Soil Amoeba Research Articles

Exploring the mechanisms of cadmium tolerance and bioaccumulation in a soil amoeba.

Cadmium (Cd) pollution is a global concern. Protists represent a prevalent yet understudied group in soil ecosystems, but our understanding of how protists interact with Cd remains limited. This study investigates the interaction between Cd and the soil amoeba Dictyostelium discoideum, focusing on its resistance, accumulation, and molecular mechanisms. We found that D. discoideum amoebae exhibit strong Cd resistance with an EC50 (half maximal effective concentration) of 899.2mg/kg and demonstrates significant Cd enrichment capabilities, achieving concentrations up to 1094.70±310.95mg/kg in stalks and a bioconcentration factor (BCF) of 7.30. Transcriptomic analysis revealed enriched pathways related to DNA replication and identified key genes involved in metal transport, detoxification, and stress response, including abc4, abc16, mms19, gcsA, ucpB, and sodA. Notably, microRNA (miRNA) regulation was found to play a critical role in modulating the expression of these genes. Our findings provide novel insights into the Cd enrichment potential of D. discoideum amoebae and elucidate its mechanisms of heavy metal resistance, highlighting the regulatory role of miRNAs. This study not only advances our understanding of protist-Cd interactions but also opens new avenues for the bioremediation of heavy metal-contaminated soils, where soil amoebae could serve as an effective agent due to their high bioaccumulation factor and rapid growth cycle.

The community diversity and metabolic function of symbiont bacteria associated with soil amoebae (dictyostelids) in free-living habitats

The community diversity and metabolic function of symbiont bacteria associated with soil amoebae (dictyostelids) in free-living habitats

Free-Living Protozoa (Ciliates and Testate Amoebae) in the Fresh Waters of the Nakhchivan Autonomous Republic

In the period from 2017 to 2022, ciliated protozoa and testate amoebae were studied in freshwater ecosystems of the Nakhchivan Autonomous Republic. 110 species of ciliates and 65 species of amoebae were identified. The species diversity of ciliates in freshwater is almost twice as high as in soil. The differences in species diversity between aquatic and soil amoebae are relatively small. The need for standardization of the methods used by researchers is emphasized. Further studies of these groups of protozoa are necessary to obtain more accurate conclusions about their ecological role, relationships, and their importance in various biological processes.

Soil Amoebae Are Unexpected Hotspots of Environmental Antibiotics and Antibiotic Resistance Genes.

Antibiotic resistance poses a significant threat to human health. While most studies focus on bacteria, interactions between antibiotics and other crucial microbial groups like protists remain uncertain. This study investigates how protists interact with antibiotics and examines how these interactions impact the fate of resistance genes. It reveals that amoebae exhibit high resistance to eight high-risk environmental antibiotics, accumulating significant quantities within their cells. Wild amoeboid strains from distant locations carry substantial antibiotic resistance genes (ARGs) and metal resistance genes (MRGs), with significant heterogeneity within a single species. Amoeboid symbionts and pathogens predominantly carry these genes. Paraburkholderia symbionts have reduced genomes and fewer resistance genes compared to free-living strains, while amoeba-endogenous Stenotrophomonas maltophilia does not exhibit a significantly reduced genome size. This suggests that the amoeboid hosts serve as a temporary medium facilitating its transmission. In summary, the study unveils that soil amoebae represent unexpected hotspots for antibiotics and resistance genes. Future research should assess the effects of antibiotics on often-overlooked protists and explore their role in spreading ARGs and MRGs in ecosystems. Incorporating protists into broader antibiotic resistance research is recommended, highlighting their significance within a One Health perspective.

NOVEL INVENTION OF SPORE INDUCTION IN A SISTER SPECIES TO GROUP 4 DICTYOSTELIA.

