Size dependent uptake and trophic transfer of polystyrene microplastics in unicellular freshwater eukaryotes.

Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 µm, 3.6 × 108 particles/mL; 6 µm, 2.1 × 105 particles/mL) and fragments (<30 µm, 5 × 103 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 µm, while T. pyriformis ingests particles up to 10 µm. In A. proteus, small MP (PS0.5µm and PS6µm) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30µm) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30µm), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum.

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

The Effect of the Agonist Cholecystokinin-4 D-GB-115 On the Character of Motor Activity of Paramecium caudatum.

The study investigated the effect of GABA in various concentrations and D-GB-115 at a concentration of 10-7 M on the behavior of Paramecium caudatum. It was shown that GABA increases motor activity and changes the movement strategy of these protozoans, and the dose-effect relationship is domed, which can be explained by the presence of two types of GABA receptors in the outer membrane of paramecia: GABA-A and GABA-B. The active concentrations of GABA range from 10-3 to 10-13 M. The effect of pharmacological agents interacting with the GABA system on the behavior of ciliates (nembutal and D-GB-115) was studied.

Integral Study of Paramecium caudatum Acute and Chronic Toxicity, Sites of Entry and Distribution, Bioconcentration and Body Burdens of Five Metals.

An integral analysis of the acute and chronic toxicity, bioaccumulation, sites of entry, and distribution of four trace metals: copper, iron, lead, and nickel, and the non-trace metal mercury were performed in the ciliate Paramecium caudatum. Mercury was the fastest metal accumulated, and the most toxic. The sensitivity of Paramecium caudatum to the five metals tested (Cu, Fe, Hg, Ni, and Zn) falls in the range of other ciliate species. We observed similarities between the toxicity of the five metals to the ciliate P. caudatum with the rotifer Euchlanis dilatata: (a) Mercury was the most toxic metal in terms of acute and body burdens. (b) Acute values were very similar in both species, Hg as the most toxic and Fe as the less toxic, (c) the vacuole/ingestion chronic tests were more sensitive than growth inhibition chronic tests. These analyses would ideally help generate safer guidelines for protecting aquatic biota.

Toxicological impacts and likely protein targets of bisphenol a in Paramecium caudatum.

Bisphenol A (BPA) is a widely used plasticizer agent and a well-known ubiquitous endocrine disruptor, which is frequently associated with a series of reproductive, developmental, and transgenerational effects over wildlife, livestocks, and humans. Although extensive toxicological data is available for metazoans, the impact of BPA over unicellular eukaryotes, which represents a considerable proportion of eukaryotic diversity, remains largely overlooked. Here, we used acute end-point toxicological assay and an inverted virtual-screening (IVS) approach to evaluate cellular impairments infringed by BPA over the cosmopolitan ciliated protist, Paramecium caudatum. Our data indicate a clear time-dependent effect over P. caudatum survival, which seems to be a consequence of disruptions to multiple core cellular functions, such as DNA and cell replication, transcription, translation and signaling pathways. Finally, the use of this ciliate as a biosensor to monitor BPA within environments and the relevance of bioinformatic methods to leverage our current knowledge on the impacts of emerging contaminants to biological systems are discussed.

In vivo intrinsic atomic interaction infer molecular eco-toxicity of industrial TiO2 nanoparticles via oxidative stress channelized steatosis and apoptosis in Paramecium caudatum.

The ecotoxicological effect of after-usage released TiO2 nanoparticles in aquatic resources has been a major concern owing to their production and utilization in different applications. Addressing the issue, this study investigates the detailed in vivo molecular toxicity of TiO2 nanoparticles with Paramecium caudatum. TiO2 nanoparticles were synthesized at a lab scale using high energy ball milling technique; characterized for their physicochemical properties and investigated for their ecotoxicological impact on oxidative stress, steatosis, and apoptosis of cells through different biochemical analysis, flow cytometry, and fluorescent microscopy. TiO2 nanoparticles; TiO2 (N15); of size 36 ± 12 nm were synthesized with a zeta potential of - 20.2 ± 8.8 mV and bandgap of 4.6 ± 0.3 eV and exhibited a blue shift in UV-spectrum. Compared to the Bulk TiO2, the TiO2 (N15) exhibited higher cytotoxicity with a 24 h LC50 of 202.4 µg/ml with P. Caudatum. The mechanism was elucidated as the size and charge-dependent internalization of nanoparticles leading to abnormal physiological metabolism in oxidative stress, steatosis, and apoptosis because of their influential effect on the activity of metabolic proteins like SOD, GSH, MDA, and catalase. The study emphasized the controlled usage TiO2 nanoparticles in daily activity with a concern for ecological and biomedical aspects.

Method for Primary Screening of Pharmaceuticals on the Paramecium caudatum Eukaryotic Cell Model.

