Exploring the impact of urban pollution on ciliate diversity along the Sapucaí River (Minas Gerais, Brazil) via DNA metabarcoding.

BACKGROUND: Protists are diverse single-celled eukaryotes found in various habitats. They exhibit a wide range of forms and functions, representing a significant portion of the eukaryotic tree of life, which also includes animals, plants, and fungi. Due to their high sensitivity to environmental changes, these organisms are widely used as biological indicators of organic pollution. METHODS AND RESULTS: We investigated the molecular diversity of ciliate protists at seven strategic points along the Sapucaí River (Itajubá, Minas Gerais State, Brazil), to assess the impact of urban pollution on the richness, abundance, and diversity indexes of these communities. For each sampling point, values of physicochemical parameters were also recorded. DNA sequences were obtained by high-throughput sequencing (HTS) and analyzed using the V4 18S-rRNA molecular marker, employing the DNA metabarcoding method. We recorded 125 ciliate taxonomic units (OTUs), with nearly 80% corresponding to the classes Spirotrichea, Oligohymenophorea, and Litostomatea. At the genus level, 54 OTUs (43.2%) were identified, spanning 28 genera. CONCLUSIONS: The composition of ciliates varied significantly along the river's course, from upstream to downstream of Itajubá city. Samples collected from the urban area displayed the lowest richness and diversity, corroborating the influence of the pollution gradient on these communities. The physicochemical parameters showed little variation among the samples and were not linked to the observed changes in ciliate communities, revealing that these organisms are strongly affected by environmental changes and respond more sensitively to these disturbances than physicochemical parameters, emphasizing their potential as bioindicators.

The burden of intestinal parasitic infections in Antioquia, Colombia: Impact in childhood growth development and nutritional status.

Gastrointestinal protist (GP) and soil-transmitted helminth (STH) infections cause significant morbidity among children in poor-resource settings of tropical and sub-tropical countries including Colombia. Few prospective transversal studies investigating how GP and STH infections affect growth development and nutritional status during childhood have been conducted in this country, none of them in the Antioquia Department. This microscopy-based study estimated the prevalence of GP and helminth (including STH) infections in faecal samples from schoolchildren (n = 384) collected during April-May 2019 in three municipalities of the Antioquia Department. Demographic, epidemiological, and household data were elicited through face-to-face interviews. Parasite detection was carried out by direct microscopic examination of both fresh smears and concentrated faecal material. Children (aged 6-15 years) also had their haemoglobin (Hb) levels, height and weight data collected, and BMI estimated. Data were analysed using bivariate and multivariate logistic regression analysis. Overall, 60.7 % (233/384) of schoolchildren were infected by at least one intestinal parasitic species. Among GPs, Blastocystis sp. was the most common species found (47.7 %, 95 % CI: 42.6-52.8), followed by G. duodenalis (15.9 %, 95 % CI: 12.4-19.9). Cryptosporidium spp. and Cyclospora cayetanensis were sporadically identified (0.3 %, 95 % CI: 0.1-1.4 each). Among helminths, the most prevalent species found were Trichuris trichiura (6.0 %, 95 % CI: 3.8-8.9) and Enterobius vermicularis (1.0 %, 95 % CI: 0.3-2.6). Hookworms, Ascaris lumbricoides, and Strongyloides stercoralis were found at prevalence rates <1 %. Underweight, overweigh, or obese schoolchildren had 1.2 times greater chance of being infected with intestinal parasites than their counterparts with a healthy weight (P-value: 0.015). Variables significantly associated with an increased likelihood of being infected by intestinal parasites include living in a household with unfinished flouring, not wearing shoes, being in close proximity to rodents, and having improper waste disposal. Relatively simple interventional measures directed towards the improvement of household conditions, access to sanitary toilets, and promoting shoe wearing can significantly reduce childhood infections by GP and helminths in the Antioquia Department.

Monitoring potentially pathogenic protists in sewage sludge using Metataxonomics.

Intestinal parasites continue to pose a significant threat to human health worldwide, particularly among children. Contaminated water and soil serve as major transmission vehicles for these parasites and intestinal protists are among the most prevalent parasites in both developed and developing nations. Traditionally, parasites have been studied using human or animal fecal samples, while studying them in environmental samples has been challenging due to technical limitations. However, advancements in Next-Generation Sequencing (NGS) and bioinformatic approaches now enable the detection of parasite DNA in environmental samples. In this study, we applied a metataxonomic and phylogenetic strategy to detect and classify DNA of protists present in sewage sludge from two major cities in Colombia: Medellin and Cali. We successfully detected several human pathogenic parasites including Giardia intestinalis, Entamoeba histolytica, and Blastocystis sp., among other protists, in all sludge samples examined. We also investigated the entry and exit of parasite DNA from the San Fernando wastewater treatment plant (WWTP). We observed a higher number of parasite DNA sequences in the plant's influent wastewater, but we also detected the discharge of DNA from pathogenic parasites in both effluent waters and biosolids.

Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens-Factors Linked to Virulence and Pathogenicity.

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens' virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Checklist, diversity and biogeography of ciliates (Ciliophora) from Chile.

There is a significant gap in research and knowledge on the diversity and distribution of Chilean ciliates. To tackle these issues, we used cultures and protargol preparations to describe the ciliates present in poorly explored areas. At these sites, we identified 45 ciliate morphospecies, 35 of which represent unprecedent records to Chile. Then, we brought together our records with literature data to construct a species checklist. This checklist summarises 132 years of data and describes the identity, habitat and distribution of 207 species, including 15 species potentially endemic to Chile. This checklist is far from complete: a diversity estimate suggests that at least two-thirds of the ciliate species occurring in Chile have yet to be described. The checklist is dominated by freshwater taxa because ciliates from marine, brackish and terrestrial environments have rarely been investigated in Chile. Finally, after controlling for sampling artefacts, we found that ciliates exhibit a bell-shaped latitudinal diversity gradient in Chile. This peculiar biogeographical pattern is common in Chile. Plants, animals and testate amoebae also exhibit a bell-shaped latitudinal diversity gradient in Chile. This finding suggests that the historical contingencies that drove the biogeography of the Chilean biota also shaped ciliate biogeography.

Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d'Ivoire, Sierra Leone, and Peru.

Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d'Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys.

Microbial eukaryote assemblages and potential novel diversity in four tropical East African Great Lakes.

East African Great Lakes are old and unique natural resources heavily utilized by their bordering countries. In those lakes, ecosystem functioning is dominated by pelagic processes, where microorganisms are key components; however, protistan diversity is barely known. We investigated the community composition of small eukaryotes (<10 µm) in surface waters of four African Lakes (Kivu, Edward, Albert and Victoria) by sequencing the 18S rRNA gene. Moreover, in the meromictic Lake Kivu, two stations were vertically studied. We found high protistan diversity distributed in 779 operational taxonomic units (OTUs), spanning in 11 high-rank lineages, being Alveolata (31%), Opisthokonta (20%) and Stramenopiles (17%) the most represented supergroups. Surface protistan assemblages were associated with conductivity and productivity gradients, whereas depth had a strong effect on protistan community in Kivu, with higher contribution of heterotrophic organisms. Approximately 40% of OTUs had low similarity (<90%) with reported sequences in public databases; these were mostly coming from deep anoxic waters of Kivu, suggesting a high extent of novel diversity. We also detected several taxa so far considered exclusive of marine ecosystems. Our results unveiled a complex and largely undescribed protistan community, in which several lineages have adapted to different niches after crossing the salinity boundary.

Exposure to Parasitic Protists and Helminths Changes the Intestinal Community Structure of Bacterial Communities in a Cohort of Mother-Child Binomials from a Semirural Setting in Mexico.

An estimated 3.5 billion people are colonized by intestinal parasites worldwide. Intestinal parasitic eukaryotes interact not only with the host but also with the intestinal microbiota. In this work, we studied the relationship between the presence of multiple enteric parasites and the community structures of gut bacteria and eukaryotes in an asymptomatic mother-child cohort from a semirural community in Mexico. Fecal samples were collected from 46 mothers and their respective children, with ages ranging from 2 to 20 months. Mothers and infants were found to be multiparasitized by Blastocystis hominis, Entamoeba dispar, Endolimax nana, Chilomastix mesnili, Iodamoeba butshlii, Entamoeba coli, Hymenolepis nana, and Ascaris lumbricoides. Sequencing of bacterial 16S rRNA and eukaryotic 18S rRNA genes showed a significant effect of parasite exposure on bacterial beta-diversity, which explained between 5.2% and 15.0% of the variation of the bacterial community structure in the cohort. Additionally, exposure to parasites was associated with significant changes in the relative abundances of multiple bacterial taxa, characterized by an increase in Clostridiales and decreases in Actinobacteria and Bacteroidales. Parasite exposure was not associated with changes in intestinal eukaryote relative abundances. However, we found several significant positive correlations between intestinal bacteria and eukaryotes, including Oscillospira with Entamoeba coli and Prevotella stercorea with Entamoeba hartmanni, as well as the co-occurrence of the fungus Candida with Bacteroides and Actinomyces, Bifidobacterium, and Prevotella copri and the fungus Pichia with Oscillospira. The parasitic exposure-associated changes in the bacterial community structure suggest effects on microbial metabolic routes, host nutrient uptake abilities, and intestinal immunity regulation in host-parasite interactions. IMPORTANCE The impact of intestinal eukaryotes on the prokaryotic microbiome composition of asymptomatic carriers has not been extensively explored, especially in infants and mothers with multiple parasitic infections. In this work, we studied the relationship between protist and helminth parasite colonization and the intestinal microbiota structure in an asymptomatic population of mother-child binomials from a semirural community in Mexico. We found that the presence of parasitic eukaryotes correlated with changes in the bacterial gut community structure in the intestinal microbiota in an age-dependent way. Parasitic infection was associated with an increase in the relative abundance of the class Clostridia and decreases of Actinobacteria and Bacteroidia. Parasitic infection was not associated with changes in the eukaryote community structure. However, we observed strong positive correlations between bacterial and other eukaryote taxa, identifying novel relationships between prokaryotes and fungi reflecting interkingdom interactions within the human intestine.

