Trackoscope: A low-cost, open, autonomous tracking microscope for long-term observations of microscale organisms.

Cells and microorganisms are motile, yet the stationary nature of conventional microscopes impedes comprehensive, long-term behavioral and biomechanical analysis. The limitations are twofold: a narrow focus permits high-resolution imaging but sacrifices the broader context of organism behavior, while a wider focus compromises microscopic detail. This trade-off is especially problematic when investigating rapidly motile ciliates, which often have to be confined to small volumes between coverslips affecting their natural behavior. To address this challenge, we introduce Trackoscope, a 2-axis autonomous tracking microscope designed to follow swimming organisms ranging from 10µm to 2mm across a 325cm2 area (equivalent to an A5 sheet) for extended durations-ranging from hours to days-at high resolution. Utilizing Trackoscope, we captured a diverse array of behaviors, from the air-water swimming locomotion of Amoeba to bacterial hunting dynamics in Actinosphaerium, walking gait in Tardigrada, and binary fission in motile Blepharisma. Trackoscope is a cost-effective solution well-suited for diverse settings, from high school labs to resource-constrained research environments. Its capability to capture diverse behaviors in larger, more realistic ecosystems extends our understanding of the physics of living systems. The low-cost, open architecture democratizes scientific discovery, offering a dynamic window into the lives of previously inaccessible small aquatic organisms.

Copper stress shapes the dynamic behavior of amoebae and their associated bacteria.

Amoeba-bacteria interactions are prevalent in both natural ecosystems and engineered environments. Amoebae, as essential consumers, hold significant ecological importance within ecosystems. Besides, they can establish stable symbiotic associations with bacteria. Copper plays a critical role in amoeba predation by either killing or restricting the growth of ingested bacteria in phagosomes. However, certain symbiotic bacteria have evolved mechanisms to persist within the phagosomal vacuole, evading antimicrobial defenses. Despite these insights, the impact of copper on the symbiotic relationships between amoebae and bacteria remains poorly understood. In this study, we investigated the effects of copper stress on amoebae and their symbiotic relationships with bacteria. Our findings revealed that elevated copper concentration adversely affected amoeba growth and altered cellular fate. Symbiont type significantly influenced the responses of the symbiotic relationships to copper stress. Beneficial symbionts maintained stability under copper stress, but parasitic symbionts exhibited enhanced colonization of amoebae. Furthermore, copper stress favored the transition of symbiotic relationships between amoebae and beneficial symbionts toward the host's benefit. Conversely, the pathogenic effects of parasitic symbionts on hosts were exacerbated under copper stress. This study sheds light on the intricate response mechanisms of soil amoebae and amoeba-bacteria symbiotic systems to copper stress, providing new insights into symbiotic dynamics under abiotic factors. Additionally, the results underscore the potential risks of copper accumulation in the environment for pathogen transmission and biosafety.

Biomonitoring tool for New Zealand peatlands: Testate amoebae and vascular plants as promising bioindicators.

In the last two centuries, a high proportion of peatlands have been lost or severely degraded across the world. The value of peatlands is now well-recognised for biodiversity conservation, flood management, and carbon mitigation, with peatland restoration now central to many government policies for climate action. A challenge, however, is to determine 'natural' and 'disturbed' conditions of peatlands to establish realistic baselines for assessing degradation and setting restoration targets. This requires a tool or set of tools that can rapidly and reliably capture peatland condition across space and time. Our aim was to develop such a tool based on combined analysis of plant and testate amoebae; a group of shelled protists commonly used as indicators of ecological change in peatlands. The value of testate amoebae is well established in Northern Hemisphere Sphagnum-dominated peatlands; however, relatively little work has been undertaken for Southern Hemisphere peat forming systems. Here we provide the first assessment and comparison of the bioindicator value of testate amoebae and vascular plants in the context of Southern Hemisphere peatlands. Our results further demonstrate the unique ecohydrological dynamics at play in New Zealand peat forming systems that set them apart from Northern Hemisphere peatlands. Our results show that plant and testate amoeba communities provided valuable information on peatland condition at different scales, we found that testate amoebae tracked changes in the abiotic variables (depth to water table, pH, and conductivity) more closely than vascular plants. Our results further demonstrate that functional traits of testate amoebae showed promising relationships with disturbance. Amoeba test compression, aperture position and test size were linked to changes in hydrology driven by fluctuations in ground water tables; however, trait responses manifested differently in ombrotrophic and minerotrophic peatlands. Overall, testate amoebae provide a promising bioindicator for tracking degradation in New Zealand peatlands and a potential additional tool to assess peatland condition.

