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The breakdown of symbiotic mutualism between cnidarian hosts and dinoflagellate algae partners (i.e., bleaching) has been linked to an immune-like response pathway brought on by a nitro-oxidative burst, a symptom of thermal stress. Stress induced by reactive oxygen species (ROS)/reactive nitrogen species is a problem common to aerobic systems. In this study, we tested the antioxidant effects of engineered poly(acrylic acid)-coated cerium dioxide nanoparticles (CeO 2 , nanoceria) on free-living Symbiodiniaceae ( Breviolum minutum ), a dinoflagellate alga that forms symbiotic relationships with reef-building corals and anemones. Results show that poly(acrylic acid)-coated CeO 2 with hydrodynamic diameters of ~4 nm are internalized by B. minutum in under 30 min and subsequently localized in the cytosol. Nanoceria exposure does not inhibit cell growth over time, with the treated cultures showing a similar growth trend over the 25-day exposure. Aerobic activity and thermal stress when held at 34°C for 1 h (+6°C above control) led to increased intracellular ROS concentration with time. A clear ROS scavenging effect of the nanoceria was observed, with a 5-fold decrease in intracellular ROS levels during thermal stress. The nitric oxide (NO) concentration decreased by ~17% with thermal stress, suggesting the rapid involvement of NO scavenging enzymes or proteins within 1 h of stress onset. The presence of nanoceria did not appear to influence NO concentration. Furthermore, aposymbiotic anemones ( Exaiptasia diaphana , ex Aiptasia pallida ) were successfully infected with nanoceria-loaded B. minutum , demonstrating that inoculation could serve as a delivery method. The ability of nanoceria to be taken up by Symbiodiniaceae and reduce ROS production could be leveraged as a potential mitigation strategy to reduce coral bleaching. 

The application of established cell viability assays such as the commonly used trypan blue staining method to coral cells is not straightforward due to different culture parameters and different cellular features specific to mammalian cells compared to marine invertebrates. UsingPocillopora damicornisas a model, we characterized the autofluorescence and tested different fluorescent dye pair combinations to identify alternative viability indicators. The cytotoxicity of different representative molecules, namely small organic molecules, proteins and nanoparticles (NP), was measured after 24 h of exposure using the fluorescent dye pair Hoechst 33342 and SYTOX orange. Our results show that this dye pair can be distinctly measured in the presence of fluorescent proteins plus chlorophyll.P. damicorniscells exposed for 24 h to Triton-X100, insulin or titanium dioxide (TiO₂) NPs, respectively, at concentrations ranging from 0.5 to 100 µg/mL, revealed a LC50 of 0.46 µg/mL for Triton-X100, 6.21 µg/mL for TiO₂NPs and 33.9 µg/mL for insulin. This work presents the approach used to customize dye pairs for membrane integrity-based cell viability assays considering the species- and genotype-specific autofluorescence of scleractinian corals, namely: endogenous fluorescence characterization followed by the selection of dyes that do not overlap with endogenous signals.

 

Abstract Coral reef ecosystems support significant biological activities and harbor huge diversity, but they are facing a severe crisis driven by anthropogenic activities and climate change. An important behavioral trait of the coral holobiont is coral motion, which may play an essential role in feeding, competition, reproduction, and thus survival and fitness. Therefore, characterizing coral behavior through motion analysis will aid our understanding of basic biological and physical coral functions. However, tissue motion in the stony scleractinian corals that contribute most to coral reef construction are subtle and may be imperceptible to both the human eye and commonly used imaging techniques. Here we propose and apply a systematic approach to quantify and visualize subtle coral motion across a series of light and dark cycles in the scleractinian coral Montipora capricornis . We use digital image correlation and optical flow techniques to quantify and characterize minute coral motions under different light conditions. In addition, as a visualization tool, motion magnification algorithm magnifies coral motions in different frequencies, which explicitly displays the distinctive dynamic modes of coral movement. Specifically, our assessment of displacement, strain, optical flow, and mode shape quantify coral motion under different light conditions, and they all show that M. capricornis exhibits more active motions at night compared to day. Our approach provides an unprecedented insight into micro-scale coral movement and behavior through macro-scale digital imaging, thus offering a useful empirical toolset for the coral research community. 

Model systems approaches search for commonality in patterns underlying biological diversity and complexity led by common evolutionary paths. The success of the approach does not rest on the species chosen but on the scalability of the model and methods used to develop the model and engage research. Fine-tuning approaches to improve coral cell cultures will provide a robust platform for studying symbiosis breakdown, the calcification mechanism and its disruption, protein interactions, micronutrient transport/exchange, and the toxicity of nanoparticles, among other key biological aspects, with the added advantage of minimizing the ethical conundrum of repeated testing on ecologically threatened organisms. The work presented here aimed to lay the foundation towards development of effective methods to sort and culture reef-building coral cells with the ultimate goal of obtaining immortal cell lines for the study of bleaching, disease and toxicity at the cellular and polyp levels. To achieve this objective, the team conducted a thorough review and tested the available methods (i.e. cell dissociation, isolation, sorting, attachment and proliferation). The most effective and reproducible techniques were combined to consolidate culture methods and generate uncontaminated coral cell cultures for ~7 days (10 days maximum). The tests were conducted on scleractinian corals Pocillopora acuta of the same genotype to harmonize results and reduce variation linked to genetic diversity. The development of cell separation and identification methods in conjunction with further investigations into coral cell-type specific metabolic requirements will allow us to tailor growth media for optimized monocultures as a tool for studying essential reef-building coral traits such as symbiosis, wound healing and calcification at multiple scales.