Paramecium (Oligohymenophorea, Ciliophora) diversity in Thailand sheds light on the genus biogeography and reveals new phylogenetic lineages.

Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard-to-reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi. Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium.

Presence of Cryptosporidium parvum in pre-washed vegetables from different supermarkets in South East England: A pilot study.

Cryptosporidium is an important water-borne and food-borne parasite with a high burden of disease. This organism has been shown to contaminate various leafy vegetables; however, studies assessing the presence of Cryptosporidium spp in pre-washed and ready-to-eat vegetables are limited. Routine surveillance in the UK revealed a nationwide exceedance of human cases of Cryptosporidium. Therefore, this study aims to assess the presence of this parasite in pre-washed vegetables from supermarkets in the UK. A total of 36 samples were purchased from four different supermarkets. A nested PCR targeting the SSU rRNA was carried out on 24 samples, 58% were PCR-positive for Cryptosporidium. Sanger sequencing confirmed that, of these sequences, 4/24 (17%) produced significant similarities to Cryptosporidium parvum. This study provides evidence for the presence of C. parvum in pre-washed and ready-to-eat vegetables. Future work to identify the point of contamination is required.

Integrative approach to species delimitation in Rhizophydiales: Novel species of Angulomyces, Gorgonomyces, and Terramyces from northern Thailand.

The Chytridiomycota is a phylum of zoosporic eufungi that inhabit terrestrial, freshwater, and oceanic habitats. Within the phylum, the Rhizophydiales contains several monotypic families theorized to hold a diverse assemblage of fungi yet to be discovered and properly described. Based on morphology alone, many species in this order are difficult or impossible to identify. In this study, we isolated three chytrids from northern Thailand. Phylogenetic analyses placed the isolates in three monotypic genera within Rhizophydiales. Intrageneric genetic distances in the internal transcribed spacer (ITS) ranged between 1.5 and 8.5%. Angulomyces solicola sp. nov. is characterized by larger sporangia, spores, and fewer discharge papilla than A.argentinensis; Gorgonomyces thailandicus sp. nov. has larger zoospores and fewer discharge papillae in culture compared to G. haynaldii; Terramyces chiangraiensis sp. nov. produces larger sporangia than T. subangulosum. We delimited species of Angulomyces, Gorgonomyces and Terramyces using a tripartite approach that employed phylogeny, ITS genetic distances and Poisson tree processes (PTP). Results of these approaches suggest more than one species in each genus. This study contributes to the knowledge of chytrids, an understudied group in Thailand and worldwide.

Comparative genomic analysis illustrates evolutionary dynamics of multisubunit tethering complexes across green algal diversity.

The chlorophyte algae are a dominant group of photosynthetic eukaryotes. Although many are photoautotrophs, there are also mixotrophs, heterotrophs, and even parasites. The physical characteristics of green algae are also highly diverse, varying greatly in size, shape, and habitat. Given this morphological and trophic diversity, we postulated that diversity may also exist in the protein components controlling intracellular movement of material by vesicular transport. One such set is the multisubunit tethering complexes (MTCs)-components regulating cargo delivery. As they span endomembrane organelles and are well-conserved across eukaryotes, MTCs should be a good proxy for assessing the evolutionary dynamics across the diversity of Chlorophyta. Our results reveal that while green algae carry a generally conserved and unduplicated complement of MTCs, some intriguing variation exists. Notably, we identified incomplete sets of TRAPPII, exocyst, and HOPS/CORVET components in all Mamiellophyceae, and what is more, not a single subunit of Dsl1 was found in Cymbomonas tetramitiformis. As the absence of Dsl1 has been correlated with having unusual peroxisomes, we searched for peroxisome biogenesis machinery, finding very few components in Cymbomonas, suggestive of peroxisome degeneration. Overall, we demonstrate conservation of MTCs across green algae, but with notable taxon-specific losses suggestive of unusual endomembrane systems.

