High-frequency imagery to capture coral tissue (Montipora capricornis) response to environmental stress, a pilot study.

Environment stress is a major threat to the existence of coral reefs and has generated a lot of interest in the coral research community. Under the environmental stress, corals can experience tissue loss and/or the breakdown of symbiosis between the cnidarian host and its symbiotic algae causing the coral tissue to appear white as the skeleton can be seen by transparency. Image analysis is a common method used to assess tissue response under the environmental stress. However, the traditional approach is limited by the dynamic nature of the coral-algae symbiosis. Here, we observed coral tissue response in the scleractinian coral, Montipora capricornis, using high frequency image analysis throughout the experiment, as opposed to the typical start/end point assessment method. Color analysis reveals that the process can be divided into five stages with two critical stages according to coral tissue morphology and color ratio. We further explore changes to the morphology of individual polyps by means of the Pearson correlation coefficient and recurrence plots, where the quasi-periodic and nonstationary dynamics can be identified. The recurrence quantification analysis also allows the comparison between the different polyps. Our research provides a detailed visual and mathematical analysis of coral tissue response to environmental stress, which potentially shows universal applicability. Moreover, our approach provides a robust quantitative advancement for improving our insight into a suite of biotic responses in the perspective of coral health evaluation and fate prediction.

The trace metal economy of the coral holobiont: supplies, demands and exchanges.

The juxtaposition of highly productive coral reef ecosystems in oligotrophic waters has spurred substantial interest and progress in our understanding of macronutrient uptake, exchange, and recycling among coral holobiont partners (host coral, dinoflagellate endosymbiont, endolithic algae, fungi, viruses, bacterial communities). By contrast, the contribution of trace metals to the physiological performance of the coral holobiont and, in turn, the functional ecology of reef-building corals remains unclear. The coral holobiont's trace metal economy is a network of supply, demand, and exchanges upheld by cross-kingdom symbiotic partnerships. Each partner has unique trace metal requirements that are central to their biochemical functions and the metabolic stability of the holobiont. Organismal homeostasis and the exchanges among partners determine the ability of the coral holobiont to adjust to fluctuating trace metal supplies in heterogeneous reef environments. This review details the requirements for trace metals in core biological processes and describes how metal exchanges among holobiont partners are key to sustaining complex nutritional symbioses in oligotrophic environments. Specifically, we discuss how trace metals contribute to partner compatibility, ability to cope with stress, and thereby to organismal fitness and distribution. Beyond holobiont trace metal cycling, we outline how the dynamic nature of the availability of environmental trace metal supplies can be influenced by a variability of abiotic factors (e.g. temperature, light, pH, etc.). Climate change will have profound consequences on the availability of trace metals and further intensify the myriad stressors that influence coral survival. Lastly, we suggest future research directions necessary for understanding the impacts of trace metals on the coral holobiont symbioses spanning subcellular to organismal levels, which will inform nutrient cycling in coral ecosystems more broadly. Collectively, this cross-scale elucidation of the role of trace metals for the coral holobiont will allow us to improve forecasts of future coral reef function.

Transfer of knowledge from model organisms to evolutionarily distant non-model organisms: The coral Pocillopora damicornis membrane signaling receptome.

With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the extension of the methodological toolbox required to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins in the two species are therefore often in the gray zone, or at least often undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to have conserved some of the functions of the human proteins. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.

Evaluation of fluorescence-based viability stains in cells dissociated from scleractinian coral Pocillopora damicornis.

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. Using Pocillopora damicornis as 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. damicornis cells 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 TiO2 NPs 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.

Applying model approaches in non-model systems: A review and case study on coral cell culture.

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.

Digital image processing to detect subtle motion in stony coral.

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.