Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

Symbiont shuffling induces differential DNA methylation responses to thermal stress in the coral Montastraea cavernosa.

Algal symbiont shuffling in favour of more thermotolerant species has been shown to enhance coral resistance to heat-stress. Yet, the mechanistic underpinnings and long-term implications of these changes are poorly understood. This work studied the modifications in coral DNA methylation, an epigenetic mechanism involved in coral acclimatization, in response to symbiont manipulation and subsequent heat stress exposure. Symbiont composition was manipulated in the great star coral Montastraea cavernosa through controlled thermal bleaching and recovery, producing paired ramets of three genets dominated by either their native symbionts (genus Cladocopium) or the thermotolerant species (Durusdinium trenchi). Single-base genome-wide analyses showed significant modifications in DNA methylation concentrated in intergenic regions, introns and transposable elements. Remarkably, DNA methylation changes in response to heat stress were dependent on the dominant symbiont, with twice as many differentially methylated regions found in heat-stressed corals hosting different symbionts (Cladocopium vs. D. trenchii) compared to all other comparisons. Interestingly, while differential gene body methylation was not correlated with gene expression, an enrichment in differentially methylated regions was evident in repetitive genome regions. Overall, these results suggest that changes in algal symbionts favouring heat tolerant associations are accompanied by changes in DNA methylation in the coral host. The implications of these results for coral adaptation, along with future avenues of research based on current knowledge gaps, are discussed in the present work.

Application of an Improved Chloroform-Free Lipid Extraction Method to Staghorn Coral (Acropora cervicornis) Lipidomics Assessments.

Lipids are excellent biomarkers for assessing coral stress, although staghorn coral data (Acropora cervicornis) is lacking. Lipid extraction is the most critical step in lipidomic assessments, usually performed using carcinogenic solvents. Efficient alternative using less toxic methods, such as the BUME method using butanol and methanol as extraction solvents, have not been applied to coral lipidomics evaluations. Thus, we aimed to develop a lipidomic approach to identify important coral health biomarkers by comparing different solvent mixtures in staghorn corals. Total lipid extraction was equivalent for both tested methods, but due to its efficiency in extracting polar lipids, the BUME method was chosen. It was then applied to different coral masses (0.33-1.00 g), resulting in non-significant differences concerning number of lipid classes and compounds. Therefore, this method can be successfully applied to coral assessments in a climate change context, with the added benefit of low sample masses, lessening coral sampling impacts.

Coral epigenetic responses to nutrient stress: Histone H2A.X phosphorylation dynamics and DNA methylation in the staghorn coral Acropora cervicornis.

Nutrient pollution and thermal stress constitute two of the main drivers of global change in the coastal oceans. While different studies have addressed the physiological effects and ecological consequences of these stressors in corals, the role of acquired modifications in the coral epigenome during acclimatory and adaptive responses remains unknown. The present work aims to address that gap by monitoring two types of epigenetic mechanisms, namely histone modifications and DNA methylation, during a 7-week-long experiment in which staghorn coral fragments (Acropora cervicornis) were exposed to nutrient stress (nitrogen, nitrogen + phosphorus) in the presence of thermal stress. The major conclusion of this experiment can be summarized by two main results: First, coral holobiont responses to the combined effects of nutrient enrichment and thermal stress involve the post-translational phosphorylation of the histone variant H2A.X (involved in responses to DNA damage), as well as nonsignificant modifications in DNA methylation trends. Second, the reduction in H2A.X phosphorylation (and the subsequent potential impairment of DNA repair mechanisms) observed after prolonged coral exposure to nitrogen enrichment and thermal stress is consistent with the symbiont-driven phosphorus limitation previously observed in corals subject to nitrogen enrichment. The alteration of this epigenetic mechanism could help to explain the synergistic effects of nutrient imbalance and thermal stress on coral fitness (i.e., increased bleaching and mortality) while supporting the positive effect of phosphorus addition to improving coral resilience to thermal stress. Overall, this work provides new insights into the role of epigenetic mechanisms during coral responses to global change, discussing future research directions and the potential benefits for improving restoration, management and conservation of coral reef ecosystems worldwide.