Three-component contour dynamics model to simulate and analyze amoeboid cell motility in two dimensions.

Amoeboid cell motility is relevant in a wide variety of biomedical processes such as wound healing, cancer metastasis, and embryonic morphogenesis. It is characterized by pronounced changes of the cell shape associated with expansions and retractions of the cell membrane, which result in a crawling kind of locomotion. Despite existing computational models of amoeboid motion, the inference of expansion and retraction components of individual cells, the corresponding classification of cells, and the a priori specification of the parameter regime to achieve a specific motility behavior remain challenging open problems. We propose a novel model of the spatio-temporal evolution of two-dimensional cell contours comprising three biophysiologically motivated components: a stochastic term accounting for membrane protrusions and two deterministic terms accounting for membrane retractions by regularizing the shape and area of the contour. Mathematically, these correspond to the intensity of a self-exciting Poisson point process, the area-preserving curve-shortening flow, and an area adjustment flow. The model is used to generate contour data for a variety of qualitatively different, e.g., polarized and non-polarized, cell tracks that visually resemble experimental data very closely. In application to experimental cell tracks, we inferred the protrusion component and examined its correlation to common biomarkers: the F-actin density close to the membrane and its local motion. Due to the low model complexity, parameter estimation is fast, straightforward, and offers a simple way to classify contour dynamics based on two locomotion types: the amoeboid and a so-called fan-shaped type. For both types, we use cell tracks segmented from fluorescence imaging data of the model organism Dictyostelium discoideum. An implementation of the model is provided within the open-source software package AmoePy, a Python-based toolbox for analyzing and simulating amoeboid cell motility.

Testate amoebae response and vegetation composition after plantation removal on a former raised bog.

Extensive drainage of peatlands in north-west Europe for the purposes of afforestation for timber production and harvesting has altered the carbon balance and biodiversity value. Large-scale restoration projects aim to reinstate hydrological conditions to keep carbon locked up in the peat and to restart active peat growth. Testate amoebae are an informal grouping of well-studied protists in peatland environments and as microbial consumers play an important role in nutrient and carbon cycling. Using a space for time substitution approach, this study investigated the response of testate amoebae assemblages and vegetation composition after tree removal on a drained raised bog. There was a clear difference in microbial assemblages between open and a chronosequenceof restoration areas. Results suggest microbial recovery after rewetting is a slow process with plant composition showing a faster response than the microbial assemblage. Mixotrophic testate amoebae had not recovered seventeen years following plantation removal and the establishment of Sphagnum mosses in the wetter microforms. These results suggest that vegetation composition and Testate amoeba assemblages respond differently to environmental drivers at forest-to-bog restoration areas. Local physicochemical peat properties were a stronger driver of the testate assemblage compared with vegetation. Complete recovery of microbial assemblages may take place over decadal timescales.

Protist Diversity Responses to Experimental N Deposition in Biological Crusts of a Semiarid Mediterranean Ecosystem.

Biological soil crusts (BSC) are associations of different macro and microorganisms and aggregated soil particles located on the surface of soils in many different habitats. BSC harbour a diverse and complex community of ciliates and testate amoebae. These phagotrophic protists play an important role in C and N recycling in soil ecosystems but have not been frequently studied in BSC. In this context, the effects of three increasing N inputs on ciliates and testate amoebae in crusts from a semi-arid Mediterranean ecosystem were evaluated. A field experiment with artificial N-deposition was designed to mimic the effects caused by anthropogenic N depositions. The results have shown that the protist populations of these semi-arid Mediterranean environments have lower species richness than other soil environments. The increase in N produces a net loss of diversity in the populations studied and shifts in the community structure. It has also been shown that some ciliates and testate amoebae, due to their population responses to increased N concentrations, could potentially be used as bio-indicators of N contamination in these BSCs.

A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment.

Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae.

Amoebae as Targets for Toxins or Effectors Secreted by Mammalian Pathogens.

Numerous microorganisms, pathogenic for mammals, come from the environment where they encounter predators such as free-living amoebae (FLA). The selective pressure due to this interaction could have generated virulence traits that are deleterious for amoebae and represents a weapon against mammals. Toxins are one of these powerful tools that are essential for bacteria or fungi to survive. Which amoebae are used as a model to study the effects of toxins? What amoeba functions have been reported to be disrupted by toxins and bacterial secreted factors? Do bacteria and fungi effectors affect eukaryotic cells similarly? Here, we review some studies allowing to answer these questions, highlighting the necessity to extend investigations of microbial pathogenicity, from mammals to the environmental reservoir that are amoebae.