Dictyostelia are soil amoebas that aggregate to form fruiting bodies with spores and stalk cells in response to starvation. Where known, species across the dictyostelid phylogeny use secreted cAMP, detected by cAMP receptors (cARs) to induce the differentiation of spores and to organize fruiting body construction. However, recent deletion of the single cAR of Polyspondylium violaceum (Pvio) left both its fruiting bodies and spores intact. To investigate whether Pvio sporulation can occur in the absence of secreted cAMP and to explore alternative inducers in a bioassay , three prespore genes were identified and gene fusions of their promoters with the LacZ reporter gene were transformed into Pvio cells. After assessing the spatial expression pattern of the genes and the stage at which prespore gene expression initiated, the effect of cAMP and other Dictyostelium discoideum ( Ddis) signal molecules were tested on prespore gene expression in vitro. Pvio genes g4562 (psp1), g2696 (psp2) and g2380 (psp3) were identified as homologs of Ddis spore coat genes. They were first expressed around 4 h of starvation in aggregation centres and later in the posterior 4/5 th of emerging sorogens and the spore head of early fruiting bodies. Cells from dissociated 4 h aggregates and shaken in suspension for 6 h increased prespore- LacZ reporter activity 4-fold for psp1 and 6-fold for psp2, but this increase was at least 5-fold higher when cells were plated on solid substratum for 6 h to develop normally. cAMP had no effect on prespore gene induction and neither had the Pvio chemoattractant glorin nor the Ddis chemoattractants and differentiation inducers folate, c-di-GMP, DIF-1, GABA, cGMP and 8Br-cAMP. The Pvio lineage uniquely evolved a novel genetic network for synthesis, detection and processing of the signal that triggers its main survival strategy.

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.

Predation-resistant Pseudomonas bacteria engage in symbiont-like behavior with the social amoeba Dictyostelium discoideum

The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.

Soil protists are more resilient to the combined effect of microplastics and heavy metals than bacterial communities

Heavy metals and micro-/nanoplastic pollution seriously threaten the environment and ecosystems. While many studies investigated their effects on diverse microbes, few studies have focused on soil protists, and it is unclear how soil protists respond to the combined effect of micro-/nanoplastics and heavy metals. This study investigated how soil protistan and bacterial communities respond to single or combined copper and micro-/nanoplastics. The bacterial community exhibited an instantaneous response to single copper pollution, whereas the combined pollution resulted in a hysteresis effect on the protistan community. Single and combined pollution inhibited the predation of protists and changed the construction of ecological networks. Though single and combined pollution did not significantly affect the overall community structure, the exposure experiment indicated that combined pollution harmed soil amoeba's fitness. These findings offer valuable new insights into the toxic effects of single and combined pollution of copper and plastics on soil protistan and bacterial communities. Additionally, this study shows that sequencing-based analyses cannot fully reflect pollutants' adverse effects, and both culture-independent and dependent methods are needed to reveal the impact of pollutants on soil microbes.

Soil physical structure drives N-glycan mediated trophic interactions in soil amoebae: Mechanisms and environmental implications

Soil protozoa are an essential part of the terrestrial ecosystem, playing a vital role in the global element cycling and energy flow. However, one research gap is what are the key factors driving their diversity and environmental fates. In this study, we hypothesized that soil texture could affect soil protozoa's predation and their interactions with environmental pollutants, and we tested it by using a soil amoeba Dictyostelium discoideum as a model system. We found that soil texture affected amoeba's growth and development. In addition, environmental factors cannot explain the variation of amoeba's fitness in different soil textures. Soil sandy particles and water content rather than particle size contribute to amoeba's fitness. Furthermore, different soil textures induced distinct transcriptional responses to amoebae, especially N-glycan-related and multiple signaling pathways and the expression of key genes (e.g., Ras superfamily, cxgE, trap1). The expression of N-glycan-related pathways, which is positively correlated with amoeba predation, was inhibited in sand soil, decreasing amoeba's fitness. Finally, the results showed that soil texture also affects amoeba's interaction with environmental pollutants. In conclusion, this study shows that soil physical structures affect amoeba's interactions with bacteria and environmental pollutants. SynopsisSoil texture affects soil protozoa's growth and development and their interactions with environmental pollutants.

