Relationships between phenotypic plasticity and epigenetic variation in two Caribbean Acropora corals.

The plastic ability for a range of phenotypes to be exhibited by the same genotype allows organisms to respond to environmental variation and may modulate fitness in novel environments. Differing capacities for phenotypic plasticity within a population, apparent as genotype by environment interactions (GxE), can therefore have both ecological and evolutionary implications. Epigenetic gene regulation alters gene function in response to environmental cues without changes to the underlying genetic sequence and likely mediates phenotypic variation. DNA methylation is currently the most well described epigenetic mechanism and is related to transcriptional homeostasis in invertebrates. However, evidence quantitatively linking variation in DNA methylation with that of phenotype is lacking in some taxa, including reef-building corals. In this study, spatial and seasonal environmental variation in Bonaire, Caribbean Netherlands was utilized to assess relationships between physiology and DNA methylation profiles within genetic clones across different genotypes of Acropora cervicornis and A. palmata corals. The physiology of both species was highly influenced by environmental variation compared to the effect of genotype. GxE effects on phenotype were only apparent in A. cervicornis. DNA methylation in both species differed between genotypes and seasons and epigenetic variation was significantly related to coral physiological metrics. Furthermore, plastic shifts in physiology across seasons were significantly positively correlated with shifts in DNA methylation profiles in both species. These results highlight the dynamic influence of environmental conditions and genetic constraints on the physiology of two important Caribbean coral species. Additionally, this study provides quantitative support for the role of epigenetic DNA methylation in mediating phenotypic plasticity in invertebrates.

Evolution of Methyltransferase-Like (METTL) Proteins in Metazoa: A Complex Gene Family Involved in Epitranscriptomic Regulation and Other Epigenetic Processes.

The methyltransferase-like (METTL) proteins constitute a family of seven-beta-strand methyltransferases with S-adenosyl methionine-binding domains that modify DNA, RNA, and proteins. Methylation by METTL proteins contributes to the epigenetic, and in the case of RNA modifications, epitranscriptomic regulation of a variety of biological processes. Despite their functional importance, most investigations of the substrates and functions of METTLs within metazoans have been restricted to model vertebrate taxa. In the present work, we explore the evolutionary mechanisms driving the diversification and functional differentiation of 33 individual METTL proteins across Metazoa. Our results show that METTLs are nearly ubiquitous across the animal kingdom, with most having arisen early in metazoan evolution (i.e., occur in basal metazoan phyla). Individual METTL lineages each originated from single independent ancestors, constituting monophyletic clades, which suggests that each METTL was subject to strong selective constraints driving its structural and/or functional specialization. Interestingly, a similar process did not extend to the differentiation of nucleoside-modifying and protein-modifying METTLs (i.e., each METTL type did not form a unique monophyletic clade). The members of these two types of METTLs also exhibited differences in their rates of evolution. Overall, we provide evidence that the long-term evolution of METTL family members was driven by strong purifying selection, which in combination with adaptive selection episodes, led to the functional specialization of individual METTL lineages. This work contributes useful information regarding the evolution of a gene family that fulfills a variety of epigenetic functions, and can have profound influences on molecular processes and phenotypic traits.

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.

Protamines from liverwort are produced by post-translational cleavage and C-terminal di-aminopropanelation of several male germ-specific H1 histones.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.

Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization.

High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance-most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates.

Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus.

Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.

The comparative study of five sex-determining proteins across insects unveils high rates of evolution at basal components of the sex determination cascade.