Environmental Free-Living Amoebae Can Predate on Diverse Antibiotic-Resistant Human Pathogens.

Here, we sought to test the resistance of human pathogens to unaltered environmental free-living amoebae. Amoebae are ubiquitous eukaryotic microorganisms and important predators of bacteria. Environmental amoebae have also been proposed to serve as both potential reservoirs and training grounds for human pathogens. However, studies addressing their relationships with human pathogens often rely on a few domesticated amoebae that have been selected to feed on rich medium, thereby possibly overestimating the resistance of pathogens to these predatory phagocytes. From an open-air composting site, we recovered over 100 diverse amoebae that were able to feed on Acinetobacter baumannii and Klebsiella pneumoniae. In a standardized and quantitative assay for predation, the isolated amoebae showed a broad predation spectrum, killing clinical isolates of A. baumannii, K. pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Interestingly, A. baumannii, which was previously reported to resist predation by laboratory strains of Acanthamoeba, was efficiently consumed by closely related environmental amoebae. The isolated amoebae were capable of feeding on highly virulent carbapenem-resistant or methicillin-resistant clinical isolates. In conclusion, the natural environment is a rich source of amoebae with broad-spectrum bactericidal activities, including against antibiotic-resistant isolates. IMPORTANCE Free-living amoebae have been proposed to play an important role in hosting and disseminating various human pathogens. The resistance of human pathogens to predation by amoebae is often derived from in vitro experiments using model amoebae. Here, we sought to isolate environmental amoebae and to test their predation on diverse human pathogens, with results that challenge conclusions based on model amoebae. We found that the natural environment is a rich source of diverse amoebae with broad-spectrum predatory activities against human pathogens, including highly virulent and antibiotic-resistant clinical isolates.

Evolution of Multicellular Complexity in The Dictyostelid Social Amoebas.

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as "proto-cell types" for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the "proto-cell type" gene regulatory networks.

Excitable actin dynamics and amoeboid cell migration.

Amoeboid cell migration is characterized by frequent changes of the direction of motion and resembles a persistent random walk on long time scales. Although it is well known that cell migration is typically driven by the actin cytoskeleton, the cause of this migratory behavior remains poorly understood. We analyze the spontaneous dynamics of actin assembly due to nucleation promoting factors, where actin filaments lead to an inactivation of these factors. We show that this system exhibits excitable dynamics and can spontaneously generate waves, which we analyze in detail. By using a phase-field approach, we show that these waves can generate cellular random walks. We explore how the characteristics of these persistent random walks depend on the parameters governing the actin-nucleator dynamics. In particular, we find that the effective diffusion constant and the persistence time depend strongly on the speed of filament assembly and the rate of nucleator inactivation. Our findings point to a deterministic origin of the random walk behavior and suggest that cells could adapt their migration pattern by modifying the pool of available actin.

Lipopeptide-mediated bacterial interaction enables cooperative predator defense.

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

Effect of asymmetric deformation dynamics in amoeboid organism on its search ability.

We investigate the effect of asymmetric deformation dynamics in an amoeboid organism on its search ability using a model amoeba. The model represents the behaviours of the amoeboid organism and its search ability is evaluated by searching for the solution to a Boolean satisfiability problem (SAT). We found that the efficiency of the search is significantly improved by implementing asymmetric delays in response to the feedback signals that increase and decrease the variable under appropriate errors. The results indicate that the model could search around the variable vector space by means of the appropriate combination of the inherent local search in the model and the error-induced global search. The results also show that the asymmetric response delays bias the variable to the values that can satisfy the SAT. We also demonstrate that an analog electronic system implementing the amoeba model with asymmetric dynamics possesses the search characteristics of the model.

Evolution and Adaptation of Legionella pneumophila to Manipulate the Ubiquitination Machinery of Its Amoebae and Mammalian Hosts.

The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.

Free-living amoebae promote Candida auris survival and proliferation in water.

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.

The Ecology and Evolution of Amoeba-Bacterium Interactions.