The  effect  of  adrenaline  in  various  concentrations  and  dopamine  at  a  concentration  of 10-10 mol/mL on the behavior of Paramecium caudatum was studied. It is shown that adrenaline reduces motor activity and changes the movement strategy of these protozoans; a dose-dependent behavioral response on the drug concentration was observed. This effect can be explained by the presence of adrenaline receptors located on the surface of the cell membrane. To study the direct effect of adrenaline on alpha and beta adrenergic receptors, the effect of non-selective adrenoblockers nicergoline and timolol is considered in this paper. At the same time, dopamine at a concentration of 10-10 mol/mL does not have a significant effect on the nature and magnitude of motor activity during the entire registration time, since this organism does not have receptors for this mediator. The proposed method makes it possible to quickly and objectively assess the nature of the effects of various pharmaceuticals acting on the catecholamine system.

Clarification of the Taxonomic Position of Paramecium caudatum Micronucleus Symbionts.

Bacteria of genus Holospora (order Holosporales, class Alphaproteobacteria) are obligate intranuclear symbionts of ciliates Paramecium spp. with strict host species and nuclear (macronucleus or micronucleus) specificity. However, three species under study Holospora undulata, Holospora elegans and 'Holospora recta' occupy the same ecological niche-micronucleus of Paramecium caudatum and demonstrate some differences in morphology of infectious form. The genetic diversity of holosporas by rrs and rpoB sequence analysis was determined. Phylogenetic and phylogenomic analysis of Holospora spp., as well as some phenotypic features indicate that there is no distinctive difference supporting studied micronuclear endosymbionts as distinct species. Therefore, Holospora elegans and 'Holospora recta' should be considered subspecies of Holospora undulata (ex Haffkine 1890) Gromov and Ossipov 1981, which was described first. Thus, we confirmed the evolutionary aspects of the development of symbiotic relationships: holosporas have a strict specificity to the host species and the type of nucleus.

Parasitism and host dispersal plasticity in an aquatic model system.

Dispersal is a central determinant of spatial dynamics in communities and ecosystems, and various ecological factors can shape the evolution of constitutive and plastic dispersal behaviours. One important driver of dispersal plasticity is the biotic environment. Parasites, for example, influence the internal condition of infected hosts and define external patch quality. Thus, state-dependent dispersal may be determined by infection status and context-dependent dispersal by the abundance of infected hosts in the population. A prerequisite for such dispersal plasticity to evolve is a genetic basis on which natural selection can act. Using interconnected microcosms, we investigated dispersal in experimental populations of the freshwater protist Paramecium caudatum in response to the bacterial parasite Holospora undulata. For a collection of 20 natural host strains, we found substantial variation in constitutive dispersal and to a lesser degree in dispersal plasticity. First, infection tended to increase or decrease dispersal relative to uninfected controls, depending on strain identity, indicative of state-dependent dispersal plasticity. Infection additionally decreased host swimming speed compared to the uninfected counterparts. Second, for certain strains, there was a weak negative association between dispersal and infection prevalence, such that uninfected hosts dispersed less when infection was more frequent in the population, indicating context-dependent dispersal plasticity. Future experiments may test whether the observed differences in dispersal plasticity are sufficiently strong to be picked up by natural selection. The evolution of dispersal plasticity as a strategy to mitigate parasite effects spatially may have important implications for epidemiological dynamics.

In situ remediation efficacy of hybrid aerogel adsorbent in model aquatic culture of Paramecium caudatum exposed to Hg(II).

Silica-gelatin hybrid aerogel of 24 wt% gelatin content is an advanced functional material suitable for the high performance selective adsorption of aqueous Hg(II). The remediation efficacy of this adsorbent was tested under realistic aquatic conditions by exposing cultures of Paramecium caudatum to Hg(II) and monitoring the model cultures by time-lapse video microscopy. The viability of Paramecium was quantified by analyzing the pixel differences of the sequential images caused by the persistent movement (motility) of the cells. The viability of Paramecium displays a clear exposure-response relationship with Hg(II) concentration. Viability decreases with increasing Hg(II) concentration when the latter is higher than 125 µg L-1. In the presence of 0.1 mg mL-1 aerogel adsorbent, the viability of the cells decreases only at Hg(II) concentrations higher than 500 µg L-1, and 220 min survival time was measured even at 1000 µg L-1 Hg(II). The effective toxicity of Hg(II) is lower in the presence of the aerogel, because the equilibrium concentration of aqueous Hg(II) is low due to adsorption, thus Paramecium cells do not uptake as much Hg(II) as in the un-remediated cultures. Video imaging of Paramecium cultures offers a simple, robust and flexible method for providing quantitative information on the effectiveness of advanced materials used in adsorption processes for water treatment.

An evolutionary trade-off between parasite virulence and dispersal at experimental invasion fronts.

