Intersection of coral molecular responses to a localized mortality event and ex situ deoxygenation.

In July 2016, East Bank of Flower Garden Banks (FGB) National Marine Sanctuary experienced a localized mortality event (LME) of multiple invertebrate species that ultimately led to reductions in coral cover. Abiotic data taken directly after the event suggested that acute deoxygenation contributed to the mortality. Despite the large impact of this event on the coral community, there was no direct evidence that this LME was driven by acute deoxygenation, and thus we explored whether gene expression responses of corals to the LME would indicate what abiotic factors may have contributed to the LME. Gene expression of affected and unaffected corals sampled during the mortality event revealed evidence of the physiological consequences of the LME on coral hosts and their algal symbionts from two congeneric species (Orbicella franksi and Orbicella faveolata). Affected colonies of both species differentially regulated genes involved in mitochondrial regulation and oxidative stress. To further test the hypothesis that deoxygenation led to the LME, we measured coral host and algal symbiont gene expression in response to ex situ experimental deoxygenation (control = 6.9 ± 0.08 mg L-1, anoxic = 0.083 ± 0.017 mg L-1) in healthy O. faveolata colonies from the FGB. However, this deoxygenation experiment revealed divergent gene expression patterns compared to the corals sampled during the LME and was more similar to a generalized coral environmental stress response. It is therefore likely that while the LME was connected to low oxygen, it was a series of interconnected stressors that elicited the unique gene expression responses observed here. These in situ and ex situ data highlight how field responses to stressors are unique from those in controlled laboratory conditions, and that the complexities of deoxygenation events in the field likely arise from interactions between multiple environmental factors simultaneously.

Associations between DNA methylation and gene regulation depend on chromatin accessibility during transgenerational plasticity.

BACKGROUND: Epigenetic processes are proposed to be a mechanism regulating gene expression during phenotypic plasticity. However, environmentally induced changes in DNA methylation exhibit little-to-no association with differential gene expression in metazoans at a transcriptome-wide level. It remains unexplored whether associations between environmentally induced differential methylation and expression are contingent upon other epigenomic processes such as chromatin accessibility. We quantified methylation and gene expression in larvae of the purple sea urchin Strongylocentrotus purpuratus exposed to different ecologically relevant conditions during gametogenesis (maternal conditioning) and modeled changes in gene expression and splicing resulting from maternal conditioning as functions of differential methylation, incorporating covariates for genomic features and chromatin accessibility. We detected significant interactions between differential methylation, chromatin accessibility, and genic feature type associated with differential expression and splicing. RESULTS: Differential gene body methylation had significantly stronger effects on expression among genes with poorly accessible transcriptional start sites while baseline transcript abundance influenced the direction of this effect. Transcriptional responses to maternal conditioning were 4-13 × more likely when accounting for interactions between methylation and chromatin accessibility, demonstrating that the relationship between differential methylation and gene regulation is partially explained by chromatin state. CONCLUSIONS: DNA methylation likely possesses multiple associations with gene regulation during transgenerational plasticity in S. purpuratus and potentially other metazoans, but its effects are dependent on chromatin accessibility and underlying genic features.

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Genetic variation underlies plastic responses to global change drivers in the purple sea urchin, Strongylocentrotus purpuratus.

Phenotypic plasticity and adaptive evolution enable population persistence in response to global change. However, there are few experiments that test how these processes interact within and across generations, especially in marine species with broad distributions experiencing spatially and temporally variable temperature and pCO2. We employed a quantitative genetics experiment with the purple sea urchin, Strongylocentrotus purpuratus, to decompose family-level variation in transgenerational and developmental plastic responses to ecologically relevant temperature and pCO2. Adults were conditioned to controlled non-upwelling (high temperature, low pCO2) or upwelling (low temperature, high pCO2) conditions. Embryos were reared in either the same conditions as their parents or the crossed environment, and morphological aspects of larval body size were quantified. We find evidence of family-level phenotypic plasticity in response to different developmental environments. Among developmental environments, there was substantial additive genetic variance for one body size metric when larvae developed under upwelling conditions, although this differed based on parental environment. Furthermore, cross-environment correlations indicate significant variance for genotype-by-environment interactive effects. Therefore, genetic variation for plasticity is evident in early stages of S. purpuratus, emphasizing the importance of adaptive evolution and phenotypic plasticity in organismal responses to global change.

The role of gene expression and symbiosis in reef-building coral acquired heat tolerance.