Escherichia coli Strains Display Varying Susceptibility to Grazing by the Soil Amoeba Dictyostelium discoideum.

Recent studies have shown that Escherichia coli can survive in different environments, including soils, and they can maintain populations in sterile soil for a long period of time. This indicates that growth-supporting nutrients are available; however, when grown in non-sterile soils, populations decline, suggesting that other biological factors play a role in controlling E. coli populations in soil. Free-living protozoa can affect the bacterial population by grazing. We hypothesized that E. coli strains capable of surviving in non-sterile soil possess mechanisms to protect themselves from amoeba predation. We determined the grazing rate of E. coli pasture isolates by using Dictyostelium discoideum. Bacterial suspensions applied to lactose agar as lines were allowed to grow for 24 h, when 4 μL of D. discoideum culture was inoculated in the center of each bacterial line. Grazing distances were measured after 4 days. The genomes of five grazing-susceptible and five grazing-resistant isolates were sequenced and compared. Grazing distance varied among isolates, which indicated that some E. coli are more susceptible to grazing by protozoa than others. When presented with a choice between grazing-susceptible and grazing-resistant isolates, D. discoideum grazed only on the susceptible strain. Grazing susceptibility phenotype did not align with the phylogroup, with both B1 and E strains found in both grazing groups. They also did not align by core genome phylogeny. Whole genome comparisons revealed that the five most highly grazed strains had 389 shared genes not found in the five least grazed strains. Conversely, the five least grazed strains shared 130 unique genes. The results indicate that long-term persistence of E. coli in soil is due at least in part to resistance to grazing by soil amoeba.

Long-Term Persistence of <i>Yersinia pestis</i> in Association with Acanthamoeba castellanii in Experiment

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods.

The neonicotinoid insecticide imidacloprid has unexpected effects on the growth and development of soil amoebae

Neonicotinoid pesticides are the most widely used insecticides worldwide and have become a global environmental issue. Previous studies have shown that imidacloprid, the most used neonicotinoid, can negatively affect a wide range of organisms, including non-target insects, fish, invertebrates, and mammals. Imidacloprid can also accumulate and persist in soils, posing threats to the terrestrial ecosystem. However, we know little about one ecologically important group of organisms, the single-celled soil protists. In this study, we used a soil amoeba, Dictyostelium discoideum, to test whether and how imidacloprid affects the growth and development of soil amoebae. We provide the first empirical evidence that environmental concentrations of imidacloprid negatively impact the fitness and development of soil amoebae. In addition, the adverse effects did not show a dose-response relationship with increased imidacloprid concentrations, where no significant difference was observed among the treatment groups. Further transcriptome analyses showed that imidacloprid affected amoeba's key DEGs related to phagocytosis, cell division, morphogenesis, and cytochrome P450. Moreover, soil amoebae show both conserved and novel transcriptional responses to imidacloprid. In conclusion, this study has expanded the non-target list of imidacloprid from animals and plants to single-celled protists, and we believe the impact of neonicotinoid pesticides on the microbiome is significantly underestimated and deserves more studies.

Short-Term Evaluation of the Spatial Distribution of Trophic Groups of Amoebae in the Rhizosphere of Zea mays Inoculated with Rhizophagus intraradices.

