Microbial Eukaryotes Research Articles

Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves.

SummaryThe purpose of this study was to survey the eukaryotic microbiome of two karst caves in the Valley and Ridge physiographic region of the Appalachian Mountains. Caves are known to harbour eukaryotic microbes but their very low densities and small cell size make them difficult to collect and identify. Microeukaryotes were surveyed using two methodologies, filtering water and submerging glass microscope slides mounted in periphytometers in cave pools. The periphyton sampling yielded 13.5 times more unique amplicon sequence variants (ASVs) than filtered water. The most abundant protist supergroup was Alveolata with large proportions of the ASVs belonging to dinoflagellate, ciliate and apicomplexan clades. The next most abundant were Rhizarians followed by Stramenopiles (diatoms and chrysophytes) and Ameobozoans. Very few of the ASVs, 1.5%, matched curated protist sequences with greater than 99% identity and only 2.5% could be identified from surface plankton samples collected in the same region. The overall composition of the eukaryotic microbiome appears to be a combination of bacterial grazers and parasitic species that could possibly survive underground as well as cells, cysts and spores probably transported from the surface.

Autophagy as a Survival Strategy for Eukaryotic Microbes Living in the Phyllosphere.

Autophagy is an intracellular degradation process that is highly conserved among eukaryotes at the molecular level. The process was originally revealed in the budding yeast, but the physiological role of autophagy in yeast cells had remained unknown as autophagy-deficient yeast mutants did now show a clear growth phenotype in laboratory conditions. In this review, we introduce the role of autophagy in the methylotrophic yeast Candida boidinii grown on the leaf surface of Arabidopsis thaliana. Autophagy is shown to be required for proliferation in the phyllosphere, and selective autophagic pathways such as pexophagy and cytoplasm-to-vacuole targeting (Cvt) pathway are strictly regulated during both the daily cycle and the host plant life cycle. This review describes our current understanding of the role of autophagy as a survival strategy for phyllosphere fungi. Critical functions of autophagy for pathogen invasions are also discussed.

Antimicrobial Volatiles of the Insect Pathogen Metarhizium brunneum.

Fungal volatile organic compounds (VOCs) represent promising candidates for biopesticide fumigants to control crop pests and pathogens. Herein, VOCs produced using three strains of the entomopathogenic fungus Metarhizium brunneum were identified via GC-MS and screened for antimicrobial activity. The VOC profiles varied with fungal strain, development state (mycelium, spores) and culture conditions. Selected VOCs were screened against a range of rhizosphere and non-rhizosphere microbes, including three Gram-negative bacteria (Escherichia coli, Pantoea agglomerans, Pseudomonas aeruginosa), five Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, B. megaterium, B. thuringiensis), two yeasts (Candida albicans, Candida glabrata) and three plant pathogenic fungi (Pythium ultimum, Botrytis cinerea, Fusarium graminearum). Microbes differed in their sensitivity to the test compounds, with 1-octen-3-ol and isovaleric acid showing broad-spectrum antimicrobial activity. Yeasts and bacteria were inhibited by the same VOCs. Cryo-SEM showed that both yeasts and bacteria underwent some form of “autolysis”, where all components of the cell, including the cell wall, disintegrated with little evidence of their presence in the clear, inhibition zone. The oomycete (P. ultimum) and ascomycete fungi (F. graminearum, B. cinerea) were sensitive to a wider range of VOCs than the bacteria, suggesting that eukaryotic microbes are the main competitors to M. brunneum in the rhizosphere. The ability to alter the VOC profile in response to nutritional cues may assist M. brunneum to survive among the roots of a wide range of plant species. Our VOC studies provided new insights as to how M. brunneum may protect plants from pathogenic microbes and correspondingly promote healthy growth.