Predicting how reef-building corals will respond to accelerating ocean warming caused by climate change requires knowledge of how acclimation and symbiosis modulate heat tolerance in coral early life-history stages. We assayed transcriptional responses to heat in larvae and juveniles of 11 reproductive crosses of Acropora tenuis colonies along the Great Barrier Reef. Larvae produced from the warmest reef had the highest heat tolerance, although gene expression responses to heat were largely conserved by cross identity. Juvenile transcriptional responses were driven strongly by symbiosis - when in symbiosis with heat-evolved Symbiodiniaceae, hosts displayed intermediate expression between its progenitor Cladocopium and the more stress tolerant Durusdinium, indicating the acquisition of tolerance is a conserved evolutionary process in symbionts. Heat-evolved Symbiodiniaceae facilitated juvenile survival under heat stress, although host transcriptional responses to heat were positively correlated among those hosting different genera of Symbiodiniaceae. These findings reveal the relative contribution of parental environmental history as well as symbiosis establishment in coral molecular responses to heat in early life-history stages.

Energetic and reproductive costs of coral recovery in divergent bleaching responses.

Mass thermal bleaching events are a primary threat to coral reefs, yet the sublethal impacts, particularly on energetics and reproduction, are poorly characterized. Given that the persistence of coral populations is contingent upon the reproduction of individuals that survive disturbances, there is an urgent need to understand the sublethal effects of bleaching on reproductive output to accurately predict coral recovery rates. In 2019, the French Polynesian island of Mo'orea experienced a severe mass bleaching event accompanied by widespread coral mortality. At the most heavily impacted sites, we observed Acropora hyacinthus individuals that were resistant to bleaching, alongside colonies that bleached but showed signs of symbiont recovery shortly after the bleaching event. We collected fragments from A. hyacinthus colonies five months post-bleaching and, using energetic assays and histological measurements, examined the physiological and reproductive consequences of these two distinct heat stress responses. Despite healthy appearances in both resistant and recovered corals, we found that recovered colonies had significantly reduced energy reserves compared to resistant colonies. In addition, we detected compound effects of stress on reproduction: recovered colonies displayed both a lower probability of containing gametes and lower fecundity per polyp. Our results indicate that bleaching inflicts an energetic constraint on the concurrent re-accumulation of energy reserves and development of reproductive material, with decreased reproductive potential of survivors possibly hampering overall reef resilience. These findings highlight the presence of intraspecific responses to bleaching and the importance of considering multiple trajectories for individual species when predicting population recovery following disturbance.

Ocean acidification promotes broad transcriptomic responses in marine metazoans: a literature survey.

For nearly a decade, the metazoan-focused research community has explored the impacts of ocean acidification (OA) on marine animals, noting that changes in ocean chemistry can impact calcification, metabolism, acid-base regulation, stress response and behavior in organisms that hold high ecological and economic value. Because OA interacts with several key physiological processes in marine organisms, transcriptomics has become a widely-used method to characterize whole organism responses on a molecular level as well as inform mechanisms that explain changes in phenotypes observed in response to OA. In the past decade, there has been a notable rise in studies that examine transcriptomic responses to OA in marine metazoans, and here we attempt to summarize key findings across these studies. We find that organisms vary dramatically in their transcriptomic responses to pH although common patterns are often observed, including shifts in acid-base ion regulation, metabolic processes, calcification and stress response mechanisms. We also see a rise in transcriptomic studies examining organismal response to OA in a multi-stressor context, often reporting synergistic effects of OA and temperature. In addition, there is an increase in studies that use transcriptomics to examine the evolutionary potential of organisms to adapt to OA conditions in the future through population and transgenerational experiments. Overall, the literature reveals complex organismal responses to OA, in which some organisms will face more dramatic consequences than others. This will have wide-reaching impacts on ocean communities and ecosystems as a whole.

Effects of thermal stress on amount, composition, and antibacterial properties of coral mucus.

The surface mucus layer of reef-building corals supports feeding, sediment clearing, and protection from pathogenic invaders. As much as half of the fixed carbon supplied by the corals' photosynthetic symbionts is incorporated into expelled mucus. It is therefore reasonable to expect that coral bleaching (disruption of the coral-algal symbiosis) would affect mucus production. Since coral mucus serves as an important nutrient source for the entire reef community, this could have substantial ecosystem-wide consequences. In this study, we examined the effects of heat stress-induced coral bleaching on the composition and antibacterial properties of coral mucus. In a controlled laboratory thermal challenge, stressed corals produced mucus with higher protein (ß = 2.1, p < 0.001) and lipid content (ß = 15.7, p = 0.02) and increased antibacterial activity (likelihood ratio = 100, p < 0.001) relative to clonal controls. These results are likely explained by the expelled symbionts in the mucus of bleached individuals. Our study suggests that coral bleaching could immediately impact the nutrient flux in the coral reef ecosystem via its effect on coral mucus.

Relationship between Acropora millepora juvenile fluorescence and composition of newly established Symbiodinium assemblage.