Circulation and colonisation of Blastocystis subtypes in schoolchildren of various ethnicities in rural northern Thailand.

Blastocystis is a protist of controversial pathogenicity inhabiting the gut of humans and other animals. Despite a century of intense study, understanding of the epidemiology of Blastocystis remains fragmentary. Here, we aimed to explore its prevalence, stability of colonisation and association with various factors in a rural elementary school in northern Thailand. One hundred and forty faecal samples were collected from 104 children at two time points (tp) 105 days apart. For tp2, samples were also obtained from 15 animals residing on campus and seven water locations. Prevalence in children was 67% at tp1 and 89% at tp2, 63% in chickens, 86% in pigs, and 57% in water. Ten STs were identified, two of which were shared between humans and animals, one between animals and water, and three between humans and water. Eighteen children (out of 36) carried the same ST over both time points, indicating stable colonisation. Presence of Blastocystis (or ST) was not associated with body mass index, ethnicity, birth delivery mode, or milk source as an infant. This study advances understanding of Blastocystis prevalence in an understudied age group, the role of the environment in transmission, and the ability of specific STs to stably colonise children.

Blastocystis subtypes in raw vegetables from street markets in northern Thailand.

Blastocystis is a stramenopile protist of controversial pathogenicity. The organism colonizes a broad range of vertebrate and invertebrate hosts. Blastocystis has also been found in the environment both in water and soil. Several routes of transmission have been proposed including human to human, animal to human, and via contaminated food and water. In recent years, the presence of Blastocystis in vegetables has started to be explored. However, most studies have focused on microscopic detection. Moreover, works of this type from Asia are barely available. Hence, the aim of this preliminary study was to examine the occurrence of Blastocystis in raw vegetables sold in markets in northern Thailand. Fresh produce (n = 20) commonly used in Thai cuisine (Lanna) was purchased from two street markets and screened for Blastocystis using qPCR. Blastocystis was detected in 45% of the samples with the dominant subtype being ST3. Produce growing underground, such as galangal, carrot, and beetroot, were positive for the organism suggesting soil or inadequately composted manure as the source of contamination. To our knowledge, our study is the first to perform subtyping of Blastocystis in vegetables. Our results hint toward fresh produce being a, as yet, not widely explored, transmission route of Blastocystis in the studied community. Looking forward, large-scale investigations on the prevalence of this and other organisms under the One Health umbrella should be undertaken.

Resistance of Blastocystis to chlorine and hydrogen peroxide.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1-ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including fecal-oral (e.g., zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water, and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1-ST9 to various chlorine and hydrogen peroxide concentrations for 24 h, and performed recovery assays along with re-exposure. Following the treatment with both compounds, all subtypes showed increased resistance, and viability could be visualized at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide. As such, this pilot study can be the platform for developing guidelines for water treatment processes.

Distinct gut microbiota profiles of Asian honey bee (Apis cerana) foragers.

Bee gut microbial communities have been studied extensively and linked to honey bee biology in terms of stages of bee development and behavior. Associations of bee gut microbiota in health and disease have also been explored. A large number of studies have centered on the gut microbiome of Apis mellifera, with similar investigations lagging far behind in Asian honey bee foragers. In this study, we characterized and compared the gut bacterial profiles of foragers and nurse bees of A. cerana and A. mellifera. Analysis of 16S rRNA partial gene sequences revealed significant differences in gut bacterial communities between the two honey bee species. Despite sharing dominant taxa, Bacteroides was more abundant in A. cerana, while Proteobacteria was higher in A. mellifera. Specific gut members are distinctly associated with hosts performing different tasks (i.e. nurse bees versus foragers). An exclusive abundance of Apibacter detected in Asian honey bee seemed to be a microbial signature of A. cerana foragers. Overall, our study highlights that variations in gut microbiota could be linked to task-specific (nurse bees and foragers) bacterial species associated with honey bees. Future investigations on the symbiotic relationship between host and the resident microbiota would be beneficial for improving honey bee health.