Global soil microbiomes: A new frontline of biome‐ecology research

AbstractAimOrganisms on our planet form spatially congruent and functionally distinct communities, which at large geographical scales are called “biomes”. Understanding their pattern and function is vital for sustainable use and protection of biodiversity. Current global terrestrial biome classifications are based primarily on climate characteristics and functional aspects of plant community assembly. These and other existing biome schemes do not take account of soil organisms, including highly diverse and functionally important microbial groups. We aimed to define large‐scale structure in the diversity of soil microbes (soil microbiomes), pinpoint the environmental drivers shaping it and identify resemblance and mismatch with existing terrestrial biome schemes.LocationGlobal.Time periodCurrent.Major taxa studiedSoil eukaryotes and prokaryotes.MethodsWe collected soil samples from natural environments world‐wide, incorporating most known terrestrial biomes. We used high‐throughput sequencing to characterize soil biotic communities and k‐means clustering to define soil microbiomes describing the diversity of microbial eukaryotic and prokaryotic groups. We used climatic data and soil variables measured in the field to identify the environmental variables shaping soil microbiome structure.ResultsWe recorded strong correlations among fungal, bacterial, archaeal, plant and animal communities, defined a system of global soil microbiomes (producing seven biome types for microbial eukaryotes and six biome types for prokaryotes) and showed that these are typically structured by pH alongside temperature. None of the soil microbiomes are directly paralleled by any current terrestrial biome scheme, with mismatch most substantial for prokaryotes and for microbial eukaryotes in cold climates; nor do they consistently distinguish grassland and forest ecosystems.Main conclusionsExisting terrestrial biome classifications represent a limited surrogate for the large‐scale diversity patterns of microbial soil organisms. We show that empirically defined soil microbiomes are attainable using metabarcoding and statistical clustering approaches and suggest that they can have wide application in theoretical and applied biodiversity research.

Microbiomes of Healthy and Bleached Corals During a 2016 Thermal Bleaching Event in the Andaman Sea of Thailand

As seawater temperature rises, repeated thermal bleaching events have negatively affected the reefs of the Andaman Sea for over decades. Studies on the coral-associated microbial diversity of prokaryotes and microbial eukaryotes (microbiome) in healthy and bleached corals are important to better understand the coral holobionts that involved augmented resistance to stresses, and this information remains limited in the Andaman Sea of Thailand. The present study thereby described the microbiomes of healthy (unbleached) and bleached colonies of four prevalent corals, Acropora humilis, Platygyra sp., Pocillopora damicornis, and Porites lutea, along with the surrounding seawater and sediments, that were collected during a 2016 thermal bleaching event, using 16S and 18S rRNA genes next-generation sequencing (NGS). Both prokaryotic and eukaryotic microbes showed isolated community profiles among sample types (corals, sediment, and seawater) [analysis of similarities (ANOSIM): p = 0.038 for prokaryotes, p < 0.001 for microbial eukaryotes] and among coral genera (ANOSIM: p < 0.001 for prokaryotes and microbial eukaryotes). In bleached state corals, we found differences in microbial compositions from the healthy state corals. Prevalent differences shared among bleached coral genera (shared in at least three coral genera) included a loss of reported coral-beneficial microbes, such as Pseudomonadales, Alteromonadales, and Symbiodinium; meanwhile an increase of putative coral-pathogenic Malassezia and Aspergillus. This difference could affect carbon and nitrogen availability for coral growth, reflective of a healthy or bleached state. Our findings in part supported previously microbial dysbiosis knowledge of thermal bleaching coral microbiomes around South East Asia marine geography, and together ongoing efforts are to support the understanding and management of microbial diversity to reduce the negative impacts to corals in massive thermal bleaching events.

Epigenetic influences of mobile genetic elements on ciliate genome architecture and evolution.

Mobile genetic elements (MGEs) are transient genetic material that can move either within a single organism's genome or between individuals or species. While historically considered "junk" DNA (i.e., deleterious or at best neutral), more recent studies reveal the potential adaptive advantages MGEs provide in lineages across the tree of life. Ciliates, a group of single-celled microbial eukaryotes characterized by nuclear dimorphism, exemplify how epigenetic influences from MGEs shape genome architecture and patterns of molecular evolution. Ciliate nuclear dimorphism may have evolved as a response to transposon invasion and ciliates have since co-opted transposons to carry out programmed DNA deletion. Another example of the effect of MGEs is in providing mechanisms for lateral gene transfer (LGT) from bacteria, which introduces genetic diversity and, in several cases, may drive ecological specialization in ciliates. As a third example, the integration of viral DNA, likely through transduction, provides new genetic materials and can change the way host cells defend themselves against other viral pathogens. We argue that the acquisition of MGEs through non-Mendelian patterns of inheritance, coupled with their effects on ciliate genome architecture and persistence throughout evolutionary history, exemplify how the transmission of mobile elements should be considered a mechanism of transgenerational epigenetic inheritance.

