Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Influence of Nano- and Small Microplastics on Ciliated Protozoan Spirostomum ambiguum (Müller, 1786) Ehrenberg, 1835

This study evaluated the uptake of secondary nano- and small microparticles by the protozoan Spirostomum ambiguum, comparing edible (baker’s yeasts) and inedible (red latex) particles. Secondary nano- and microplastic particles were prepared from household materials made of four different polymers and served to the protozoans separately and as two-component mixtures in different proportions. The number and content of food vacuoles formed by the protozoan were analyzed using a digital microscope. The microscopic results showed that the protozoans ingested the secondary microplastic particles to a similar degree as the latex microspheres but to a lesser extent compared to the nutritional food—baker’s yeasts. At the microplastic concentrations of 1000 and 10,000 particles mL–1, no food vacuoles were observed inside the cells, which may be a finding of great ecological importance. In the protozoans served two-component mixtures, both microplastics and yeasts were found in the vacuoles formed by the organisms. The egestion of two-component vacuoles by the protozoans was slower than that of vacuoles containing a single component.

Chloroquine and Hydroxychloroquine: The History Revisited

The medicinal properties of the bark of the Cinchona tree have been known for centuries. It was known to cure fever and malaria. The active alkaloid was first isolated by the French chemists Pelletier and Coventou in 1820. The organized use of a large amount of quinine to prevent and treat malaria was largely done by the colonial military units in the tropics, especially in Africa and Southeast Asia. Scientists soon learned to synthesize quinine-like compounds which included chloroquine and hydroxychloroquine. Quinine (C18H26CIN3) was first synthesized successfully in 1934. In 1946, hydroxychloroquine (C18H26CIN3O) was developed as its less toxic alternative. Quinine derivatives, CQ and HCQ, are weak bases chemically. They are accumulated in acidic food vacuoles of intraerythrocytic trophozoites. Its antimalarial action is due to the induction of selective toxicity to lysosomes in parasites, thereby preventing hemoglobin degradation. For a long time the role of CQ as an anti-inflammatory agent has been investigated. It is used as a disease-modifying agent against rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It has antitumor activities also and has been studied in glioblastoma, colon, and pancreatic cancers. In the recent outbreak of the corona pandemic, there is a resurgence of interest in its use as an antiviral agent. Its mechanism of action is not fully understood; it may involve multiple pathways to act as an antiviral agent. The research activities to explore its efficacy and new applications in various diseases have never ceased. Despite its long history the drug and its derivatives have attracted scientists world-wide.

Microplastics and phagotrophic soil protists: evidence of ingestion

Microplastics (MPs) can now be found in all the Earth’s biomes, thereby representing a global change phenomenon with largely unknown consequences for biodiversity and ecosystem functioning. Soil protists are eukaryotic, primarily single celled organisms that play important roles in the soil food web. Microplastics have been shown to affect protist populations in freshwater and marine environments, yet the interactions between soil protists and MPs remains largely unknown. Here we examined whether phagotrophic soil protists can ingest MPs and experience declines in abundance. We exposed protists to soil treatments with different concentrations of MPs using commercial polymer fluorescent microspheres and used fluorescence microscopy to find evidence of MP ingestion. In addition, we quantified the total number of active phagotrophic protists over time. We show that most soil protists (>75% individuals) can readily ingest and keep MP within their food vacuoles, even at relatively small MP concentrations (0.1% w/w). There was a trend for higher prevalence of ingestion and for declines in protist abundance at the highest concentration of MPs (1% w/w). However, more data are necessary to further ascertain cause-effect relationships. This is the first report indicating that soil protists can play an important role in the transport and uptake of MPs in the soil food web.

Biosynthetic heme of malaria parasite induces cerebral pathogenesis by regulating hemozoin formation and griseofulvin can prevent cerebral malaria

ABSTRACTHeme-biosynthetic pathway of malaria parasite is dispensable for asexual stages, but essential for sexual and liver stages. Despite having backup mechanisms to acquire hemoglobin-heme, pathway intermediates and/or enzymes from the host, asexual parasites express heme pathway enzymes and synthesize heme. Here we show heme synthesized in asexual stages promotes cerebral pathogenesis by enhancing hemozoin formation. Hemozoin is a parasite molecule associated with inflammation, aberrant host-immune responses, disease severity and cerebral pathogenesis. The heme pathway knockout parasites synthesize less hemozoin, and mice infected with knockout parasites are completely protected from cerebral malaria and death due to anaemia is delayed. Biosynthetic heme regulates food vacuole integrity and the food vacuoles from knockout parasites are compromised in pH, lipid unsaturation and proteins, essential for hemozoin formation. Targeting parasite heme synthesis by griseofulvin - a FDA-approved drug, prevents cerebral malaria in mice and provides a new adjunct therapeutic option for cerebral and severe malaria.

