusomonads are cosmopolitan bacterivorous biflagellate protists usually gliding on freshwater and marine sediment or wet soils. These nanoflagellates form a sister lineage to opisthokonts and may have retained ancestral features helpful to understanding the early evolution of this large supergroup. Although molecular environmental analyses indicate that apusomonads are genetically diverse, few species have been described. Here, we morphologically characterize 11 new apusomonad strains. Based on molecular phylogenetic analyses of the rRNA gene operon, we describe four new strains of the known species Multimonas media, Podomonas capensis, Apusomonas proboscidea, and Apusomonas australiensis, and rename Thecamonas oxoniensis as Mylnikovia oxoniensis n. gen., n. comb. Additionally, we describe four new genera and six new species: Catacumbia lutetiensis n. gen. n. sp., Cavaliersmithia chaoae n. gen. n. sp., Singekia montserratensis n. gen. n. sp., Singekia franciliensis n. gen. n. sp., Karpovia croatica n. gen. n. sp., and Chelonemonas dolani n. sp. Our comparative analysis suggests that apusomonad ancestor was a fusiform biflagellate with a dorsal pellicle, a plastic ventral surface, and a sleeve covering the anterior flagellum, that thrived in marine, possibly oxygen-poor, environments. It likely had a complex cell cycle with dormant and multiple fission stages, and sex. Our results extend known apusomonad diversity, allow updating their taxonomy, and provide elements to understand early eukaryotic evolution.

True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia).

Lophomonas blattarum is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of L. blattarum and its sole congener, Lophomonas striata, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of L. striata, but not consistent with sequences from human samples attributed to L. blattarum.

Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

Paraburkholderia flagellata sp. nov. and Paraburkholderia adhaesiva sp. nov., two novel species isolated from forest soil in Dinghushan Biosphere Reserve in Guangdong, China.

Two Gram-stain-negative, aerobic, motile and short rod strains, designated 4D117T and ZD32-2T, were isolated from the forest soils. Strains 4D117T and ZD32-2T grew optimally at pH 4.0-6.5, 20-33 °C and pH 4.5-7.0, 33 °C, respectively, and both at 0.5% (w/v) NaCl concentration. Strains 4D117T and ZD32-2T shared the highest 16S rRNA gene sequence similarity with P. acidiphila 7Q-K02T (99.1%) and P. ferrariae NBRC 106233T (98.7%), respectively. The genome size and G + C contents of strains 4D117T and ZD32-2T were 9,002,095 bp, 62.9% and 6,974,420 bp, 61.7%, respectively. The dDDH and ANI values between strains 4D117T, ZD32-2T and closely related Paraburkholderia species were in the ranges of 21.9-51.6% and 82.9-94.4%, and 81.7% and 25.4% between themself, respectively. Functional genomic analysis showed both strains were capable of degrading contaminants, such as benzoate, anthranilic acid and catechol for 4D117T, and benzene and catechol for ZD32-2T, indicating that they may have potentials for soil pollutant treatment. The main polar lipids of strains 4D117T and ZD32-2T were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain 4D117T contained C16:0, C19:0 cyclo ω8c and C18:1 ω7c and/or C18:1 ω6c, while strain ZD32-2T had C16:0 and C17:0 cyclo as their major cellular fatty acids (> 10%). Based on the phenotypic characters and genomic data, strains 4D117T and ZD32-2T represent two novel species of genus Paraburkholderia, for which the names Paraburkholderia flagellata sp. nov. (type strain 4D117T = GDMCC 1.2617T = NBRC 115278T) and Paraburkholderia adhaesiva sp. nov. (type strain ZD32-2T = GDMCC 1.2622T = NBRC 115282T) are proposed.

Channelrhodopsins: From Phototaxis to Optogenetics.

Channelrhodopsins stand out among other retinal proteins because of their capacity to generate passive ionic currents following photoactivation. Owing to that, channelrhodopsins are widely used in neuroscience and cardiology as instruments for optogenetic manipulation of the activity of excitable cells. Photocurrents generated by channelrhodopsins were first discovered in the cells of green algae in the 1970s. In this review we describe this discovery and discuss the current state of research in the field.

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

Description of Imasa heleensis, gen. nov., sp. nov. (Imasidae, fam. nov.), a Deep-Branching Marine Malawimonad and Possible Key Taxon in Understanding Early Eukaryotic Evolution.

Malawimonadida is a deep-level (arguably "kingdom-scale") lineage of eukaryotes whose phylogenetic affinities are uncertain but of great evolutionary interest, as the group is suspected to branch close to the root of the tree of eukaryotes. Part of the difficulty in placing Malawimonadida phylogenetically is its tiny circumscription: at present, it comprises only two described and one cultured but undescribed species, all of them are freshwater suspension-feeding nanoflagellates. In this study, we cultivated and characterised Imasa heleensis gen. nov., sp. nov. (Imasidae fam. nov.), the first marine malawimonad to be described. Light and electron microscopy observations show that Imasa is largely similar to other malawimonads, but more frequently adheres to the substrate, often by means of a pliable posterior extension. Phylogenetic analyses based on two ribosomal RNA genes and four translated protein-coding genes using three different taxon sets place Imasa as sister to the three freshwater malawimonad strains with strong support. Imasa's mitochondrial genome is circular-mapping and shows a similar gene complement to other known malawimonads. We conclude that Imasa represents an important expansion of the range of taxa available for future evolutionary study.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Flagellate Dermatitis After Ingestion of Shiitake Mushrooms in a Healthy Female Living in the Southeastern United States.