Adaptations of microbial communities and dissolved organics to seasonal pressures in a mesotrophic coastal Mediterranean lake

In lake ecosystems, changes in eukaryotic and prokaryotic microbes and the concentration and availability of dissolved organic matter (DOM) produced within or supplied to the system by allochthonous sources are components that characterize complex processes in the microbial loop. We address seasonal changes of microbial communities and DOM in the largest Croatian lake, Vrana. This shallow lake is connected to the Adriatic Sea and is impacted by agricultural activity. Microbial community and DOM structure were driven by several environmental stressors, including drought, seawater intrusion and heavy precipitation events. Bacterial composition of different lifestyles (free-living and particle-associated) differed and only a part of the particle-associated bacteria correlated with microbial eukaryotes. Oscillations of cyanobacterial relative abundance along with chlorophyll a revealed a high primary production season characterized by increased levels of autochthonous DOM that promoted bacterial processes of organic matter degradation. From our results, we infer that in coastal freshwater lakes dependent on precipitation-evaporation balance, prolonged dry season coupled with heavy irrigation impact microbial communities at different trophic levels even if salinity increases only slightly and allochthonous DOM inputs decrease. These pressures, if applied more frequently or at higher concentrations, could have the potential to overturn the trophic state of the lake.

Freshwater Microbial Eukaryotic Core Communities, Open-Water and Under-Ice Specialists in Southern Victoria Island Lakes (Ekaluktutiak, NU, Canada).

Across much of the Arctic, lakes and ponds dominate the landscape. Starting in late September, the lakes are covered in ice, with ice persisting well into June or early July. In summer, the lakes are highly productive, supporting waterfowl and fish populations. However, little is known about the diversity and ecology of microscopic life in the lakes that influence biogeochemical cycles and contribute to ecosystem services. Even less is known about the prevalence of species that are characteristic of the seasons or whether some species persist year-round under both ice cover and summer open-water conditions. To begin to address these knowledge gaps, we sampled 10 morphometrically diverse lakes in the region of Ekaluktutiak (Cambridge Bay), on southern Victoria Island (NU, Canada). We focused on Greiner Lake, the lakes connected to it, isolated ponds, and two nearby larger lakes outside the Greiner watershed. The largest lakes sampled were Tahiryuaq (Ferguson Lake) and the nearby Spawning Lake, which support commercial sea-run Arctic char (Salvelinus alpinus) fisheries. Samples for nucleic acids were collected from the lakes along with limnological metadata. Microbial eukaryotes were identified with high-throughput amplicon sequencing targeting the V4 region of the 18S rRNA gene. Ciliates, dinoflagellates, chrysophytes, and cryptophytes dominated the lake assemblages. A Bray–Curtis dissimilarity matrix separated communities into under-ice and open-water clusters, with additional separation by superficial lake area. In all, 133 operational taxonomic units (OTUs) occurred either in all under-ice or all open-water samples and were considered “core” microbial species or ecotypes. These were further characterized as seasonal indicators. Ten of the OTUs were characteristic of all lakes and all seasons sampled. Eight of these were cryptophytes, suggesting diverse functional capacity within the lineage. The core open-water indicators were mostly chrysophytes, with a few ciliates and uncharacterized Cercozoa, suggesting that summer communities are mixotrophic with contributions by heterotrophic taxa. The core under-ice indicators included a dozen ciliates along with chrysophytes, cryptomonads, and dinoflagellates, indicating a more heterotrophic community augmented by mixotrophic taxa in winter.