The Secret Life of Giant Viruses in the California Current.

In the last few decades, the virology field has experienced a revolution in knowledge related to viral richness, diversity, and distribution in the oceans. Metagenomics associated with virus isolation methods have contributed to outstanding discoveries in marine virology. Giant viruses and other protist-infecting viruses belonging to the phylum Nucleocytoviricota have raised fundamental questions such as "what are the limits of virion size?", "what is a viral genome able to encode?", and "what is the ecological role of giant viruses in the ocean?" In a recent paper published in mSystems by Ha, Moniruzzaman, and Aylward (mSystems 6:e00293-21, 2021, https://doi.org/10.1128/mSystems.00293-21), the authors demonstrated by metatranscriptomic-related analyses that giant viruses are active members of the California Current microbial community, replicating, modulating, and exchanging genes with their protist hosts. This work not only explores the dynamics of giant virus gene expression in a natural environment but also reveals that nucleocytoviricotal abundance and ecological importance are underestimated.

Soil biota shift with land use change from pristine rainforest and Savannah (Cerrado) to agriculture in southern Amazonia.

Southern Amazonia is currently experiencing extensive land use change from forests to agriculture caused by increased local and global demand for agricultural products. However, little is known about the impacts of deforestation and land use change on soil biota. We investigated two regions in southern Amazonia (rainforest and Savannah/Cerrado biomes), analysing soil biota community turnover based on 16S (Archaea and Bacteria) and 18S rRNA genes (Eukaryotes, including Fungi, Protists and Animalia) and correlating them with soil chemistry and land use intensity. We found that soil biota community structure is driven by land use change in both Cerrado and rainforest. Crop fields approximatively doubled the richness of soil Archaea, Bacteria and Protists. We propose that crop systems not only increase soil pH and fertility, but also create continued disturbance (crop seasons) that stimulates soil diversity, as predicted by the dynamic equilibrium model (DEM) and the intermediate disturbance hypothesis (IDH). Even though agricultural fields had higher soil biota richness, some taxa were suppressed by agriculture (6/31 operational taxonomic units of Archaea, 245/1790 of Bacteria, 12/74 of Animalia, 20/144 of Fungi and 25/310 of Protists). Consequently, land use change in this region should proceed with caution. In the southern Amazonia region of Brazil, current laws require farmers to keep 20%-80% pristine vegetation areas on their property. Our data support the relevance of this law: since there are unique soil taxa under native vegetation, keeping these pristine areas adjacent to the agricultural fields should maximize soil biodiversity protection in these regions.

Identification of Metabolically Quiescent Leishmania mexicana Parasites in Peripheral and Cured Dermal Granulomas Using Stable Isotope Tracing Imaging Mass Spectrometry.

