Understanding the response of the coral holobiont to environmental change is crucial to inform conservation efforts. The most pressing problem is “coral bleaching,” usually precipitated by prolonged thermal stress. We used untargeted, polar metabolite profiling to investigate the physiological response of the coral species Montipora capitata and Pocillopora acuta to heat stress. Our goal was to identify diagnostic markers present early in the bleaching response. From the untargeted UHPLC-MS data, a variety of co-regulated dipeptides were found that have the highest differential accumulation in both species. The structures of four dipeptides were determined and showed differential accumulation in symbiotic and aposymbiotic (alga-free) populations of the sea anemone Aiptasia (Exaiptasia pallida), suggesting the deep evolutionary origins of these dipeptides and their involvement in symbiosis. These and other metabolites may be used as diagnostic markers for thermal stress in wild coral.

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Background Corals, which form the foundation of biodiverse reef ecosystems, are under threat from warming oceans. Reefs provide essential ecological services, including food, income from tourism, nutrient cycling, waste removal, and the absorption of wave energy to mitigate erosion. Here, we studied the coral thermal stress response using network methods to analyze transcriptomic and polar metabolomic data generated from the Hawaiian rice coral Montipora capitata. Coral nubbins were exposed to ambient or thermal stress conditions over a 5-week period, coinciding with a mass spawning event of this species. The major goal of our study was to expand the inventory of thermal stress-related genes and metabolites present in M. capitata and to study gene-metabolite interactions. These interactions provide the foundation for functional or genetic analysis of key coral genes as well as provide potentially diagnostic markers of pre-bleaching stress. A secondary goal of our study was to analyze the accumulation of sex hormones prior to and during mass spawning to understand how thermal stress may impact reproductive success in M. capitata. Methods M. capitata was exposed to thermal stress during its spawning cycle over the course of 5 weeks, during which time transcriptomic and polar metabolomic data were collected. We analyzed these data streams individually, and then integrated both data sets using MAGI (Metabolite Annotation and Gene Integration) to investigate molecular transitions and biochemical reactions. Results Our results reveal the complexity of the thermal stress phenome in M. capitata, which includes many genes involved in redox regulation, biomineralization, and reproduction. The size and number of modules in the gene co-expression networks expanded from the initial stress response to the onset of bleaching. The later stages involved the suppression of metabolite transport by the coral host, including a variety of sodium-coupled transporters and a putative ammonium transporter, possibly as a response to reduction in algal productivity. The gene-metabolite integration data suggest that thermal treatment results in the activation of animal redox stress pathways involved in quenching molecular oxygen to prevent an overabundance of reactive oxygen species. Lastly, evidence that thermal stress affects reproductive activity was provided by the downregulation of CYP-like genes and the irregular production of sex hormones during the mass spawning cycle. Overall, redox regulation and metabolite transport are key components of the coral animal thermal stress phenome. Mass spawning was highly attenuated under thermal stress, suggesting that global climate change may negatively impact reproductive behavior in this species.

Spatial genetic structure (SGS) is important to a population's ability to adapt to environmental change. For species that reproduce both sexually and asexually, the relative contribution of each reproductive mode has important ecological and evolutionary implications because asexual reproduction can have a strong effect on SGS. Reef building corals reproduce sexually, but many species also propagate asexually under certain conditions. In order to understand SGS and the relative importance of reproductive mode across environmental gradients, we evaluated genetic relatedness in almost 600 colonies of Montipora capitata across 30 environmentally characterized sites in Kaneohe Bay, Oahu, Hawaii using low-depth restriction digest associated sequencing. Clonal colonies were relatively rare overall but influenced SGS. Clones were located significantly closer to one another spatially than average colonies and were more frequent on sites where wave energy was relatively high, suggesting a strong role of mechanical breakage in their formation. Excluding clones, we found no evidence of isolation by distance within sites or across the bay. Several environmental characteristics were significant predictors of the underlying genetic variation (including degree heating weeks, time spent above 30°C, depth, sedimentation rate and wave height); however, they only explained 5% of this genetic variation. Our results show that colony fragmentation contributes to the ecology of M. capitata at local scales and that genetic diversity is maintained despite strong environmental gradients in a highly impacted ecosystem, suggesting potential for broad adaptation or acclimatization in this population.

23Coral reef systems are under global threat due to warming and acidifying oceans 1 . 24Understanding the response of the coral holobiont to environmental change is crucial to aid 25 conservation efforts. The most pressing problem is "coral bleaching", usually precipitated 26 by prolonged thermal stress that disrupts the algal symbiosis sustaining the holobiont 2,3 . We 27 used metabolomics to understand how the coral holobiont metabolome responds to heat 28 stress with the goal of identifying diagnostic markers prior to bleaching onset. We studied 29 the heat tolerant Montipora capitata and heat sensitive Pocillopora acuta coral species from 30 the Hawaiian reef system in Kāne'ohe Bay, O'ahu. Untargeted LC-MS analysis uncovered 31 both known and novel metabolites that accumulate during heat stress. Among those showing 32 the highest differential accumulation were a variety of co-regulated dipeptides present in 33 both species. The structures of four of these compounds were determined (Arginine-34 Glutamine, Lysine-Glutamine, Arginine-Valine, and Arginine-Alanine). These dipeptides 35 also showed differential accumulation in symbiotic and aposymbiotic (alga free) individuals 36 of the sea anemone model Aiptasia 4 , suggesting their animal provenance and algal symbiont 37 related function. Our results identify a suite of metabolites associated with thermal stress 38 that can be used to diagnose coral health in wild samples. 39 40 The exchange of metabolites, either between organism-environment or organism-organism, gave 41 rise not only to early life, but complex life systems on Earth. Examples include bacterial deep vent 42communities 5 , the plant rhizosphere 6 , the human microbiome 7 , and the coral holobiont 8 . Exchange 43 of metabolites between stony corals (Scleractinia), their dinoflagellate algal photosymbionts 44 (Symbiodiniaceae), and associated microbes is the foundation for modern coral reef ecosystems 1,9 45 that cover ca. 255,000 km 2 of the planet surface 10 . Under ambient conditions, the algal cells 46 provide 90-95% of host energy needs in the form of lipids, carbohydrates, amino acids, and O2 11 . 47