In insects, the sex determination cascade is composed of genes that interact with each other in a strict hierarchical manner, constituting a coadapted gene complex built in reverse order from bottom to top. Accordingly, ancient elements at the bottom are expected to remain conserved ensuring the correct functionality of the cascade. In the present work, we have studied the levels of variation displayed by five key components of the sex determination cascade across 59 insect species, including Sex-lethal, transformer, transformer-2, fruitless, doublesex, and sister-of-Sex-lethal (a paralog of Sxl encompassing sex-independent functions). Surprisingly, our results reveal that basal components of the cascade (doublesex, fruitless) seem to evolve more rapidly than previously suspected. Indeed, in the case of Drosophila, these proteins evolve more rapidly than the master regulator Sex-lethal. These results agree with the notion suggesting that genes involved in early aspects of development will be more constrained due to the large deleterious pleiotropic effects of mutations, resulting in increased levels of purifying selection at top positions of the cascade. The analyses of the selective episodes involved in the recruitment of Sxl into sex-determining functions further support this idea, suggesting the presence of bursts of adaptive selection in the common ancestor of drosophilids, followed by the onset of purifying selection preserving the master regulatory role of this protein on top of the Drosophila sex determination cascade. Altogether, these results underscore the importance of the position of sex determining genes in the cascade, constituting a major constraint shaping the molecular evolution of the insect sex determination pathway.

In Vitro Analysis of Early Genotoxic and Cytotoxic Effects of Okadaic Acid in Different Cell Types of the Mussel Mytilus galloprovincialis.

Okadaic acid (OA) is the predominant biotoxin responsible for diarrhetic shellfish poisoning (DSP) syndrome in humans. While its harmful effects have been extensively studied in mammalian cell lines, the impact on marine organisms routinely exposed to OA is still not fully known. Few investigations available on bivalve molluscs suggest less genotoxic and cytotoxic effects of OA at high concentrations during long exposure times. In contrast, no apparent information is available on how sublethal concentrations of OA affect these organisms over short exposure times. In order to fill this gap, this study addressed for the first time in vitro analysis of early genotoxic and cytotoxic effects attributed to OA in two cell types of the mussel Mytilus galloprovincialis. Accordingly, hemocytes and gill cells were exposed to low OA concentrations (10, 50, 100, 200, or 500 nM) for short periods of time (1 or 2 h). The resulting DNA damage, as apoptosis and necrosis, was subsequently quantified using the comet assay and flow cytometry, respectively. Data demonstrated that (1) mussel hemocytes seem to display a resistance mechanism against early genotoxic and cytotoxic OA-induced effects, (2) mussel gill cells display higher sensitivity to early OA-mediated genotoxicity than hemocytes, and (3) mussel gill cells constitute more suitable systems to evaluate the genotoxic effect of low OA concentrations in short exposure studies. Taken together, this investigation provides evidence supporting the more reliable suitability of mussel gill cells compared to hemocytes to evaluate the genotoxic effect of low short-duration exposure to OA.

Ecological forensic testing: Using multiple primers for eDNA detection of marine vertebrates in an estuarine lagoon subject to anthropogenic influences.

Many critical aquatic habitats are in close proximity to human activity (i.e., adjacent to residences, docks, marinas, etc.), and it is vital to monitor biodiversity in these and similar areas that are subject to ongoing urbanization, pollution, and other environmental disruptions. Environmental DNA (eDNA) metabarcoding is an accessible, non-invasive genetic technique used to detect and monitor species diversity and is a particularly useful approach in areas where traditional biodiversity monitoring methods (e.g., visual surveys or video surveillance) are challenging to conduct. In this study, we implemented an eDNA approach that used a combination of three distinct PCR primer sets to detect marine vertebrates within a canal system of Biscayne Bay, Florida, an ecosystem representative of challenging sampling conditions and a myriad of impacts from urbanization. We detected fish species from aquarium, commercial, and recreational fisheries, as well as invasive, cryptobenthic, and endangered vertebrate species, including charismatic marine mammals such as the protected West Indian manatee, Trichechus manatus. Our results support the potential for eDNA analyses to supplement traditional biodiversity monitoring methods and ultimately serve as an important tool for ecosystem management. This approach minimizes stress or disturbance to organisms and removes the intrinsic risk and logical limitations of SCUBA diving, snorkeling, or deploying sensitive equipment in areas that are subject to high vessel traffic and/or low visibility. Overall, this work sets the framework to understand how biodiversity may change over different spatial and temporal scales in an aquatic ecosystem heavily influenced by urbanization and validates the use of eDNA as a complementary approach to traditional ecological monitoring methods.