Amoebae are protists that have complicated relationships with bacteria, covering the whole spectrum of symbiosis. Amoeba-bacterium interactions contribute to the study of predation, symbiosis, pathogenesis, and human health. Given the complexity of their relationships, it is necessary to understand the ecology and evolution of their interactions. In this paper, we provide an updated review of the current understanding of amoeba-bacterium interactions. We start by discussing the diversity of amoebae and their bacterial partners. We also define three types of ecological interactions between amoebae and bacteria and discuss their different outcomes. Finally, we focus on the implications of amoeba-bacterium interactions on human health, horizontal gene transfer, drinking water safety, and the evolution of symbiosis. In conclusion, amoeba-bacterium interactions are excellent model systems to investigate a wide range of scientific questions. Future studies should utilize advanced techniques to address research gaps, such as detecting hidden diversity, lack of amoeba genomes, and the impacts of amoeba predation on the microbiome.

Characterization of the bacterial microbiomes of social amoebae and exploration of the roles of host and environment on microbiome composition.

As predators of bacteria, amoebae select for traits that allow bacteria to become symbionts by surviving phagocytosis and exploiting the eukaryotic intracellular environment. Soil-dwelling social amoebae can help us answer questions about the natural ecology of these amoeba-bacteria symbioses along the pathogen-mutualist spectrum. Our objective was to characterize the natural bacterial microbiome of phylogenetically and morphologically diverse social amoeba species using next-generation sequencing of 16S rRNA amplicons directly from amoeba fruiting bodies. We found six phyla of amoeba-associated bacteria: Proteobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, Firmicutes, and Acidobacteria. The most common associates of amoebae were classified to order Chlamydiales and genus Burkholderia-Caballeronia-Paraburkholderia. These bacteria were present in multiple amoeba species across multiple locations. While there was substantial intraspecific variation, there was some evidence for host specificity and differentially abundant taxa between different amoeba hosts. Amoebae microbiomes were distinct from the microbiomes of their soil habitat, and soil pH affected amoeba microbiome diversity. Alpha-diversity was unsurprisingly lower in amoebae samples compared with soil, but beta-diversity between amoebae samples was higher than between soil samples. Further exploration of social amoebae microbiomes may help us understand the roles of bacteria, host, and environment on symbiotic interactions and microbiome formation in basal eukaryotic organisms.

A High Throughput Viability Screening Method for the Marine Ectoparasite Neoparamoeba perurans.

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.

Differential gene expression analysis and cytological evidence reveal a sexual stage of an amoeba with multiparental cellular and nuclear fusion.

Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated. Recent genomic studies revealed microbial eukaryotes possess a genetic toolkit necessary for sexual reproduction. However, the mechanism of sexual development in a majority of microbial eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in microbial eukaryotes is a lack of observational evidence, primarily due to its cryptic nature. In this study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development using stage-specific Differential Gene Expression (DGE) and cytological analyses. Both DGE and cytological results showed that most of the meiosis and sex-related genes are upregulated in Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category representations in unfused and fused cells revealed a functional skew of the fused transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment to sexual development. However, the GO categories of unfused cells were dominated by metabolic pathways and other processes indicative of a vegetative phase. Our study provides strong evidence that the fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in understanding the evolution and mechanism of inheritance involving multiparents in other eukaryotes with a similar reproductive strategy.

Are paradoxes of amoeboid cognition, memristors, and memory mandating a re-conceptualization of actions and behaviors?

Background From intelligent behaviors of individual molecules and primitive organisms to those with a nervous system and brain, nature displays a fascinating collective memory across time and space. Using certain electronic circuits called memristors, it has been possible to emulate several processes of memory and learning, including a predictive form of intelligence of simple amoebas. Purpose What is the core of such intelligent performances? Although memristors are able to effectively describe puzzling biologic behaviors, recent results challenge their very existence altogether. This paper asks what can be gleaned from the underlying controversy itself, which will be argued is analogous to the observer principle in quantum physics. It is hypothesized that the material phenomena of resonance and sync have a counterpart involving behavior and memory. This questions the prominent role of a causative agent, and which type of behavior or memory is being occasioned. Results Building on the theory of memristors, the challenge of their actualization, and puzzling behaviors of amoebas, this paper proposes that 'resonating actions' - that is, similar or interlinked behaviors beyond space and time - are enabled by an underlying field of potentiation or domain of influence, and not by a cause-effect relationship. Such linked behaviors resemble a form of 'action coherence.' Habituated actions, memory, and behavior may arise out of a similar field of consciousness (here, modeled as memristors). These principles may be at the core of many hitherto unexplained mysteries of collective learning and behavior beyond space and time. Although this study is based on amoebas, the key postulates are also of great relevance to human thinking, behavior, and performance (including the representation of illness), and have independently been confirmed in the fields of consciousness research and spirituality.