Primary production in terrestrial ecosystems is sustained by plants, microbiota, and fungi, which are the major organic matter providers in the root zone, setting in motion the soil food webs. Predators like soil amoebae voraciously feed on bacteria, fungi, and microbial eukaryotes releasing the nutrients sequestered in their biomass. Early food web setting up is crucial for seedling nutrition and its further development after establishment. Mycorrhizal fungi are more than phosphorus providers, and we wonder what their role is in structuring the predators' trophic groups in the root zone. We evaluated the effect of Rhizophagus intraradices inoculated in Zea mays (mycorrhizosphere), on the structuration of amoebae trophic groups along vertical and horizontal (3, 6, and 9cm) soil distribution when compared to un-inoculated plants, after 20days in microcosms. Amoebae species richness was highest in non-mycorrhizal seedlings in the root zone at 6- to 9-cm depth, and 3cm away from plants. More bacterial species are needed when plants are devoid of mycorrhiza, and their influence is constrained 3cm away from roots. Higher diversity of trophic groups was recorded at mycorrhizal seedlings and at the compartment influenced by the mycelium at 6- to 9-cm depth. The highest bacterivorous diversity, higher number of rare species and protozoa-eating amoebae, and the absence of fungivorous group recorded at the mycorrhizosphere of Z. mays, indicate that the community was very different from the non-mycorrhizal plants. We conclude that the arbuscular mycorrhizal fungus exerts significant changes on the community of trophic groups of amoebae.

Interaction of Talaromyces marneffei with free living soil amoeba as a model of fungal pathogenesis.

Talaromyces (Penicillium) marneffei is an important dimorphic mycosis endemic in Southeast Asia and Southern China, but the origin and maintenance of virulence traits in this organism remains obscure. Several pathogenic fungi, including Cryptococcus neoformans, Aspergillus fumigatus, Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum and Paracoccidioides spp. interact with free living soil amoebae and data suggests that fungal pathogenic strategies may emerge from environmental interactions of these fungi with ubiquitous phagocytic microorganisms. In this study, we examined the interactions of T. marneffei with the soil amoeba Acanthamoeba castellanii. T. marneffei was rapidly ingested by A. castellanii and phagocytosis of fungal cells resulted in amoeba death after 24 h of contact. Co-culture also resulted in a rapid transition for conidia to the fission-yeast form. In addition, well-established virulence factors such as melanin and a yeast specific mannoprotein of T. marneffei were expressed during interaction with A. castellanii at 37°C. Our findings support the assumption that soil amoebae environmental predators play a role in the selection and maintenance of particular features in T. marneffei that impart virulence to this clinically important dimorphic fungus in mammalian hosts.

First isolation of Leptomyxa (Amoebozoa, Leptomyxida) from endozoic conditions: an interaction between two common soil organisms

We report the first isolation of a widely distributed free-living soil amoeba Leptomyxa (Amoebozoa, Leptomyxida) from endozoic conditions. The amoebae were detected after 14 days following inoculation of the intestine of the earthworm Lumbricus terrestris on an agar plate with Escherichia coli. The earthworm was collected from Calcaric Fluvisol (pH 7) in the Upper Váh region, north-west Slovakia. Observed amoebae were uninucleate, polypodial with typically branched pseudopodia, and morphologically resembled Leptomyxa australiensis. This study enlarges the range of amphizoic tendency in leptomyxid amoebae and indicates their interactions with Oligochaeta.

Soil Amoebae Affect Iron and Chromium Reduction through Preferential Predation between Two Metal-Reducing Bacteria.

Soil protists are essential but often overlooked in soil and could impact microbially driven element cycling in natural ecosystems. However, how protists influence heavy metal cycling in soil remains poorly understood. In this study, we used a model protist, Dictyostelium discoideum, to explore the effect of interactions between soil amoeba and metal-reducing bacteria on the reduction of soil Fe(III) and Cr(VI). We found that D. discoideum could preferentially prey on the Fe(III)-reducing bacterium Shewanella decolorationis S12 and significantly decrease its biomass. Surprisingly, this predation pressure also stimulated the activity of a single S. decolorationis S12 bacterium to reduce Fe(III) by enhancing the content of electron-transfer protein cyt c, intracellular ATP synthesis, and reactive oxygen species (e.g., H2O2). We also found that D. discoideum could not prey on the Cr(VI)-reducing bacterium Brevibacillus laterosporus. In contrast, B. laterosporus became edible to amoebae in the presence of S. decolorationis S12, and their Cr(VI) reduction ability decreased under amoeba predation pressure. This study provides direct evidence that protists can affect the Cr and Fe cycling via the elective predation pressure on the metal-reducing bacteria, broadening our horizons of predation of protists on soil metal cycling.

