Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G. and IT838-13 to A.A.), and by the Swiss National Science Foundation

Oribatid communities and heavy metal bioaccumulation in selected species associated with lichens in a heavily contaminated habitat

The study examines oribatid communities and heavy metal bioaccumulation in selected species associated with different microhabitats of a post-smelting dump, i.e. three lichen species of Cladonia with various growth forms and the slag substrate. The abundance of oribatids collected from the substrate was significantly lower than observed in lichen thalli. The morphology and chemical properties of lichens, and to some extent varying concentrations of heavy metals in thalli, are probably responsible for significant differences in oribatid communities inhabiting different Cladonia species. Some oribatids demonstrate the ability to accumulate zinc and cadmium with unusual efficiency, whereas lead is the most effectively regulated element by all species. A positive correlation was found between Zn content in all studied oribatids and their microhabitats. Oribatids exploring different food resources, i.e. fungivorous and non-fungivorous grazers, show considerable differences in bioconcentrations of certain elements

Microscopic analysis of the microbiota of three commercial Phytoseiidae species (Acari: Mesostigmata)

Microbes associated with the external and internal anatomy of three commercially available predatory mite species, Phytoseiulus persimilis, Typhlodromips (=Amblyseius) swiskii, and Neoseiulus (=Amblyseius) cucumeris were examined using light microscopy, confocal laser scanning microscopy and fluorescence in-situ hybridization (FISH). Four microbe morphotypes were observed on external body regions. These included three microfungi-like organisms (named T1, T2 and T3) and rod-shaped bacteria (T4). Morphotypes showed unique distributions on the external body regions and certain microbes were found only on one host species. Microfungi-like T1 were present in all three species whereas T2 and T3 were present in only P. persimilis and T. swirskii respectively. T1 and T2 microbes were most abundant on the ventral structures of the idiosoma and legs, most frequently associated with coxae, coxal folds, ventrianal shields and epigynal shields. T3 microbes were most abundant on legs and dorsal idiosoma. T4 microbes were less abundant and were attached to epigynal shields of N. cucumeris and T. swirskii. Significant differences in distribution between seasons (spring and winter) suggest that there are fluctuations in the microbiota of phytoseiids in mass reared systems. FISH using the EUB338 (I-III) probes showed bacteria within the alimentary tract, in Malpighian tubules and anal atria. It is possible these have a role in absorbing excretory products or maintaining gut physiology. We suggest how microbes might be transmitted to offspring and throughout populations. The implications of these findings for commercial mass rearing are discussed. This study highlights the necessity of understanding the intrinsic microbiota of Phytoseiidae and other Acari

Gut contents, digestive half-lives and feeding state prediction in the soil predatory mite Pergamasus longicornis (Mesostigmata: Parasitidae)

Mid- and hind-gut lumenal changes are described in the free-living predatory soil mite Pergamasus longicornis (Berlese) from a time series of histological sections scored during and after feeding on fly larval prey. Three distinct types of tangible material are found in the lumen. Bayesian estimation of the change points in the states of the gut lumenal contents over time is made using a time-homogenous first order Markov model. Exponential processes within the gut exhibit ’stiff’ dynamics. A lumen is present throughout the midgut from 5 min after the start of feeding as the gut rapidly expands. It peaks at about 21.5 h - 1.5 days and persists post-feeding (even when the gut is contracted) up until fasting/starvation commences 10 days post start of feeding. The disappearance of the lumen commences 144 h after the start of feeding. Complete disappearance of the gut lumen make take 5-9 weeks from feeding commencing. Clear watery prey material arrives up to 10 min from the start of feeding - driving gut lumen expansion. Intracellular digestion triggered by maximum gut expansion is indicated. Detectable granular prey material appears in the lumen during the concentrative phase of coxal droplet production and, despite a noticeable collapse around 12 h, lasts in part for 52.5 h. Posterior midgut regions differ slightly from anterior regions in their main prey food dynamics being somewhat faster in processing yet being slightly delayed. Posterior regions are confirmed as Last-In-Last-Out depots, anterior regions confirmed as First-In-First-Out conveyor belt processes. Evidence for differential lability of prey fractions is found. A scheme of granular imbibed prey material being first initially rapidly absorbed (t andfrac12; = 23 min), and also being quickly partly converted to globular material extra-corporeally/extracellularly (t andfrac12; = 36 min) - which then rapidly disappears (t andfrac12; =1.1 h, from a peak around 4 h) is presented. This is then followed by slow intracellular digestion (t andfrac12; = 6.9 h) of the resultant resistant prey residue matching the slow rate of appearance of opaque pre-excretory egestive refractive grains (overall t andfrac12; = 4.5 days). The latter confirmed latent ’catabolic fraction’ (along with Malpighian tubule produced guanine crystals) drives rectal vesicle expansion as ’faeces’ during the later phases of gut emptying/contraction. Catabolic half-lives are of the order of 6.3-7.8 h. Membraneous material is only present in the lumen of the gut in starving mites. No obvious peritrophic membrane was observed. The total feeding cycle time may be slightly over 52.5 h. Full clearance in the gut system of a single meal including egestive and excretory products may take up to 3 weeks. Independent corroborative photographs are included and with posterior predictive densities confirm the physiological sequence of:- ingestion/digestion; egestion; excretion; defecation; together with their timings. Visually dark midguts almost certainly indicate egestive refractive grains (?xanthine) production. Nomograms to diagnose the feeding state of P.longicornis in field samples are presented and show that the timing of these 4 phases in the wild could be inferred by scoring 10-12 mites out of a sample of 20. Suggestions to critically confirm or refute the conclusions are included