Invasive annual cheatgrass enhances the abundance of native microbial and microinvertebrate eukaryotes but reduces invasive earthworms

Invasive annual cheatgrass enhances the abundance of native microbial and microinvertebrate eukaryotes but reduces invasive earthworms

An elevation transect study of testate amoeba communities up to 4000 m a.s.l. on Mount Kinabalu, Borneo

Testate amoebae are a polyphyletic assemblage of unicellular eukaryotes with a specific shell. They occur frequently in soil and freshwater habitats all over the world, important for ecosystem functioning and useful for bio-indication. Terrestrial amoeba data from Borneo or Malaysia are rarely available. In this study, data on species composition and relative abundance were presented along a transect from 1730 m up to 4000 m above sea level from 10 elevational levels on Mt. Kinabalu, Malaysian Borneo. In total 78 morphotypes of testate amoebae were recorded. Most were small (under 100 µm) and supposedly cosmopolitan. However, Certesella certesi with limited geographical distribution and some rare taxa (e.g., Padaungiella lageniformis cordiformis, Placocista jurassica, Trinema chardezi) and two undescribed morphospecies were also observed. Presence/absence and relative abundance data of testate amoebae were associated to elevation with a pronounced change at the tree line, as revealed by non-metric multidimensional scaling ordination analysis. Our data from Southeast Asia contribute to the actual discussion on distributional patterns considering elevation gradients and biogeography of eukaryotic microbes.

Isolation of Estuary Soil Fungi and Screening Antibacterial Activity Against Staphylococcus aureus

Infectious disease is one of the causes of death in the world. Antibiotics are often used in the treatment of bacterial infections but are currently experiencing resistance. Based on this, the search for alternative antibiotics from natural sources needs to be done, one of which is from fungi. Fungi are eukaryotic microbes which have been an eminent sources for many currently available antibiotics including penicillin. This study was conducted to determine the potential antibacterial activity of ethyl acetate extract of estuarine fungi isolates against gram-positive, Staphylococcus aureus bacteria. Phytochemical screening of the extract using the TLC method while antibacterial testing was carried through a standard microdilution protocol. Antibacterial activity was reported in percent inhibition. The antibacterial test resulted in seven extracts possessed significant activity, with the highest percent inhibition of the IS-IB-T2 isolate code is 66.5 ± 1.1% and the lowest of the IS-IB-B2 isolate code is 12.2 ± 0.7% at concentration of 100 µg/mL. Majority compounds detected in each extract was terpenoids which suggested the contribution of antibacterial mechanisms. Therefore, estuary soil fungi have the potential to be further explored by antibiotic compounds.Keywords: antibacterial, estuarine fungi, staphylococcus aureus

Mitochondrial Cytochrome Oxidase Subunit 1: A Promising Molecular Marker for Species Identification in Foraminifera

Traditional morphological methods for species identification are highly time consuming, especially for small organisms, such as Foraminifera, a group of shell-building microbial eukaryotes. To analyze large amounts of samples more efficiently, species identification methods have extended to molecular tools in the last few decades. Although a wide range of phyla have good markers available, for Foraminifera only one hypervariable marker from the ribosomal region (18S) is widely used. Recently a new mitochondrial marker cytochrome oxidase subunit 1 (COI) has been sequenced. Here we investigate whether this marker has a higher potential for species identification compared to the ribosomal marker. We explore the genetic variability of both the 18S and COI markers in 22 benthic foraminiferal morphospecies (orders Miliolida and Rotaliida). Using single-cell DNA, the genetic variability within specimens (intra) and between specimens (inter) of each species was assessed using next-generation sequencing. Amplification success rate was twice as high for COI (151/200 specimens) than for 18S (73/200 specimens). The COI marker showed greatly decreased intra- and inter-specimen variability compared to 18S in six out of seven selected species. The 18S phylogenetic reconstruction fails to adequately cluster multiple species together in contrast to COI. Additionally, the COI marker helped recognize misclassified specimens difficult to morphologically identify to the species level. Integrative taxonomy, combining morphological and molecular characteristics, provides a robust picture of the foraminiferal species diversity. Finally, we suggest the use of a set of sequences (two or more) to describe species showing intra-genomic variability additionally to using multiple markers. Our findings highlight the potential of the newly discovered mitochondrial marker for molecular species identification and metabarcoding purposes.

Reproduction dynamics of planktonic microbial eukaryotes in the open ocean.