BACKGROUND: Shiitake flagellate dermatitis is a shiitake mushroom (SM)-induced toxic dermatosis that is a widely recognized clinical phenomenon in Japan, China, and Korea but is rarely reported outside of Asia. Typically, 24-48 hours after consumption of SM, patients develop severely pruritic, linear, often parallel, infiltrated erythematous lesions, commonly described as being in a "whip-like" pattern. The dermatosis is noted preferentially on the trunk, extremities, and neck. Shiitake flagellate dermatitis is self-limited and typically resolves within days to weeks of its appearance. CASE REPORT: A healthy 36-year-old woman living in the southeastern United States presented with a 48-hour history of pruritic, truncal, morbilliform, flagellate-like efflorescences that extended to her buttocks and thighs. Her hard and soft palates, palms and soles, hair-bearing scalp, and face were also involved. Two days before presentation, the patient ate a store-bought salad preparation that contained raw mushrooms including SMs. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Shiitake dermatitis has a characteristic whip-like pattern that, if identified early by first-line physicians, can relieve concerns for more serious etiologies and expedite appropriate therapy.

DETECTION OF AN ARENAVIRUS IN A GROUP OF CAPTIVE WAGLER'S PIT VIPERS (TROPIDOLAEMUS WAGLERI).

A group of eight Wagler's pit vipers (Tropidolaemus wagleri) from a private collection died with respiratory signs within 6 mo of one another. The group consisted of an adult breeding pair that was wild caught and six offspring from this pair. Four of the dead snakes were submitted for gross and histopathology. Signs of bacterial pneumonia were detected in all four examined snakes. No inclusion bodies suggestive of viral infection were found in any of the examined tissues. Polymerase chain reactions for the detection of ferla-, adeno-, reo-, and nidoviruses were all negative, but reptarenaviruses closely related to viruses previously described in boa constrictors (Boa constrictor) with inclusion body disease were detected in two of the four snakes. This is the first description of reptarenaviruses in viperid snakes. The pathogenic role of the virus in illness is unknown.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

A Revised Taxonomy of Diplonemids Including the Eupelagonemidae n. fam. and a Type Species, Eupelagonema oceanica n. gen. & sp.

Recent surveys of marine microbial diversity have identified a previously unrecognized lineage of diplonemid protists as being among the most diverse heterotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. Recently, we documented morphology and molecular sequences from several DSPDs, one of which is particularly widespread and abundant in environmental sequence data. To simplify the communication of future work on this important group, here we formally propose to erect the family Eupelagonemidae to encompass this clade, as well as a formal genus and species description for the apparently most abundant phylotype, Eupelagonema oceanica, for which morphological information and single-cell amplified genome data are currently available.

Differential Response of Cafeteria roenbergensis to Different Bacterial and Archaeal Prey Characteristics.

In the marine environment, the abundance of Bacteria and Archaea is either controlled bottom-up via nutrient availability or top-down via grazing. Heterotrophic nanoflagellates (HNF) are mainly responsible for prokaryotic grazing losses besides viral lysis. However, the grazing specificity of HNF on specific bacterial and archaeal taxa is under debate. Bacteria and Archaea might have different nutritive values and surface properties affecting the growth rates of HNF. In this study, we offered different bacterial and archaeal strains with different morphologic and physiologic characteristics to Cafeteria roenbergensis, one of the most abundant and ubiquitous species of HNF in the ocean. Two Nitrosopumilus maritimus-related strains isolated from the northern Adriatic Sea (Nitrosopumilus adriaticus, Nitrosopumilus piranensis), two Nitrosococcus strains, and two fast growing marine Bacteria (Pseudoalteromonas sp. and Marinobacter sp.) were fed to Cafeteria cultures. Cafeteria roenbergensis exhibited high growth rates when feeding on Pseudoalteromonas sp., Marinobacter sp., and Nitrosopumilus adriaticus, while the addition of the other strains resulted in minimal growth. Taken together, our data suggest that the differences in growth of Cafeteria roenbergensis associated to grazing on different thaumarchaeal and bacterial strains are likely due to the subtle metabolic, cell size, and physiological differences between different bacterial and thaumarchaeal taxa. Moreover, Nitrosopumilus adriaticus experienced a similar grazing pressure by Cafeteria roenbergensis as compared to the other strains, suggesting that other HNF may also prey on Archaea which might have important consequences on the global biogeochemical cycles.

Toxicity of BDE-47, BDE-99 and BDE-153 on swimming behavior of the unicellular marine microalgae Platymonas subcordiformis and implications for seawater quality assessment.