Early-diverging fungal phyla: taxonomy, species concept, ecology, distribution, anthropogenic impact, and novel phylogenetic proposals.

The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota, Aphelidiomycota, Rozellomycota, Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga (Chytridiomycota: Nephridiophagales), a new type species, Nephridiophaga blattellae, is proposed.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

Entamoeba chiangraiensis n. sp. (Amoebozoa: Entamoebidae) isolated from the gut of Asian swamp eel (Monopterus albus) in northern Thailand.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 µm in length and 9-13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.

EukRef-Ciliophora: a manually curated, phylogeny-based database of small subunit rRNA gene sequences of ciliates.

High-throughput sequencing (HTS) surveys, among the most common approaches currently used in environmental microbiology, require reliable reference databases to be correctly interpreted. The EukRef Initiative (eukref.org) is a community effort to manually screen available small subunit (SSU) rRNA gene sequences and produce a public, high-quality and informative framework of phylogeny-based taxonomic annotations. In the context of EukRef, we present a database for the monophyletic phylum Ciliophora, one of the most complex, diverse and ubiquitous protist groups. We retrieved more than 11 500 sequences of ciliates present in GenBank (28% from identified isolates and 72% from environmental surveys). Our approach included the inference of phylogenetic trees for every ciliate lineage and produced the largest SSU rRNA tree of the phylum Ciliophora to date. We flagged approximately 750 chimeric or low-quality sequences, improved the classification of 70% of GenBank entries and enriched environmental and literature metadata by 30%. The performance of EukRef-Ciliophora is superior to the current SILVA database in classifying HTS reads from a global marine survey. Comprehensive outputs are publicly available to make the new tool a useful guide for non-specialists and a quick reference for experts.

Genetic diversity of Blastocystis in non-primate animals.

Blastocystis is an anaerobic protist, commonly inhabiting the intestinal tract of both humans and other animals. Blastocystis is extremely diverse comprising 17 genetically distinct subtypes in mammals and birds. Pathogenicity of this enteric microbe is currently disputed and knowledge regarding its distribution, diversity and zoonotic potential is fragmentary. Most research has focused on Blastocystis from primates, while sampling from other animals remains limited. Herein, we investigated the prevalence and distribution of Blastocystis in animals held within a conservation park in South East England. A total of 118 samples were collected from 27 vertebrate species. The barcoding region of the small-subunit ribosomal RNA was used for molecular identification and subtyping. Forty one per cent of the species were sequence positive for Blastocystis indicating a high prevalence and wide distribution among the animals in the park. Six subtypes were identified, one of which is potentially novel. Moreover, the majority of animals were asymptomatic carriers, suggesting that Blastocystis is not pathogenic in animals. This study provides a thorough investigation of Blastocystis prevalence within a wildlife park in the UK and can be used as a platform for further investigations on the distribution of other eukaryotic gut microbes.

Antibacterial and Antioxidant Metabolites of Diaporthe spp. Isolated from Flowers of Melodorum fruticosum.

Fifty-two strains of endophytic fungi were isolated from flowers of the medicinal plant Melodorum fruticosum. Seven genera were identified including Alternaria, Aspergillus, Colletotrichum, Diaporthe, Fusarium, Greeneria and Nigrospora. All strains were cultured for 30 days and further macerated in ethyl acetate solvent for 3 days. The obtained fungal extracts were examined for antibacterial activity using agar disc diffusion against nine pathogenic bacteria: Staphylococcus aureus, Bacillus subtilis, B. cereus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, Vibrio cholerae and V. parahaemolyticus. Forty-three fungal extracts exhibited antibacterial activity against at least one tested pathogen. The antioxidant properties of all extracts were also investigated by DPPH scavenging assay. Sixteen extracts displayed high antioxidant capacity (IC50 ranging from 10 to 50 µg/mL) when compared to the gallic acid and trolox standards (IC50 of 12.46 and 2.55 µg/mL, respectively). The crude extracts of Diaporthe sp. MFLUCC16-0682 and Diaporthe sp. MFLUCC16-0693 exhibited notable antibacterial and antioxidant activities. Analysis of chemical composition using gas chromatography-mass spectrometry suggested that the observed antibacterial activity of the two Diaporthe spp. was possibly due to the presence of abienol, 4-methoxy stilbene, phenethyl cinnamate and 2Z,6Z-farnesal, while their potential antioxidant activity could be attributed to phenolic compounds, such as benzene acetaldehyde, benzyl benzoate, salicylaldehyde, benzoin and benzyl cinnamate. The results suggest that the genus Diaporthe is a potential source of metabolites that can be used in a variety of applications.