A role for myosin II clusters and membrane energy in cortex rupture for Dictyostelium discoideum.

Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum, white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cortex in locations where blebs are expected to form. In this work, we examine the role of membrane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-agarose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at positions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy.

Complex Bilateral Interactions Determine the Fate of Polystyrene Micro- and Nanoplastics and Soil Protists: Implications from a Soil Amoeba.

Nano- and microplastics have become a serious global concern, threatening our living environments. Previous studies have shown that many organisms, including bacteria, animals, and plants, can be affected by microplastics. However, little is known about one ecologically important group of soil organisms, the protists. In this study, we investigated how polystyrene micro- and nanoplastics interacted with a soil amoeba Dictyostelium discoideum. The results showed that environmental concentrations of nano- and microplastics could negatively affect the soil amoeba's fitness and development. D. discoideum ingested both nano- and microplastics through phagocytosis but packed and excreted them during slug migration, which also promoted their biodegradation. Fourier transform infrared spectroscopy analyses revealed the formation of new oxygen-containing functional groups and the sign of possible oxidation of polystyrene. Also, nano- and microplastic exposure disrupted the nutrient and energy metabolisms of D. discoideum and affected the expression of key genes (e.g., cf45-1, dcsA, aprA, dymB, and gefB) related to morphogenesis and phagocytosis. In conclusion, our results show that nano- and microplastics have complex bilateral interactions with the soil amoeba, affecting each other's fate in the soil environment. This study provides new insights into how soil protists interact with nano- and microplastics in the soil ecosystem.

Natural History and Ecology of Interactions Between Bordetella Species and Amoeba.

A variety of bacteria have evolved the ability to interact with environmental phagocytic predators such as amoebae, which may have facilitated their subsequent interactions with phagocytes in animal hosts. Our recent study found that the animal pathogen Bordetella bronchiseptica can evade predation by the common soil amoeba Dictyostelium discoideum, survive within, and hijack its complex life cycle as a propagation and dissemination vector. However, it is uncertain whether the mechanisms allowing interactions with predatory amoebae are conserved among Bordetella species, because divergence, evolution, and adaptation to different hosts and ecological niches was accompanied by acquisition and loss of many genes. Here we tested 9 diverse Bordetella species in three assays representing distinct aspects of their interactions with D. discoideum. Several human and animal pathogens retained the abilities to survive within single-celled amoeba, to inhibit amoebic plaque expansion, and to translocate with amoebae to the fruiting body and disseminate along with the fruiting body. In contrast, these abilities were partly degraded for the bird pathogen B. avium, and for the human-restricted species B. pertussis and B. parapertussis. Interestingly, a different lineage of B. parapertussis only known to infect sheep retained the ability to interact with D. discoideum, demonstrating that these abilities were lost in multiple lineages independently, correlating with niche specialization and recent rapid genome decay apparently mediated by insertion sequences. B. petrii has been isolated sporadically from diverse human and environmental sources, has acquired insertion sequences, undergone genome decay and has also lost the ability to interact with amoebae, suggesting some specialization to some unknown niche. A genome-wide association study (GWAS) identified a set of genes that are potentially associated with the ability to interact with D. discoideum. These results suggest that massive gene loss associated with specialization of some Bordetella species to a closed life cycle in a particular host was repeatedly and independently accompanied by loss of the ability to interact with amoebae in an environmental niche.

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence

SummaryOpportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.