Leishmania are sandfly-transmitted protists that induce granulomatous lesions in their mammalian host. Although infected host cells in these tissues can exist in different activation states, the extent to which intracellular parasites stages also exist in different growth or physiological states remains poorly defined. Here, we have mapped the spatial distribution of metabolically quiescent and active subpopulations of Leishmania mexicana in dermal granulomas in susceptible BALB/c mice, using in vivo heavy water labeling and ultra high-resolution imaging mass spectrometry. Quantitation of the rate of turnover of parasite and host-specific lipids at high spatial resolution, suggested that the granuloma core comprised mixed populations of metabolically active and quiescent parasites. Unexpectedly, a significant population of metabolically quiescent parasites was also identified in the surrounding collagen-rich, dermal mesothelium. Mesothelium-like tissues harboring quiescent parasites progressively replaced macrophage-rich granuloma tissues following treatment with the first-line drug, miltefosine. In contrast to the granulomatous tissue, neither the mesothelium nor newly deposited tissue sequestered miltefosine. These studies suggest that the presence of quiescent parasites in acute granulomatous tissues, together with the lack of miltefosine accumulation in cured lesion tissue, may contribute to drug failure and nonsterile cure.IMPORTANCE Many microbial pathogens switch between different growth and physiological states in vivo in order to adapt to local nutrient levels and host microbicidal responses. Heterogeneity in microbial growth and metabolism may also contribute to nongenetic mechanisms of drug resistance and drug failure. In this study, we have developed a new approach for measuring spatial heterogeneity in microbial metabolism in vivo using a combination of heavy water (2H2O) labeling and imaging mass spectrometry. Using this approach, we show that lesions contain a patchwork of metabolically distinct parasite populations, while the underlying dermal tissues contain a large population of metabolically quiescent parasites. Quiescent parasites also dominate drug-depleted tissues in healed animals, providing an explanation for failure of some first line drugs to completely eradicate parasites. This approach is broadly applicable to study the metabolic and growth dynamics in other host-pathogen interactions.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses.

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7-22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

Ciliate Diversity From Aquatic Environments in the Brazilian Atlantic Forest as Revealed by High-Throughput DNA Sequencing.

Rainforest aquatic ecosystems include complex habitats with scarce information on their unicellular eukaryote diversity and community structure. We have investigated the diversity of ciliates in freshwater and brackish environments along the Brazilian Atlantic Forest, based on the hypervariable V4 region of the 18S-rDNA obtained by high-throughput DNA sequencing. Our analyses detected 409 ciliate taxonomic units (OTUs), mostly attributed to the classes Oligohymenophorea and Spirotrichea. A total of 11 classes, 12 subclasses, 112 genera, and 144 species were reported. We found the following: (a) the ciliate communities are more diverse in freshwater- than in Atlantic Forest-associated brackish environments; (b) the ciliate communities are composed by a small amount of highly abundant OTUs, but a high number of low-abundant or rare OTUs; (c) nearly one-third of the ciliate OTUs share less than 97% sequence identity to reference sequences and (d) phylogenetic inference supports the hypothesis that the V4 region of the Ciliophora 18S-rDNA is a suitable marker for accurate evolutionary inferences at class level. Our results showed that a considerable fraction of the HTS-detected diversity of ciliates from Brazilian Atlantic Forest is not represented in the currently available molecular databases.

Heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao, the Netherlands Antilles.

Recent progress in understanding the early evolution of eukaryotes was tied to morphological identification of flagellates and heliozoans from natural samples, isolation of their culture and genomic and ultrastructural investigations. These protists are the smallest and least studied microbial eukaryotes but play an important role in the functioning of microbial food webs. Using light and electron microscopy, we have studied the diversity of heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao (The Netherlands Antilles), and provide micrographs and morphological descriptions of observed species. Among 86 flagellates and 3 centrohelids encountered in this survey, five heterotrophic flagellates and one сentrohelid heliozoan were not identified even to the genus. Some flagellate protists have a unique morphology, and may represent undescribed lineages of eukaryotes of high taxonomic rank. The vast majority (89%) of identified flagellates is characterized by wide geographical distribution and have been reported previously from all hemispheres and various climatic regions. More than half of the species were previously observed not only from marine, but also from freshwater habitats. The parameters of the species accumulation curve indicate that our species list obtained for the Curacao study sites is far from complete, and each new sample should yield new species.

Metabarcoding advances for ecology and biogeography of Neotropical protists: what do we know, where do we go? / Ecologia e biogeografia de protistas Neotropicais usando metabarcoding: O que sabemos? Para onde iremos?

Abstract: The Neotropics is one of the most diverse regions of the globe in terms of plants and animal species. Regarding the microbial world, however, little is known about the diversity and biogeography patterns of microorganisms in the Neotropics. The biogeography of several microbial taxonomic groups is still missing and/or incomplete, such as the protists. Despite the hard taxonomic identification of protists, the advance of molecular techniques (e.g., metabarcoding) have allowed to better explore the distribution of several protistan groups. Our goal here was to summarize the available information of Neotropical protists, focusing on metabarcoding studies, to explore what these data evidence on their ecology and biogeography. For this, we reviewed the findings from all articles that focused on or included the terrestrial protists using a metabarcoding approach and identified the gaps and future perspectives in this research field. We found that Neotropical protistan diversity patterns seem to be, at least in part, congruent with that of macro-organisms and, different than plants and bacteria, just weakly explained by environmental variables. We argue that studies with standardized protocols including different ecoregions are necessary, such as temperate forests, grasslands, and savannas from Southern of South America and Northern Atlantic Forest, to fully characterize the ecology and biogeography on Neotropical protists. Furthermore, dismembering evolutionary lineages and functional guilds of protists are important to better understand the relationship between diversity, dispersal abilities, and functionality of particular taxa of protists in their habitats.