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, have been extensively applied and eventually leached into the surrounding environment. Marine microalgae are not only the dominant primary producers of marine ecosystem, but also food source for aquaculture. PBDEs have been found to remarkably inhibit growth, photosynthesis and metabolism of marine microalgae. However, whether they also affect swimming behavior of marine motile microalgae remains unknown. We chose BDE-47, BDE-99 and BDE-153 as model PBDEs, and the unicellular marine green flagellate, Platymonas subcordiformis, as test organism to figure out this issue. After two-hour exposure, motile cells proportion (MOT), swimming velocity (VCL, VAP and VSL), and swimming pattern (LIN and STR) of P. subcordiformis were measured via computer assisted cell movement tracking. Results suggest that the three PBDEs not only reduced motile cells proportion and swimming velocity, but also altered swimming pattern. BDE-47 was more toxic than BDE-99, followed by BDE-153, indicating their toxicity decreased as bromination degree increases. Swimming ability of P. subcordiformis was even completely arrested when BDE-47 and BDE-99 at 32 µg/L. The impairment of swimming ability by PBDEs might thereby hinder growth and survival of marine microalgae, and subsequently threaten marine ecosystem and aquaculture industry. More importantly, this study implies that marine microalgae swimming behavior test is more efficiency and sensitive than traditional marine microalgal bioassays, like growth and photosynthesis tests. We suggest that although future work is needed, swimming behavior analysis of P. subcordiformis with MOT, VCL and VAP as endpoints can be developed as a low-cost, convenient, fast, reliable and sensitive method for seawater quality assessment.

Protoplast preparation from enriched flagellates and resting cells of Haematococcus pluvialis.

AIMS: To establish a proper protoplast-preparation route from enriched motile flagellates and nonmotile resting cells of Haematococcus pluvialis. METHODS AND RESULTS: Through cultivations in two mixotrophic media, enriched Haematococcus flagellates and resting cells were respectively produced and applied in enzymatic protoplast preparations. Great differences of enzymatic sensitivity and osmotic-lability were identified between them. Flagellates showed the same osmotic-lability as protoplasts and the extracellular matrix-removing rate was applied for an evaluation of protoplast-releasing. During the treatment of flagellates, an addition of more than 0·2 mmol l-1 Ca2+ was found to be essential for maintenance of high cellular viability. More than 80% cellular viability and a 90% protoplast-releasing rate were obtained simultaneously after 2-3 h treatment of 0·06% proteinase K in 0·05 mol l-1 Tris-HCl (pH7·8) buffer with 0·2 mmol l-1 CaCl2 and 0·2 mol l-1 sorbitol/mannitol. For resting cells, a treatment of both 0·12% proteinase K and a combination of 2% cellulase + 1% snailase could function similarly in order to degrade the cellulosic cell wall, while the protoplast yield was limited to about 40%, due to the existence of an undegradable secondary wall in the mature resting cell. CONCLUSION: Proteinase K was efficient for protoplast-releasing from either flagellates or resting cells. Due to the great difference of enzymatic sensitivity and osmotic-lability between flagellates and resting cells, it was necessary to select a different enzymatic treating process based upon the main cell type in the culture. A better protoplast-preparing efficiency was obtained from Haematococcus cells when flagellates were in the majority. SIGNIFICANCE AND IMPACT OF THE STUDY: The protoplast preparation of H. pluvialis was firstly established based on two main cell types of H. pluvialis, motile flagellates and nonmotile resting cells. Increase of flagellate stability and viable protoplast-preparing efficiency through addition of Ca2+ in enzymatic solution was firstly reported.

Tunic extract of the host ascidian attracts the causal agent of soft tunic syndrome, Azumiobodo hoyamushi (Kinetoplastea: Neobodonida).

Azumiobodo hoyamushi, a kinetoplastid flagellate, is the causative agent of soft tunic syndrome, an infectious disease of the edible ascidian Halocynthia roretzi. The flagellate is thought to invade the tunic matrix via a damaged area of the tunic on the siphon wall. We hypothesized that the flagellate locates the tunic entry site by a chemotactic response to soluble substances diffused from the host ascidians. To investigate this hypothesis, we examined whether the flagellate shows a chemotactic response to tissue extracts (tunic and other tissues) from the host ascidian H. roretzi. We tested extracts from 5 tissues as well as hemolymph. Only the tunic extract showed significant positive chemotactic activity, and the activity decreased with increasing dilution. Furthermore, autoclaved tunic extract, extracts from diseased individuals, and extract from the styelid ascidian Styela clava also had chemotactic activity, although the activities were lower than that of tunic extract from healthy H. roretzi. Ultrafiltration of the tunic extract through a 3 kDa cutoff membrane completely abrogated the activity; the ultrafiltration retentate still showed activity. Thus, the soluble factors that attract the flagellate are present exclusively in the tunic extract, and the chemotactic factors are larger than 3 kDa. Our experiments also suggested that the tunic extract contains both heat-stable and heat-labile factors. We conclude that the flagellate locates the tunic entry site by chemotaxis toward soluble factors that diffuse from a damaged area of the tunic on the siphon wall.