Novel in vivo experimental viability assays with high sensitivity and throughput capacity using a bdelloid rotifer

Abstract

Rotifers have been used in biological research as well-characterized models of aging. Their multi-organ characters and their sensitivity for chemicals and environmental changes make them useful as in vivo toxicological and lifespan models. Our aim was to create a bdelloid rotifer model to use in high-throughput viability and non-invasive assays. In order to identify our species Philodina acuticornis odiosa (PA), 18S rDNA-based phylogenetic analysis was carried out and their species-specific morphological markers identified. To execute the rotifer-based experiments, we developed an oil-covered water-drop methodology adapted from human in vitro fertilization techniques. This enables toxicological observations of individual one-housed rotifers in a closed and controllable micro-environment for up to several weeks. Hydrogen peroxide (HO) and sodium azide (NaN) exposures were used as well-understood toxins. The toxicity and survival lifespan (TSL), the bright light disturbance (BLD) the mastax contraction frequency (MCF) and the cellular reduction capacity (CRC), indices were recorded. These newly developed assays were used to test the effects of lethal and sublethal doses of the toxins. The results showed the expected dose-dependent decrease in indices. These four different assays can either be used independently or as an integrated system for studying rotifers. These new indices render the PA invertebrate rotifer model a quantitative system for measuring viability, toxicity and lifespan (with TSL), systemic reaction capacity (with BLD), organic functionality (with MCF) and reductive capability of rotifers (with CRC), in vivo. This novel multi-level system is a reliable, sensitive and replicable screening tool with potential application in pharmaceutical science.

Neurodegeneration-related beta-amyloid as autocatabolism-attenuator in amicro-in vivosystem

“…27689, 50 nM), 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (BisANS, D4162; 50 µM), propidiume-iodide (PI, 81845; 5 µM;), AO (13000; 15 µM) and NR (N-4638; 50 mM) dyes obtained from Sigma-Aldrich, USA. The rotifers were cultured based on the following methods of our previous publications (Olah et al, 2017). For standard food of cultures, we used homogenized baker's yeast (EUstandard granulated instant form, 2-01-420674/001-Z12180/HU) which was heat-inactivated and filtered (Whatman filter with 10 µm pore; 6728-5100).…”

In vivo screening of diterpene alkaloids using bdelloid rotifer assays

“…The purity (≥95%) of the isolated compounds was confirmed by HPLC and 1 H NMR spectroscopy. The exact composition of SM, matrix and food, along with the pattern of feeding and culturing were described at length by Oláh et al [23].…”

“…Rotifers approximately 5 day after hatching (determined by body size; length 220 ± 10 µm and width 60 ± 5 µm), 1-2 days before the beginning of the reproductive stage, were chosen. The selection process followed the harvesting protocols, published in ref [23].…”

“…The methodical protocols have been reported in our previous paper [23], therefore in this manuscript we only intend to give an overview of the applied techniques.…”

“…The impact of the test compounds on the lifespan of unfed PA rotifers was assessed. The morphological viability markers, chosen for evaluation were adapted from Poeggeler et al [24] were extended and defined in our previous work [23].…”