Understanding the biology of reproduction of an organismal lineage is important for retracing key evolutionary processes, yet gaining detailed insights often poses major challenges. Planktonic Foraminifera are globally distributed marine microbial eukaryotes and important contributors to the global carbon cycle. They cannot routinely be cultured under laboratory conditions across generations, and thus details of their life cycle remain incomplete. The production of flagellated gametes has long been taken as an indication of exclusively sexual reproduction, but recent research suggests the existence of an additional asexual generation in the life cycle. To gain a better understanding of the reproductive biology of planktonic Foraminifera, we applied a dynamic, individual-based modelling approach with parameters based on laboratory and field observations to test if sexual reproduction is sufficient for maintaining viable populations. We show that temporal synchronization and potentially spatial concentration of gamete release seems inevitable for maintenance of the population under sexual reproduction. We hypothesize that sexual reproduction is likely beneficial during the adaptation to new environments, while population sustenance in stable environments can be ensured through asexual reproduction.

The Diverse World of Protists-an Ideal Community with which to Introduce Microscopy in the Microbiology Teaching Laboratory.

ABSTRACTProtists and other eukaryotes are present in diverse terrestrial and aquatic environments. They can easily be collected from local ponds and streams. Although they are typically larger in size than prokaryotes, making them easy to study with even basic microscopes, microbiology laboratory courses often do not discuss them in detail and may only dedicate a short time to observing preserved samples. This laboratory exercise allows students to develop microscope skills, experience real world application of collecting and processing field samples, and delve deeper into the diverse world of protists and other microbial eukaryotes. Students may also engage by sharing their findings on the website iNaturalist to contribute to the scientific knowledge collected around the world. This laboratory module was initially designed for in-person learning but has been successfully adapted to remote learning and can also be applied to a complete online learning environment.

Complex marine microbial communities partition metabolism of scarce resources over the diel cycle.

Complex assemblages of microbes in the surface ocean are responsible for approximately half of global carbon fixation. The persistence of high taxonomic diversity despite competition for a small suite of relatively homogeneously distributed nutrients, that is, 'the paradox of the plankton', represents a long-standing challenge for ecological theory. Here we find evidence consistent with temporal niche partitioning of nitrogen assimilation processes over a diel cycle in the North Pacific Subtropical Gyre. We jointly analysed transcript abundances, lipids and metabolites and discovered that a small number of diel archetypes can explain pervasive periodic dynamics. Metabolic pathway analysis of identified diel signals revealed asynchronous timing in the transcription of nitrogen uptake and assimilation genes among different microbial groups-cyanobacteria, heterotrophic bacteria and eukaryotes. This temporal niche partitioning of nitrogen uptake emerged despite synchronous transcription of photosynthesis and central carbon metabolism genes and associated macromolecular abundances. Temporal niche partitioning may be a mechanism by which microorganisms in the open ocean mitigate competition for scarce resources, supporting community coexistence.

A large transposable element mediates metal resistance in the fungus Paecilomyces variotii

The horizontal transfer of large gene clusters by mobile elements is a key driver of prokaryotic adaptation in response to environmental stresses. Eukaryotic microbes face similar stresses; however, a parallel role for mobile elements has not been established. A stress faced by many microorganisms is toxic metal ions in their environment. In fungi, identified mechanisms for protection against metals generally rely on genes that are dispersed within an organism's genome. Here, we discover a large (∼85 kb) region that confers tolerance to five metal/metalloid ions (arsenate, cadmium, copper, lead, and zinc) in the genomes of some, but not all, strains of a fungus, Paecilomyces variotii. We name this region HEPHAESTUS (Hφ) and present evidence that it is mobile within the P.variotii genome with features characteristic of a transposable element. HEPHAESTUS contains the greatest complement of host-beneficial genes carried by a transposable element in eukaryotes, suggesting that eukaryotic transposable elements might play a role analogous to bacteria in the horizontal transfer of large regions of host-beneficial DNA. Genes within HEPHAESTUS responsible for individual metal tolerances include those encoding a P-type ATPase transporter-PcaA-required for cadmium and lead tolerance, a transporter-ZrcA-providing tolerance to zinc, and a multicopper oxidase-McoA-conferring tolerance to copper. In addition, a subregion of Hφ confers tolerance to arsenate. The genome sequences of other fungi in the Eurotiales contain further examples of HEPHAESTUS, suggesting that it is responsible for independently assembling tolerance to a diverse array of ions, including chromium, mercury, and sodium.

Size-Fractionated Microbiome Structure in Subarctic Rivers and a Coastal Plume Across DOC and Salinity Gradients.