Exploitative parasites are predicted to evolve in highly connected populations or in expanding epidemics. However, many parasites rely on host dispersal to reach new populations, potentially causing conflict between local transmission and global spread. We performed experimental range expansions in interconnected microcosms of the protozoan Paramecium caudatum, allowing natural dispersal of hosts infected with the bacterial parasite Holospora undulata. Parasites from range front treatments facilitated host dispersal and were less virulent, but also invested less in horizontal transmission than parasites from range cores. These differences were consistent with parameter estimates derived from an epidemiological model fitted on population-level time-series data. Our results illustrate how dispersal selection can have profound consequences for the evolution of parasite life history and virulence. Decrypting the eco-evolutionary processes that shape parasite 'dispersal syndromes' may be important for the management of spreading epidemics in changing environments, biological invasions or in other spatial non-equilibrium settings.

In vitro toxicity of isolated strains and cyanobacterial bloom biomasses over Paramecium caudatum (ciliophora): Lessons from a non-metazoan model organism.

Cyanobacteria have been considered a major global threat because of their widespread ability to proliferate and contaminate inland and marine waters with toxic metabolites. For this reason, to avoid risks to humans and environmental health, regulatory legislation and guidelines have been established based on extensive toxicological data. However, most of what is known in this field come from works on microcystin (MC) variants, which effects were almost exclusively tested in metazoan models. In this work, we used acute end-point toxicological assays and high-resolution hybrid quadrupole time-of-flight mass spectrometer coupled with electrospray ionization source (ESI-Q-TOF-MS) analyses to evaluate the deleterious impact of aqueous extracts prepared from cultures of cyanobacteria and environmental bloom biomasses over a non-metazoan model organism, the cosmopolitan fresh/brackish water unicellular microeukaryote, Paramecium caudatum (Ciliophora). Our data suggest that all extracts produced time-dependent effects on P. caudatum survival, irrespective of their metabolite profile; and that this ciliate is more sensitive to extracts containing microginins than to extracts with only MCs, stressing that more toxicological investigations should be performed on the environmental impact of neglected cyanotoxins. Further, our data provide evidence that P. caudatum may be more sensitive to cyanotoxins than vertebrates, indicating that guidelines values, set on metazoans are likely to be inaccurate to protect organisms from basal food web positions. Thus, we highly recommend the widespread use of microeukaryotes, such as ciliates in environmental risk assessment frameworks for the establishment of more reliable cyanotoxin monitoring guideline values.

Cytotoxicity of Potassium Salts of (+)- and (-) Usnic Acid for Paramecium caudatum.

We analyzed cytotoxicity of water-soluble potassium salts of (+)- and (-) usnic acid (UA) for ciliates P. caudatum. The median lethal concentrations for (+)- and (-) enantiomers did not significantly differ and were 7.5±0.5 and 6.7±0.4, respectively. In a concentration of 8 µM, (+)-UA and (-)-UA salts increased the content of TBA-reactive products, which indicates the formation of oxidative stress under the action of high UA concentrations. In the presence of (+)-UA and (-)-UA salts in a concentration range from 2 to 8 µM, the number of food vacuoles in ciliates decreased, which attested to a decrease in phagocytosis activity. The concentrations of UA enantiomers >0.5 µM affected macronucleus morphology (shape and size). The cytotoxic activity of (+)-UA and (-)-UA salts against P. caudatum did not differ.

Cellulose-based hybrid glycosilicones via grafted-to metal-catalyzed hydrosilylation: "When opposites unite".

Hydrosilylation catalyzed by the rhodium(I) complex [Rh(acac)(CO)2] or platinum(0)-based Karstedt's catalyst was employed to combine hydrophilic propargylated hydroxyethyl cellulose and hydrophobic hydride-terminated polydimethylsiloxane to give polymer hybrid structures. The final polymers were characterized by FTIR, solid state 1H, 13C and 29Si NMR, contact angle, microcalorimetry and thermogravimetry measurements. The grafting degree was controlled by the catalyst choice and by the reagent load variations; an increase of the polysiloxane load and a change from Karstedt's to the rhodium catalyst led to a higher (from 2 to 7%) silicon content in the glycosilicones. The glycosilicones were insoluble in water, but swelled in organic solvents (DMSO, DMF, and chloroform). The hydrophilicity of the glycosilicones decreased with incrementing silicon content: the contact angles increased from 30 (cellulose) to 103-131° in the hybrids. The glycosilicones obtained via the hydrosilylation are less toxic toward algae Chlorella vulgaris and infusoria Paramecium caudatum than those obtained with CuAAC.

Individual and mixture toxicity of carbofuran and diuron to the protozoan Paramecium caudatum and the cladoceran Ceriodaphnia silvestrii.