Phylogenomics solves a long-standing evolutionary puzzle in the ciliate world: The subclass Peritrichia is monophyletic.

The phylum Ciliophora is one of the most broadly studied protozoan lineages. The era of molecular investigation has brought forth a major ongoing debate: is the subclass Peritrichia Stein, 1859 monophyletic? Numerous analyses mostly using the small subunit (SSU) rRNA gene have failed to recover the Mobilida and Sessilida, the two peritrich orders, as sister clades. Here we have sequenced five peritrich species - three sessilids and two mobilids. We constructed a supermatrix of 158 genes and 44,696 characters for 24 ciliate species, and as outgroup taxa, nine species from the Apicomplexa and four from the Dinophyceae. Our analyses using both maximum likelihood and Bayesian methods recover a monophyletic class Oligohymenophorea and two robust clades within it. The first clade is a monophyletic Peritrichia with the orders Sessilida and Mobilida maximally supported as sister clades. The second oligohymenophorean clade includes species of the subclasses Scuticociliatia and Hymenostomatia, which are sister clades. Our analyses resolve a long-standing debate in ciliate molecular phylogenetics and provide support for the classical view that the morphological features of the two peritrich orders Mobilida and Sessilida arose by descent from the same common ancestor and are not the result of convergence.

Fungal Endophytes: an Alternative Source for Production of Volatile Compounds from Agarwood Oil of Aquilaria subintegra.

Fungal endophytes are microorganisms that are well-known for producing a diverse array of secondary metabolites. Recent studies have uncovered the bioprospecting potential of several plant endophytic fungi. Here, we demonstrate the presence of highly bioactive fungal endophytic species in Aquilaria subintegra, a fragrant wood plant collected from Thailand. Thirty-three fungal endophytic strains were isolated and further identified to genus level based on morphological characteristics. These genera included Colletotrichum, Pestalotiopsis, Fusarium, Russula, Arthrinium, Diaporthe and Cladosporium. All strains were cultured on potato dextrose broth for 30 days prior to partitioning with ethyl acetate. The volatile compounds of all extracts were investigated by gas chromatography-mass spectrometry (GC-MS). Four strains-Arthrinium sp. MFLUCC16-0042, Colletotrichum sp. MFLUCC16-0047, Colletotrichum sp. MFLUCC16-0048 and Diaporthe sp. MFLUCC16-0051-produced a broad spectrum of volatile compounds, including ß-agarofuran, α-agarofuran, δ-eudesmol, oxo-agarospirol, and ß-dihydro agarofuran. These compounds are especially important, because they greatly resemble those originating from the host-produced agarwood oil. Our findings demonstrate the potential of endophytic fungi to produce bioactive compounds with applications in perfumery and cosmetic industries. Antioxidant activity of all extracts was also evaluated by using 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays. The ethyl acetate extract of Diaporthe sp. MFLUCC16-0051 demonstrated superior antioxidant capacity, which was comparable to that of the gallic acid standard. Our results indicate that the MFLUCC16-0051 strain is a resource of natural antioxidant with potential medicinal applications.