Resumo: A região Neotropical é uma das mais diversas regiões do globo em termos de espécies vegetais e animais. Em relação ao mundo microbiano, entretanto, pouco se sabe sobre a diversidade e os padrões biogeográficos dos microrganismos no Neotrópico. Nesse contexto, a biogeografia de diversos grupos taxonômicos microbianos ainda é escasso e/ou incompleto como os protistas, devido à difícil identificação taxonômica de tais microscópicos organismos. Neste contexto, o avanço dos dados moleculares de amostras ambientais (por exemplo, metabarcoding) permitiu explorar a distribuição de vários grupos de protistas. Nosso objetivo aqui foi resumir as informações disponíveis dos protistas neotropicais, com foco em metabarcoding, para explorar o que esses dados evidenciam sobre sua ecologia e biogeografia. Para isso, revisamos os resultados de todos os artigos que enfocavam ou incluíam os protistas terrestres usando uma abordagem de metabarcoding e identificamos as lacunas e as perspectivas futuras neste campo de pesquisa. Os padrões de diversidade dos protistas Neotropicais parecem ser, pelo menos em parte, congruentes com os de macroorganismos e, diferentes das plantas e bactérias, sendo pouco explicados por variáveis ambientais. Estudos com protocolos padronizados incluindo diferentes Ecorregiões são necessários, como em florestas temperadas, campos nativos e savanas no sul da América do Sul e no norte da Mata Atlântica, para melhor caracterizar a ecologia e biogeografia de protistas Neotropicais. Além disso, é importante diferenciar linhagens evolutivas e guildas funcionais de protistas para entender melhor a relação entre diversidade, capacidade de dispersão e funcionalidade de determinados táxons de protistas em seus habitats.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Heterotrophic flagellates (Amorpha and Diaphoretiches) in phytotelmata bromeliad (Bromeliaceae) / Flagelados heterotróficos (Amorpha e Diaphoretiches) em fitotelmata de bromélia (Bromeliaceae)

Abstract Many plants may accumulate rainwater, forming phytotelmata, aquatic microhabitats inhabited by various organisms. The aim of this study was to conduct an inventory of heterotrophic flagellates associated with phytotelmata of the bromeliad Aechmea distichantha Lem., found in rocky cliffs on the Upper Paraná River. The bromeliads were removed manually from the rocky wall, the water was removed and cultures of organisms of each plant were mounted in Petri dishes. Sixteen species of heterotrophic flagellate were recorded, drawn and described, among them one species belonging to the Amorpha Domain and 15 species to the Diaphoretiches Domain. The groups with most species were Euglenida and Kinetoplastea. The low diversity of heterotrophic flagellates recorded in this study, compared to the plankton of lakes and reservoirs, is probably related to the fact that phytotelmata are habitats with extreme environmental conditions, thus selecting organisms tolerant to these environments.

Resumo Em sua superfície externa, muitas plantas podem acumular água da chuva formando fitotelmata, ou seja, micro-habitat aquáticos colonizados por diversos organismos, entre eles, os flagelados protistas. Fez-se um inventário de flagelados heterotróficos associados ao fitotelmata da bromélia Aechmea distichantha Lem., encontrada em paredões rochosos em um trecho da bacia do alto rio Paraná. As bromélias foram retiradas manualmente de paredão rochoso e, em laboratório, a água foi retirada dos tanques de cada planta, e foram montadas culturas dos organismos em placas de Petri. Os flagelados heterotróficos foram, então, observados in vivo sob microscópio óptico Olympus BX51, com sistema de contraste de interferência diferencial (DIC) acoplado. Dezesseis espécies flagelados heterotróficos foram registradas, desenhadas e descritas, sendo uma espécie pertencente ao Domínio Amorpha e 15 espécies ao Domínio Diaphoretiches. Os grupos mais especiosos foram Euglenida e Kinetoplastea. A diversidade relativamente reduzida de flagelados heterotróficos, quando comparada ao plâncton de lagos e reservatórios, pode estar relacionada ao fato de os fitotelmata serem habitats limitados, com condições ambientais extremas, selecionando, assim, organismos tolerantes a esses ambientes.

The highly diverse TATA box-binding proteins among protists: A review.

Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.