Intraspecies Variation in Tetrahymena rostrata

Two distinct isolates of the facultative parasite, Tetrahymena rostrata were compared, identifying and utilising markers that are useful for studying clonal variation within the species were identified and utilised. The sequences of mitochondrial genomes and several nuclear genes were determined using Illumina short read sequencing. The two T. rostrata isolates had similar morphology. The linear mitogenomes had the gene content and organisation typical of the Tetrahymena genus, comprising 8 tRNA genes, 6 ribosomal RNA genes and 45 protein coding sequences (CDS), twenty-two of which had known function. The two isolates had nucleotide identity within common nuclear markers encoded within the histone H3 and H4 and small subunit ribosomal RNA genes and differed by only 2–4 nucleotides in a region of the characterised actin genes. Variation was observed in several mitochondrial genes and was used to determine intraspecies variation and may reflect the natural history of T. rostrata from different hosts or the geographic origins of the isolates.

Infection of Slugs with Theronts of the Ciliate Protozoan, Tetrahymena rostrata

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii’s response to oxidative stress. The role of the large TrxR, however, remains elusive.

Three-dimensional reconstructions of haustoria in two parasitic plant species in the Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Three-dimensional reconstructions of the internal structures of haustoria in parasitic Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides structural insights into how organs interact between hosts and parasitic plants.One-sentence summaryThree-dimensional image reconstruction was used to visualize the spatial organization of cell types in the haustoria of parasitic plants with special reference to their interaction with host roots.

Subtilase activity in the intrusive cells mediates haustorium maturation in parasitic plants

ABSTRACTParasitic plants that infect crops are devastating to agriculture throughout the world. They develop a unique inducible organ called the haustorium, which connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically towards the host vasculature. Then, some of the intrusive cells re-differentiate to form a xylem bridge that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells are unknown. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-Sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and used promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of an SBT inhibitor in the intrusive cells inhibited their development, inhibited the development of the xylem bridge, and reduced auxin response levels near the site where the xylem bridge normally develops. Therefore, we propose that subtilase activity plays an important role in haustorium development in this parasitic plant.One sentence summaryTissue-specific analysis showed that the subtilases specifically expressed in intrusive cells regulate auxin-mediated host-parasite connections in the parasitic plant Phtheirospermum japonicum.

Nematodes associated with terrestrial slugs in the Edmonton region of Alberta, Canada

A survey of nematodes associated with terrestrial slugs was conducted in residential gardens, nurseries, greenhouses and agricultural sites located in and around Edmonton, Alberta, Canada. A total of 2406 slugs were collected from 82 sites. Slugs were decapitated and cadavers were incubated for two weeks, with emerging nematodes removed and processed for identification. Nematodes were identified using molecular sequence data for the 18S ribosomal DNA. Nematodes were recovered from 20 of the 82 sites surveyed, with 24.4% of the slugs infected with nematodes. A total of seven nematodes were identified to species level, including Caenorhabditis elegans, Panagrolaimus papillosus, Pellioditis typica, Pelodera pseudoteres, Rhabditella axei, Rhabditoides inermiformis and Phasmarhabditis californica. An additional four specimens were identified to genus level, including Oscheius sp. (9), Pristionchus sp., Rhabditis sp. and Rhabditophanes sp. (1). The two most common nematode species were C. elegans and P. pseudoteres. The facultative parasite, P. californica, was recovered from a single Arion rufus specimen, collected from a seasonal nursery. To our knowledge, this study represents the first survey of slug-associated nematodes in Canada.

Nanopore sequencing improves the draft genome of the human pathogenic amoeba Naegleria fowleri

Naegleria fowleri is an environmental protist found in soil and warm freshwater sources worldwide and is known for its ability to infect humans and causing a rapid and mostly fatal primary amoebic meningoencephalitis. When contaminated water enters the nose, the facultative parasite follows the olfactory nerve and enters the brain by crossing the cribriform plate where it causes tissue damage and haemorrhagic necrosis. Although N. fowleri has been studied for several years, the mechanisms of pathogenicity are still poorly understood. Furthermore, there is a lack of knowledge on the genomic level and the current reference assembly is limited in contiguity. To improve the draft genome and to investigate pathogenicity factors, we sequenced the genome of N. fowleri using Oxford Nanopore Technology (ONT). Assembly and polishing of the long reads resulted in a high-quality draft genome whose N50 is 18 times higher than the previously published genome. The prediction of potentially secreted proteins revealed a large proportion of enzymes with a hydrolysing function, which could play an important role during the pathogenesis and account for the destructive nature of primary amoebic meningoencephalitis. The improved genome provides the basis for further investigation unravelling the biology and the pathogenic potential of N. fowleri.

Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity capable of inactivating the superoxide anion (•O2−) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34–44 kDa and with very different AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50 kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60 kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2− is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress.

Nature of the Dutch elm disease: new aspects of diagnostics, pathogenesis, and etiology

Расхождения по установлению первопричины голландской болезни вязов (ГБВ) возникли сразу после её обнаружения в 1918 г. В разные годы указывали фитопатогенные бактерии (Васillus amylovorus, Miсrососсus ulmi, Рseudomonas lignicola) и гриб Ophiostoma (Ceratostomella) ulmi (Graphium ulmi). В 1937 г. в Краснодарском крае наряду с закупоркой сосудов на вязах описано ядро бактериальной водянки, т. е. уже 80 100 лет назад отмечали смешанную этиологию ГБВ, но бактериозы посчитали недостоверной причиной, возобладала теория грибной моноинфекции. Позже открыли агрессивный патоген Ophiostoma novoulmi, близкие виды O. himalulmi, O. novoulmi subsp. novoulmi, O. novoulmi subsp. americana. Параллельно на вязах выявлены бактериальная водянка (Pectobacterium carotovorum), бактериальный ожог (Erwinia группы Amylovora), Pseudomonas amygdali pv. ulmi, связанная с поражением коры, Xylella fastidiosa, вызывающая ожог листвы. К симптоматике ГБВ причастны виды фитопатогенных бактерий. Анализ факторов патогенности гриба показывает неоднозначность их проявления в симптоматике ГБВ. Агрессивность O. novoulmi проявилась не в патогенезе, а в вытеснении O. ulmi. Продукты метаболизма бактерий (кислота, газ, ферменты) разрушают целлюлозу, лигнин клеточных стенок, срединную пластинку и крахмал, вызывая камедь, мокроту, экссудат, закупорку сосудов, некрозы, мацерацию, растрескивание коры и древесины. Graphium ulmi развивается на подготовленных тканях совместно с бактериями как факультативный паразит, не проявляя антагонизма. Предполагается, что ГБВ имеет либо полифункциональную этиологию смешанного бактериальногрибного происхождения, либо самостоятельную бактериальную. Ареалы ильмовых пород, таксонов комплекса Ophiostoma ulmi и бактериозов имеют зоны перекрытия, что предполагает смешанный или сопряженный патогенез. В проблеме ГБВ на смену теории грибной моноинфекции должна прийти парадигма фитопатологической диагностики с использованием метагеномного анализа. Discordance on establishing the prime cause of the Dutch elm disease (DED) arose soon after its discovery in 1918. Through the years different causes were specified in literature: phytopathogenic bacteria (Bacillus amylovorus, Micrococcus ulmi, Pseudomonas lignicola) and fungi Ophiostoma (Ceratostomella) ulmi (Graphium ulmi). The bacterial dropsy together with obstruction of vessels was described in 1937. Hence, even 80 100 years ago the mixed etiology was noted. However, bacterioses were considered as a doubtful reason. Later on, an aggressive pathogen Ophiostoma novoulmi and close species O. himalulmi, O. novoulmi subsp. novoulmi, O. novoulmi subsp. americana were found. At the same time, bacterial dropsy (Pectobacterium carotovorum), fire blight (Erwinia of group Amylovora), Pseudomonas amygdali pv. ulmi connected to the injuries of bark, and Xylella fastidiosa causing foliage burn, were revealed on elms. Some species of phytopathogenic bacteria are also involved in symptoms of DED. The analysis of factors of pathogenesis of G. ulmi shows ambiguity of their manifestation in DED symptoms. The aggressivity of O. novoulmi was not in pathogenesis, these fungi force another species O. ulmi out. Products of bacteria metabolism (acid, gas, and enzymes) destroy cellulose, lignin cell walls, middle plate, and starch, causing gum, sputum, exudate, blockage of vessels, necrosis, maceration, cracks of bark and wood. Graphiosis settles on the prepared tissues where G. ulmi grows as a facultative parasite together with bacteria without showing any antagonism. It is suggested that DED has polyfunctional etiology of the mixed bacterial and fungi origin or independent bacterial origin. Distribution areas of elms, taxa of the Ophiostoma ulmi complex and the main bacterioses of elms have overlapping zones, that suggests mixed or associated pathogenesis. The theory of fungal monoinfection in a problem of DED requires a change of the paradigm of phytopathological diagnostics and use of metagenomic analysis in the diagnostics.