Coral-dinoflagellate symbiosis is the key biological interaction enabling existence of modern-type coral reefs, but the mechanisms regulating initial host-symbiont attraction, recognition and symbiont proliferation thus far remain largely unclear. A common reef-building coral, Acropora millepora, displays conspicuous fluorescent polymorphism during all phases of its life cycle, due to the differential expression of fluorescent proteins (FPs) of the green fluorescent protein family. In this study, we examine whether fluorescent variation in young coral juveniles exposed to natural sediments is associated with the uptake of disparate Symbiodinium assemblages determined using ITS-2 deep sequencing. We found that Symbiodinium assemblages varied significantly when redness values varied, specifically in regards to abundances of clades A and C. Whether fluorescence was quantified as a categorical or continuous trait, clade A was found at higher abundances in redder juveniles. These preliminary results suggest juvenile fluorescence may be associated with Symbiodinium uptake, potentially acting as either an attractant to ecologically specific types or as a mechanism to modulate the internal light environment to control Symbiodinium physiology within the host.

CRISPR/Cas9-mediated genome editing in a reef-building coral.

Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in ∼50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.

Molecular characterization of larval development from fertilization to metamorphosis in a reef-building coral.

BACKGROUND: Molecular mechanisms underlying coral larval competence, the ability of larvae to respond to settlement cues, determine their dispersal potential and are potential targets of natural selection. Here, we profiled competence, fluorescence and genome-wide gene expression in embryos and larvae of the reef-building coral Acropora millepora daily throughout 12 days post-fertilization. RESULTS: Gene expression associated with competence was positively correlated with transcriptomic response to the natural settlement cue, confirming that mature coral larvae are "primed" for settlement. Rise of competence through development was accompanied by up-regulation of sensory and signal transduction genes such as ion channels, genes involved in neuropeptide signaling, and G-protein coupled receptor (GPCRs). A drug screen targeting components of GPCR signaling pathways confirmed a role in larval settlement behavior and metamorphosis. CONCLUSIONS: These results gives insight into the molecular complexity underlying these transitions and reveals receptors and pathways that, if altered by changing environments, could affect dispersal capabilities of reef-building corals. In addition, this dataset provides a toolkit for asking broad questions about sensory capacity in multicellular animals and the evolution of development.

Red fluorescence in coral larvae is associated with a diapause-like state.

Effective dispersal across environmental gradients is the key to species resilience to environmental perturbation, including climate change. Coral reefs are among the most sensitive ecosystems to global warming, but factors predicting coral dispersal potential remain unknown. In a reef-building coral Acropora millepora, larval fluorescence emerged as a possible indicator of dispersal potential since it correlates with responsiveness to a settlement cue. Here, we show that gene expression in red fluorescent larvae of A. millepora is correlated with diapause-like characteristics highly likely to be associated with extended dispersal. We compared gene expression among three larval fluorescent morphs under three coloured light treatments. While colour morphs did not differ in their gene expression responses to light colour, red larvae demonstrated gene expression signatures of cell cycle arrest and decreased transcription accompanied by elevated ribosome production and heightened defenses against oxidative stress. A meta-analysis revealed that this profile was highly similar to the signatures of elevated thermal tolerance in the same coral species, and moreover, functionally resembled diapause states in an insect and a nematode. Our results support a connection between red fluorescence and long-range dispersal, which offers a new perspective on the molecular underpinnings of coral larval dispersal and the biological function of GFP-like fluorescent proteins.

Differential responses of coral larvae to the colour of ambient light guide them to suitable settlement microhabitat.

Reef-building corals produce planktonic planula larvae that must select an appropriate habitat to settle and spend the rest of their life, a behaviour that plays a critical role in survival. Here, we report that larvae obtained from a deep-water population of Pseudodiploria strigosa settled more readily under blue light and in the dark, which aligns well with the light field characteristics of their natal habitat. By contrast, larvae of the shallow-water coral Acropora millepora settled in high proportions under blue and green light while settlement was less in the dark. Acropora millepora larvae also showed reduced settlement under red light, which should be abundant at shallow depth. Hypothesizing that this might be a mechanism preventing the larvae from settling on the exposed upwards-facing surfaces, we quantified A. millepora settlement in manipulated light chambers in situ on the reef. While A. millepora larvae naturally preferred settling on vertical rather than exposed horizontal surfaces, swapping the colours of upwards-facing and sideways-facing light fields was sufficient to invert this preference. We also tested if the variation in intrinsic red fluorescence in A. millepora larvae correlates with settlement rates, as has been suggested previously. We observed this correlation only in the absence of light, indicating that larval red fluorescent protein is probably not directly involved in light sensing. Our study reveals previously under-appreciated light-sensory capabilities in coral larvae, which could be an important axis of ecological differentiation between coral species and/or populations.

A resource for manipulating gene expression and analyzing cis-regulatory modules in the Drosophila CNS.

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.