The toxicity of the insecticide carbofuran and herbicide diuron (individually and in mixture) to the invertebrates Paramecium caudatum and Ceriodaphnia silvestrii was evaluated. Acute and chronic toxicity tests were carried out with the diuron and carbofuran active ingredients and their commercial products, Diuron Nortox® 500 SC and Furadan® 350 SC, respectively. Individual toxicity tests showed that C. silvestrii was more sensitive to both carbofuran and diuron than P. caudatum. In single exposures, both pesticides caused adverse effects to C. silvestrii in environmentally relevant concentrations (48 h EC50 = 0.001 mg L-1 and 8 d LOEC = 0.00038 mg L-1 for formulated carbofuran; 8 d LOEC < 0.05 mg L-1 for formulated diuron). For P. caudatum, carbofuran and diuron in single exposures were only slightly toxic (24 h IC50 = 5.1 mg L-1 and 6.9 mg L-1 for formulated carbofuran and diuron, respectively). Acute and chronic exposures to diuron and carbofuran mixtures caused significant deviations of the toxicity predicted by the Concentration Addition and Independent Action reference models for both test species. For the protozoan P. caudatum, a dose-dependent deviation was verified for mortality, with synergism caused mainly by carbofuran and antagonism caused mainly by diuron. For protozoan population growth, however, an antagonistic deviation was observed when the active ingredient mixtures were tested. In the case of C. silvestrii, antagonism at low concentrations and synergism at high concentrations were revealed after acute exposure to active ingredient mixtures, whereas for reproduction an antagonistic deviation was found. Commercial formulation mixtures presented significantly higher toxicity than the active ingredient mixtures. Our results showed that carbofuran and diuron interact and cause different toxic responses than those predicted by the individually tested compounds. Their mixture toxicity should therefore be considered in risk assessments as these pesticides are likely to be present simultaneously in edge-of-field waterbodies.

Local surface plasmon resonance of gold nanoparticles as a correlative light and electron microscopy (CLEM) tag for biological samples.

In this study, we investigated use of local surface plasmon resonance (LSPR) of metal nanoparticles (NPs) as a correlative light and electron microscopy (CLEM) tag for biological samples. Gold NPs in ultra-thin sections for TEM revealed that LSPR could be observed by optical microscopy at sizes of 20 nm or larger. Gold NPs at sizes less than 20 nm could be observed using the gold enhancement method. Therefore, this CLEM tag could be applied to immunoelectron microscopy using this gold enhancement method.

Combined effects of food resources and exposure to ammonium nitrogen on population growth performance in the bacterivorous ciliate Paramecium caudatum.

Ciliated protozoa (ciliates) play vital roles in biological wastewater-treatment processes, however, combined effects of abiotic and biotic factors as well as the importance of species-specificity of bacterial food organisms on population growth dynamics remain poorly understood, which are hampering the management and optimization of biological wastewater treatment processes. This study investigated the effects of food resources and ammonium nitrogen (NH4+) exposure, both independently and in combination, on the population growth of the bacterivorous ciliate Paramecium caudatum. Results showed that, when fed with two different bacterial food organisms, population growth performance of P. caudatum differed significantly and increased with the addition of protozoa pellet medium. When exposed to NH4+ population growth declined and metabolic enzyme activities were altered. The negative effects of NH4+ on population growth could be weakened by supplementing the food resource with protozoa pellet media. In brief, it was confirmed that the existence of interactive effect of food resources and ammonium nitrogen, as well as the importance of species-specificity of bacterial food organisms on the population growth performance of ciliates. These findings might lead to the development of a valuable strategy for improving the performance of biological wastewater-treatment processes.

Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds.

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.

On measuring nanoparticle toxicity and clearance with Paramecium caudatum.

As the extent to which aquatic environments are polluted with nano-scale objects is becoming known, we are presented with an urgent need to study their effects on various forms of life and to clear and/or detoxify them. A range of methods exist to these ends, but a lack of inter-study comparability arising from an absence of experimental standardisation impedes progress. Here we present experiments that demonstrate measurement of orchestrated uptake and clearance of two environmentally-relevant nano- and micromaterials by a model aquatic microoraganism, Paramecium caudatum. Experiments were based on a simple, modular, multi-chamber platform that permits standardised control of organism behaviour and measurement of variables relevant to the study of nanotoxicology, including nanomaterial chemotaxis assays, bioaccumulation and deleterious effects on cell motility systems. Uptake of internalised materials may be estimated through the addition of a low-cost fluorescence spectrometer. P. caudatum cells can clear an estimated 0.7 fg of contaminant materials (or 161 of the particles used) per cell over a 5 mm distance per 6 hour experiment, whilst suffering few short-term adverse effects, suggesting that the organism and the platform used to investigate their properties are well-suited to a range of laboratory and field-based nanotoxicological studies.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.