Mammalian cell-based assays are poorly adapted to in vitro work with scleractinian coral cells. This is in part due to the diversity and abundance of endogenous fluorescence present in reef-building corals but also due to factors such as the lack of cell attachment, the salinity (~ 35‰) or strong ionic (~ 0.7 M) nature of seawater, and other unknowns. Indeed, as our knowledge of coral cell physiology and function grows, so will the diversity of coral-specific assays. Coral cell viability assessment is key to developing other assays as cell viability is one of the most straightforward endpoints of in vitro research. In this study we developed a framework for coral scientists to tailor fluorescence-based membrane integrity assays to the coral species phenotype of their choice. This method first involves accurately determining the different fluorescent signals emitted by the coral species genotype and finding fluorescent dyes that do not overlap or that can easily be deconvoluted. The membrane integrity-based dye pair Hoechst 33342-SYTOX® Orange, avoids the endogenous fluorescent signals of P. damicornis cells and allows us to test the toxicity of TiO2 and insulin in vitro applied to cells dissociated for scleractinian coral P. damicornis (green phenotype) for the first time. Two viability assays common in mammalian research, the MTS and LDH assays, were also tested yielding unsatisfactory results. The poor reaction observed with the MTS assay can potentially be explained by the fact that, for the reduction of tetrazolium salt to occur as a function of cellular activity, reductase need to be transported across the plasma membrane into the culture medium which is not common to all organisms17. The inconsistencies in the LDH assay results on the other hand, might be dur to the presence of other dehydrogenases, such as opine dehydrogenases which are functionally analogous to lactate dehydrogenase, being used to regenerate NAD+ in many invertebrates18. Montipora capitata, another scleractinian coral, showed strombine dehydrogenase and alanopine dehydrogenase activity but no LDH activity18.
One of the complications associated with in vitro coral research is limited cell attachment. Compared to adherent mammalian cell lines which attach to the substrate, coral cells have shown limited attachment to standard culture flasks and plates3. Here, we worked with mixed, unsorted cells; therefore, we increased cell attachment by coating the well plates with poly-D-lysine to reduce cell loss during aspiration. Without poly-D-lysine, cell counts dropped drastically and reached levels below critical mass. Poly-D-lysine had the side effect of trapping the TiO2 nanoparticles which resulted in the persistence of TiO2 agglomerates after exposure medium washing, which is visibly present in the brightfield images. The entrapment of TiO2 particles could have reduced their interactions with P. damicornis cells and affected the measured cytotoxicity. TiO2 nanoparticles have strong UV photoactive properties which have led to their increased use in paints, solar cells, and sunscreens and consequently their unintentional release in the environmentt19. The toxicity of TiO2 nanoparticle exposure in various freshwater (reviewed in 20) and marine organisms (reviewed in 21) has been tested, and the toxicity has been shown to increase with UV exposure due to photoactivation20,21. It is important to note that UV conditions are not always reported making it challenging to determine UV-enhanced cytotoxicity. The light requirements of reef-building corals intrinsically involve UV exposure, and we applied a 10 hour light / 14 hour dark cycle (PAR 40 ± 2, i.e. ~ 8.71 W/m2 = 0.0871 mJ/cm2, see Materials and Methods for more details) throughout our experimentation. TiO2 has been shown to damage symbiotic dinoflagellates and induce bleaching in Acropora spp. corals22 and Montastraea faveolata23, and also to increase mortality, abnormal movements and abnormal feeding behaviors in clown fish24, and reduce marine phytoplankton growth25. In both Acropora and Montastraea, slight bleaching occurred after exposure to 6.3 mg/L TiO2 for up to 48 hours and 10 mg/L TiO2 for 17 days. Our results corroborate these findings with a reduction in cell viability starting at 10 mg/L TiO2 with LC50 at 20 mg/L under a UV intensity five times lower than that experiences in the environment. Environmental concentration of TiO2 are estimated to range between 0.02–0.9 mg/L of TiO2 per day based on a study performed along three beaches in the south of France26. These concentrations are orders of magnitude less than the LC50 found here; however, studies to date including ours represent only short term exposures.
The symbiotic mutualism between coral host and dinoflagellate endosymbionts fulfills up to 90% of the holobionts energetic needs. This energy trafficking suggests a transport and signaling system where a molecule such as insulin could come into play. Furthermore, as bleaching involves the breakdown of symbiosis, this transport and signaling system can be disrupted, presumably with similarities to the diabetic response in vertebrates. To enable future investigation of this hypothesis, we here investigated insulin cytotoxicity as an example for proteotoxicity. Insulin has been a model system for the study of proteotoxicity in protein evolution27, and different conformations and oligomerization states of insulin are relevant in the etiology and treatment of diabetes28,29. There are numerous reports of preproinsulin-like psudogenes in a variety of different organisms including insects, invertebrates, plants and microbial eukaryotes and prokaryotes30. Remarkably, human insulin has been shown to have physiological effects on other organisms, such as Acanthamoeba castellanii31, suggesting a conservation of structure and function across long evolutionary distances. Thus, the natural first step in evaluation the effects of insulin on P. damicornis is its potential cytotoxicity. Our findings suggest that insulin reduces P. damicornis cell viability (~ 20% decrease) at concentrations between 10 and 100 µg/mL. Insulin cytotoxicity is known to depend on different solvent properties, such as increased temperature and high concentrations of salts32,33, leading to insulin aggregates and misfolding. The salinity or the ionic strength of seawater could have had similar effects despite the relatively low temperature (25°C) and basic nature of the culture medium. Thus, it is possible that seawater might be modifying insulin conformation and cytotoxic behavior, and the present work lays the foundation for further research related to the effects of insulin on corals.
Regardless of organism studied, the type of cell death mechanism is highly dependent on the nature and duration of the stress applied and the ability of cells to maintain homeostasis. The positive control, Triton X-100, is a common surfactant used to lyse cells. It is cytotoxic to a number of cell types: ciliated protozoan, fish and mammalian34. Using it as a positive control for membrane integrity assays involves the understanding that cell count will decrease with increasing Triton X-100 concentration. Different mechanisms of cell death can be involved when coral cells are exposed to substances such as insulin and TiO2 NPs tested here. Therefore, the Triton X-100 serves as a positive control verifying that the assay worked and additional research is needed to identify the mechanisms of cell death. For example, in mammalian cells, the absence of caspase activation, cytochrome c release, DNA fragmentation, membrane damage and changes in cell morphology are all cell parameters that can be used to discriminate cell necrosis from apoptosis35 and other types of cell death. This distinction is also particularly relevant in the study of dysbiosis. The breakdown of symbiosis between endosymbiotic dinoflagellate algae and coral host is still not well understood despite the urgent need to characterize coral bleaching at the cellular level. Stable cultures of the endosymbiont-holding gastrodermal cells, combined with cytotoxicity and cell death mechanism assays could help better define the mechanisms of coral-dinoflagellate dysbiosis.