Little is known about the microbial diversity of rivers that flow across the changing subarctic landscape. Using amplicon sequencing (rRNA and rRNA genes) combined with HPLC pigment analysis and physicochemical measurements, we investigated the diversity of two size fractions of planktonic Bacteria, Archaea and microbial eukaryotes along environmental gradients in the Great Whale River (GWR), Canada. This large subarctic river drains an extensive watershed that includes areas of thawing permafrost, and discharges into southeastern Hudson Bay as an extensive plume that gradually mixes with the coastal marine waters. The microbial communities differed by size-fraction (separated with a 3-μm filter), and clustered into three distinct environmental groups: (1) the GWR sites throughout a 150-km sampling transect; (2) the GWR plume in Hudson Bay; and (3) small rivers that flow through degraded permafrost landscapes. There was a downstream increase in taxonomic richness along the GWR, suggesting that sub-catchment inputs influence microbial community structure in the absence of sharp environmental gradients. Microbial community structure shifted across the salinity gradient within the plume, with changes in taxonomic composition and diversity. Rivers flowing through degraded permafrost had distinct physicochemical and microbiome characteristics, with allochthonous dissolved organic carbon explaining part of the variation in community structure. Finally, our analyses of the core microbiome indicated that while a substantial part of all communities consisted of generalists, most taxa had a more limited environmental range and may therefore be sensitive to ongoing change.

Dating Microbial Evolution with MCMCtree.

This protocol explains how to use the program MCMCtree to estimate divergence times in microbial phylogenies. The main advantage of MCMCtree is the implementation of an approximation to the molecular data likelihood that dramatically speeds up computation during Bayesian MCMC sampling of divergence times and evolutionary rates. The approximation allows the analysis of large phylogenies with hundreds of taxa and molecular alignments with thousands or millions of sites. Two examples are used to illustrate Bayesian clock dating with MCMCtree. The first is a phylogeny of (mostly) microbial eukaryotes and prokaryotes encompassing the major groups of life on Earth, and for which fossil information, to calibrate the nodes of the phylogeny, is available. The second is a phylogeny of influenza viruses with known sampling times. An overview of Bayesian MCMC sampling is given as well as practical advice on issues such as construction of the time and rate prior and assessment of convergence of MCMC chains. Strategies for estimating times in microbial phylogenies for which neither fossil information nor sampling times are known are discussed.

Accessible and Insightful Scientific Learning Experiences Using the Microorganism Haloferax volcanii.

Early exposure to science is critical to incite interest in scientific careers, promote equity and retention in STEM fields, and increase the general understanding of the scientific method. For many educators, however, the myriad resources that many scientific experiments require are not readily available. Microbiology experiments in particular can often be inaccessible for a lot of classrooms. In addition, microbiological studies often involve eukaryotic microbes and bacteria while excluding an entire domain of life: archaea. Archaea are more closely related to eukaryotes than are bacteria, and although all prokaryotic cells lack a nucleus, various key aspects of the cell biology of archaea and bacteria are fundamentally different. In addition to being useful for teaching about the diversity and evolution of living organisms, these differences between archaea and bacteria can also be harnessed to teach and emphasize other important biological topics. Haloferax volcanii is a non-pathogenic model haloarchaeon that allows for safe, affordable, and accessible microbiological experiments, as the requirement of high-salt media to grow H. volcanii presents a low risk of contamination. Here, we describe how H. volcanii can be used in the classroom and outline a protocol demonstrating their resistance to a broad spectrum of antibiotics, underscoring the distinct cell biology of bacteria and archaea. Finally, we introduce strategies and protocols to perform this and other H. volcanii experiments such that they can be performed based on the resources available in a high school or undergraduate classroom.

Epulopiscium spp.

When these large, motile microbes were first discovered their unusual cell morphology and reproductive biology led to the proposal that Epulopiscium spp. were a new kind of microbial eukaryote. Later, molecular phylogeny revealed that Epulopiscium spp. are bacteria. Additional 16S rRNA gene surveys have demonstrated that epulos comprise a diverse group of closely related bacteria living inside surgeonfish from tropical reef systems around the globe, including the Great Barrier Reef, Hawaii, Guam, and other Indo-Pacific Islands, as well as surgeonfish in the Atlantic Ocean.