Vertebrate nucleoplasmin and NASP: egg histone storage proteins with multiple chaperone activities.

Recent reviews have focused on the structure and function of histone chaperones involved in different aspects of somatic cell chromatin metabolism. One of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by egg histone storage chaperones. These include members of the nucleoplasmin (NPM2/NPM3), which are preferentially associated with histones H2A-H2B in the egg and the nuclear autoantigenic sperm protein (NASP) families. Interestingly, in addition to binding and providing storage to H3/H4 in the egg and in somatic cells, NASP has been shown to be a unique genuine chaperone for histone H1. This review revolves around the structural and functional roles of these two families of chaperones whose activity is modulated by their own post-translational modifications (PTMs), particularly phosphorylation. Beyond their important role in the remodeling of paternal chromatin in the early stages of embryogenesis, NPM and NASP members can interact with a plethora of proteins in addition to histones in somatic cells and play a critical role in processes of functional cell alteration, such as in cancer. Despite their common presence in the egg, these two histone chaperones appear to be evolutionarily unrelated. In contrast to members of the NPM family, which share a common monophyletic evolutionary origin, the different types of NASP appear to have evolved recurrently within different taxa.

Okadaic acid meet and greet: an insight into detection methods, response strategies and genotoxic effects in marine invertebrates.

Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation.

Bivalve omics: state of the art and potential applications for the biomonitoring of harmful marine compounds.

The extraordinary progress experienced by sequencing technologies and bioinformatics has made the development of omic studies virtually ubiquitous in all fields of life sciences nowadays. However, scientific attention has been quite unevenly distributed throughout the different branches of the tree of life, leaving molluscs, one of the most diverse animal groups, relatively unexplored and without representation within the narrow collection of well established model organisms. Within this Phylum, bivalve molluscs play a fundamental role in the functioning of the marine ecosystem, constitute very valuable commercial resources in aquaculture, and have been widely used as sentinel organisms in the biomonitoring of marine pollution. Yet, it has only been very recently that this complex group of organisms became a preferential subject for omic studies, posing new challenges for their integrative characterization. The present contribution aims to give a detailed insight into the state of the art of the omic studies and functional information analysis of bivalve molluscs, providing a timely perspective on the available data resources and on the current and prospective applications for the biomonitoring of harmful marine compounds.

The CHROMEVALOA database: a resource for the evaluation of Okadaic Acid contamination in the marine environment based on the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis.

Okadaic Acid (OA) constitutes the main active principle in Diarrhetic Shellfish Poisoning (DSP) toxins produced during Harmful Algal Blooms (HABs), representing a serious threat for human consumers of edible shellfish. Furthermore, OA conveys critical deleterious effects for marine organisms due to its genotoxic potential. Many efforts have been dedicated to OA biomonitoring during the last three decades. However, it is only now with the current availability of detailed molecular information on DNA organization and the mechanisms involved in the maintenance of genome integrity, that a new arena starts opening up for the study of OA contamination. In the present work we address the links between OA genotoxicity and chromatin by combining Next Generation Sequencing (NGS) technologies and bioinformatics. To this end, we introduce CHROMEVALOAdb, a public database containing the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis (a sentinel model organism) in response to OA exposure. This resource constitutes a leap forward for the development of chromatin-based biomarkers, paving the road towards the generation of powerful and sensitive tests for the detection and evaluation of the genotoxic effects of OA in coastal areas.

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis.

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.

Age group DNA methylation differences in lemon sharks (Negaprion brevirostris): Implications for future age estimation tools.