Antibacterial and Antioxidant Constituents of Extracts of Endophytic Fungi Isolated from Ocimum basilicum var. thyrsiflora Leaves.

Fourteen fungal endophytes were isolated from the Ocimum basilicum var. thyrsiflora leaves collected from Northern Thailand. Eight genera were identified including Aspergillus, Ascochyta, Nigrospora, Blastomyces, Colletotrichum, Exidia, Clitopilus, and Nomuraea. The antibacterial activity of crude extracts from all endophytic fungi was tested against nine human bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. All crude extracts showed some degree of antibacterial activity, but the crude extract from Nigrospora MFLUCC16-0605 exhibited broad spectrum activity with MIC values ranging from 7.81 to 250 µg/mL. The antioxidant activity of all crude extracts was also investigated by DPPH radical scavenging assay. Crude extract from MFLUCC16-0605 had high antioxidant activity (IC50 value of 15.36 µg/mL) comparable to the trolox and gallic acid standards showing IC50 values of 2.56 and 12.89 µg/mL, respectively. The chemical composition of the crude extract from MFLUCC16-0605 was determined using GC-MS. Sixty-two compounds were identified representing 92.09% of crude extract with six major components including 5E,9E-farnesyl acetone, columellarin, totarene, laurenan-2-one, and 8S,13-cedranediol. PCR amplification and sequencing of the barcoding region identified MFLUCC16-0605 as belonging to Nigrospora genus. The notable activities of MFLUCC16-0605 indicate that the endophyte is a potent natural resource and its use as an antibacterial/antioxidant agent should be further explored.

Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists.

Ciliates comprise a highly diverse protozoan lineage inhabiting all biotopes and playing crucial roles in regulating microbial food webs. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. Here, we use the species-rich taxon Frontonia and employ both nuclear and mitochondrial loci. We attempt to assess the level of genetic diversity and evaluate the potential of each marker in delineating species of Frontonia. Morphological features and ecological characteristics are also integrated into genetic results, in an attempt to resolve conflicts of species identification based on morphological and molecular methods. Our studies reveal: (1) the mitochondrial cox1 gene, nuclear ITS1 and ITS2 as well as the hypervariable D2 region of LSU rDNA are promising candidates for species delineation; (2) the cox1 gene provides the best resolution for analyses below the species level; (3) the V2 and V4 hypervariable regions of SSU rDNA, and D1 of LSU rDNA as well as the 5.8S rDNA gene do not show distinct barcoding gap due to overlap between intra- and inter-specific genetic divergences; (4) morphological character-based analysis shows promise for delimitation of Frontonia species; and (5) all gene markers and character-based analyses demonstrate that the genus Frontonia consists of three groups and monophyly of the genus Frontonia is questionable.

First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group.

Archamoebae is an understudied group of anaerobic free-living or endobiotic protists that constitutes the major anaerobic lineage of the supergroup Amoebozoa. Hitherto, the phylogeny of Archamoebae was based solely on SSU rRNA and actin genes, which did not resolve relationships among the main lineages of the group. Because of this uncertainty, several different scenarios had been proposed for the phylogeny of the Archamoebae. In this study, we present the first multigene phylogenetic analysis that includes members of Pelomyxidae, and Rhizomastixidae. The analysis clearly shows that Mastigamoebidae, Pelomyxidae and Rhizomastixidae form a clade of mostly free-living, amoeboid flagellates, here called Pelobiontida. The predominantly endobiotic and aflagellated Entamoebidae represents a separate, deep-branching lineage, Entamoebida. Therefore, two unique evolutionary events, horizontal transfer of the nitrogen fixation system from bacteria and transfer of the sulfate activation pathway to mitochondrial derivatives, predate the radiation of recent lineages of Archamoebae. The endobiotic lifestyle has arisen at least three times independently during the evolution of the group. We also present new ultrastructural data that clarifies the primary divergence among the family Mastigamoebidae which had previously been inferred from phylogenetic analyses based on SSU rDNA.