Feeding experiments of the seep-associated foraminifer Nonionellina labradorica with a marine methanotroph from the Arctic

<p>Foraminifera on the seafloor are known to have species-specific feeding habits. Among those are deposit feeders, eating organic detritus and bacteria. Little is known about the feeding habits of foraminifera from Arctic seep environments. That is, in particular, of interest as variable δ<sup>13</sup>C values in the tests of foraminifera have been suggested to be partly linked with a diet rich in bacteria, themselves lighter in δ<sup>13</sup>C values. As there is little information on the ecology of the foraminifer <em>Nonionellina labradorica</em> (Dawson, 1860), this study examined feeding habits on bacteria and compared them to in situ collected specimens, using Transmission Electron microscopy (TEM). As bacterial food, the marine methane-oxidizing bacterium <em>Methyloprofundus sedimenti</em> was chosen, which is an important representative of methanotrophs in the marine environment near methane seeps. Sediment samples containing living N. labradorica specimens collected in close vicinity(approx. 5 m) from an active methane seep in Storfjordrenna, Barents Sea (382-m water depth).  We performed a feeding experiment on <em>N. labradorica </em>(n=17 specimen), which were incubated in the dark at in situ temperature. Specimens were fed at the beginning of the experiment, except the un-fed controls, and incubations terminated after 4, 8 and 20 h. After fixation in epoxy resin the ultrastructure of all specimens and their food vacuoles was observed and compared using a TEM. All examined specimens were living at the time of fixation, based on observation of intact mitochondrial membranes. In all specimens, inorganic detritus was preserved inside food vacuoles. Closer observation of food vacuoles also revealed that in addition to inorganic debris, such as clay, occasionally bacteria were visible. This led us to conclude that our <em>N. labradorica </em>can  generally be classified as a deposit feeder, which is rather a generalist than a specialist. Regarding uptake of <em>M. sedimenti</em>, the timing of the experimentation seemed to be critical. We did not observe methanotrophs preserved in the resin at the 4 and 8 h incubations, but found two putative methanotrophs near the apertural region after the 20-h incubation. After closer observation, we could identify one of those two putative specimen as the menthanothroph <em>M. sedimenti</em> near the foraminiferal aperture, based on presence of a typical type I stacked intracytoplasmic membrane (ICM) and storage granules (SC). We concluded that <em>N. labradorica</em> may ingest <em>M. sedimenti</em> via “untargeted grazing” in seeps. Further studies must examine the exact relationship between diet and δ<sup>13</sup>C in foraminiferal test on several different paleo-oceanographically relevant species.</p>

Metabolomics for early detection of stress in freshwater alga Poterioochromonas malhamensis exposed to silver nanoparticles

Silver nanoparticles (AgNPs) are one of the most used engineered nanomaterials. Despite progress in assessing their environmental implications, knowledge gaps exist concerning the metabolic perturbations induced by AgNPs on phytoplankton, essential organisms in global biogeochemical cycles and food-web dynamics. We combine targeted metabolomics, biouptake and physiological response studies to elucidate metabolic perturbations in alga Poterioochromonas malhamensis induced by AgNPs and dissolved Ag. We show time-dependent perturbation of the metabolism of amino acids, nucleotides, fatty acids, tricarboxylic acids, photosynthesis and photorespiration by both Ag-treatments. The results suggest that dissolved Ag ions released by AgNPs are the major toxicity driver; however, AgNPs internalized in food vacuoles contributed to the perturbation of amino acid metabolism, TCA cycle and oxidative stress. The metabolic perturbations corroborate the observed physiological responses. We highlight the potential of metabolomics as a tool for understanding the molecular basis for these metabolic and physiological changes, and for early detection of stress.

Uptake of plastic microbeads by ciliate Paramecium aurelia

Microplastics (MPs) are small fraction of plastics that are less than 5 mm in length. They are bountiful and widespread pollutants in the aquatic environment. A wide range of organisms which play an important role in the food web, ingest microplastic particles and transfer them to the higher trophic levels. In this work, ingestion of fluorescent polystyrene beads 2 µm of diameter by ciliated protozoa Paramecium aurelia in different concentrations and times of exposure was studied. We studied also the ingestion and clearance rate as well as formation of food vacuoles. The highest uptake of beads by ciliates reached 1047.2 ± 414.46 particles after 10 min of incubation. Food vacuoles formation reflected the ingestion rate of P. aurelia, which increased at higher beads concentration up to the10th minute of incubation and decreased afterwards. On the contrary, the clearance rate persisted to be higher at low concentration. These findings showed that maximum capacity of microplastics ingestion by paramecia depended on beads concentration and on time of exposure.

Cascading effects in freshwater microbial food webs by predatory Cercozoa, Katablepharidacea and ciliates feeding on aplastidic bacterivorous cryptophytes

ABSTRACT Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-µm filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-µm fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55–90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-µm treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-µm treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.

The transcriptome of Escherichia coli O157: H7 reveals a role for oxidative stress resistance in its survival from predation by Tetrahymena

ABSTRACT Pathogenic E. coli remains undigested upon phagocytosis by Tetrahymena and is egested from the ciliate as viable cells in its fecal pellets. Factors that are involved in the survival of Shiga toxin-producing E. coli serovar O157: H7 (EcO157) from digestion by Tetrahymena were identified by microarray analysis of its transcriptome in the protozoan phagosome. Numerous genes belonging to anaerobic metabolism and various stress responses were upregulated significantly ≥ 2-fold in EcO157 cells in the food vacuoles compared with in planktonic cells that remained uningested by the protist. Among these were the oxidative stress response genes, ahpF and katG. Fluorescence microscopy and staining with CellROX® Orange confirmed the presence of reactive oxygen species in food vacuoles containing EcO157 cells. Frequency distribution analysis of the percentage of EcO157 viable cells in Tetrahymena fecal pellets revealed that the ΔahpCF and ΔahpCFΔkatG mutants were less fit than the wild type strain and ΔkatG mutant after passage through the protist. Given the broad use of oxidants as sanitizers in the food industry, our observation of the oxidative stress response in EcO157 during its interaction with Tetrahymena emphasizes the importance of furthering our knowledge of the physiology of this human pathogen in environments relevant to its ecology and to food safety.