Age information is often non-existent for most shark populations due to a lack of measurable physiological and morphological traits that can be used to estimate age. Recently, epigenetic clocks have been found to accurately estimate age for mammals, birds, and fish. However, since these clocks rely, among other things, on the availability of reference genomes, their application is hampered in non-traditional model organisms lacking such molecular resources. The technique known as Methyl-Sensitive Amplified Polymorphism (MSAP) has emerged as a valid alternative for studying DNA methylation biomarkers when reference genome information is missing, and large numbers of samples need to be processed. Accordingly, the MSAP technique was used in the present study to characterize global DNA methylation patterns in lemon sharks from three different age groups (juveniles, subadults, and adults). The obtained results reveal that, while MSAP analyses lack enough resolution as a standalone approach to infer age in these organisms, the global DNA methylation patterns observed using this technique displayed significant differences between age groups. Overall, these results confer that DNA methylation does change with age in sharks like what has been seen for other vertebrates and that MSAP could be useful as part of an epigenetics pipeline to infer the broad range of ages found in large samples sizes.

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases.

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution-whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)-using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance.

Birth-and-death long-term evolution promotes histone H2B variant diversification in the male germinal cell line.

The rich diversity within each of the five histone families (H1, H2A, H2B, H3, and H4) can hardly be reconciled with the notion of homogenizing evolution. The prevalence of birth-and-death long-term evolution over concerted evolution has already been demonstrated in the linker histone H1 family as well as for the H2A, H3, and H4 core histone families. However, information about histone H2B is lacking. In the present work, we have analyzed the diversity of the members of this histone family across different eukaryotic genomes and have characterized the mechanisms involved in their long-term evolution. Our results reveal that, quite in contrast with other histones, H2B variants are subject to a very rapid process of diversification that primarily affects the male germinal cell lineage and involves their functional specialization probably as a consequence of neofunctionalization and subfunctionalization events after gene duplication. The overall parallelism observed between the molecular phylogenies and the relationships among the electrostatic potentials of the different variants suggests that the latter may have played a major structural selective constraint during H2B evolution. It thus seems that the reorganization of chromatin structure during spermiogenesis might have affected the evolutionary constraints driving histone H2B evolution, leading to an increase in diversity.

Origin and evolution of chromosomal sperm proteins.

In the eukaryotic cell, DNA compaction is achieved through its interaction with histones, constituting a nucleoprotein complex called chromatin. During metazoan evolution, the different structural and functional constraints imposed on the somatic and germinal cell lines led to a unique process of specialization of the sperm nuclear basic proteins (SNBPs) associated with chromatin in male germ cells. SNBPs encompass a heterogeneous group of proteins which, since their discovery in the nineteenth century, have been studied extensively in different organisms. However, the origin and controversial mechanisms driving the evolution of this group of proteins has only recently started to be understood. Here, we analyze in detail the histone hypothesis for the vertical parallel evolution of SNBPs, involving a "vertical" transition from a histone to a protamine-like and finally protamine types (H --> PL --> P), the last one of which is present in the sperm of organisms at the uppermost tips of the phylogenetic tree. In particular, the common ancestry shared by the protamine-like (PL)- and protamine (P)-types with histone H1 is discussed within the context of the diverse structural and functional constraints acting upon these proteins during bilaterian evolution.

Coral environmental memory: causes, mechanisms, and consequences for future reefs.

The apparent ability of corals to acquire and maintain enhanced stress tolerance through a dose-dependent environmental memory, which may persist for multiple years, has critical implications for coral reef conservation research. Such responses are variable across coral species and environmental stressors, with primed corals exhibiting a modified response to secondary stress exposures. While the mechanisms underlying coral memory responses are poorly understood, they likely involve both the coral host and microbiome. With advances in molecular technologies, it is now possible to investigate potential memory mechanisms in non-model organisms, including transcriptional regulation through epigenetic modifications. We integrate evidence of coral environmental memory and suggest future research directions to evaluate the potential for this process to enhance coral resilience under climate change.