Without mechanisms, theories and models in insect epigenetics remain a black box.

Insect epigenetics must confront the remarkable diversity of epigenomic systems in various lineages and use mechanistic approaches to move beyond vague functional explanations based on predictions and inferences. To accelerate progress, what is required now is a convergence of genomic data with biochemical and single-cell-type analyses in selected species representing contrasting evolutionary solutions in epigenetics.

Reminiscences on the honeybee genome project and the rise of epigenetic concepts in insect science.

The sequencing of the honeybee genome in 2006 was an important technological and logistic achievement experience. But what benefits have flown from the honeybee genome project? What does the annotated genomic assembly mean for the study of behavioural complexity and organismal function in honeybees? Here, I discuss several lines of research that have arisen from this project and highlight the rapidly expanding studies on insect epigenomics, emergent properties of royal jelly, the mechanism of nutritional control of development and the contribution of epigenomic regulation to the evolution of sociality. I also argue that the term 'insect epigenetics' needs to be carefully redefined to reflect the diversity of epigenomic toolkits in insects and the impact of lineage-specific innovations on organismal outcomes. The honeybee genome project helped pioneer advances in social insect molecular biology, and fuelled breakthrough research into the role of flexible epigenomic control systems in linking genotype to phenotype.

Transcriptome-based insights into gene networks controlling myopia prevention.

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.

DNA Methylation in Honey Bees and the Unresolved Questions in Insect Methylomics.

DNA methylation has been found in most invertebrate lineages except for Diptera, Placozoa and the majority of Nematoda. In contrast to the mammalian methylation toolkit that consists of one DNMT1 and several DNMT3s, some of which are catalytically inactive accessory isoforms, invertebrates have different combinations of these proteins with some using just one DNMT1 and the others, like the honey bee, two DNMT1s one DNMT3. Although the insect DNMTs show sequence similarity to mammalian DNMTs, their in vitro and in vivo properties are not well investigated. In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies that are frequently associated with active transcription. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. We argue that some of the lineage-specific properties of DNA methylation are the key to understanding the role of this genomic modification in insects. Future mechanistic work is needed to explain the relationship between insect DNMTs, genetic variation, differential DNA methylation, other epigenetic modifications, and the transcriptome in order to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.

Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development.

The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality.

DNA Methylation and Gene Regulation in Honeybees: From Genome-Wide Analyses to Obligatory Epialleles.

In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies. Importantly, this gene body methylation is frequently associated with active transcription, and studies in the honeybee have shown that there are strong links between gene body methylation and alternative splicing. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. Here we discuss the current knowledge in this emerging field and highlight both similarities and differences between DNA methylation systems in mammals and invertebrates. Finally, we argue that the relationship between genetic variation, differential DNA methylation, other epigenetic modifications and the transcriptome must be further explored to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.

DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees.

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non-protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Insects as innovative models for functional studies of DNA methylation.

The emerging field of epigenomics has the potential to bridge the gap between static genomic sequences and complex phenotypes that arise from multigenic, nonlinear and often context-dependent interactions. However, this goal can only be achieved if easily manageable experimental systems are available in which changes in epigenomic settings can be evaluated in the context of the phenotype under investigation. Recent progress in the characterization of insect DNA methylation patterns enables evaluation of the extent to which epigenetic mechanisms contribute to complex phenotypes in easily accessible organisms whose relatively small genomes are not only sparingly methylated, but the methylated sites are also found almost exclusively in gene bodies. The implementation of insect models in the study of DNA methylation will accelerate progress in understanding the functional significance of this important epigenetic mechanism in controlling gene splicing, in environmentally driven reprogramming of gene expression and in adult brain plasticity.

The honey bee epigenomes: differential methylation of brain DNA in queens and workers.

In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain.

The PWWP domain and the evolution of unique DNA methylation toolkits in Hymenoptera.

DNMT3 in Hymenoptera has a unique duplication of the essential PWWP domain. Using GST-tagged PWWP fusion proteins and histone arrays we show that these domains have gained new properties and represent the first case of PWWP domains binding to H3K27 chromatin modifications, including H3K27me3, a key modification that is important during development. Phylogenetic analyses of 107 genomes indicate that the duplicated PWWP domains separated into two sister clades, and their distinct binding capacities are supported by 3D modeling. Other features of this unique DNA methylation system include variable copies, losses, and duplications of DNMT1 and DNMT3, and combinatorial generations of DNMT3 isoforms including variants missing the catalytic domain. Some of these losses and duplications of are found only in parasitic wasps. We discuss our findings in the context of the crosstalk between DNA methylation and histone methylation, and the expanded potential of epigenomic modifications in Hymenoptera to drive evolutionary novelties.

Clinical Epigenetics on the Baltic Coast.

This report summarizes the proceedings of the inaugural Clinical Epigenetics Conference that was held in Szczecin, Poland, from 8 June 2022. With focus on epigenetic diseases whose causes, progression, and prognosis are associated with aberrant epigenomic alterations, the meeting was a timely forum to discuss recent progress in this rapidly evolving field and consider avenues for converting experimental data into clinical reality that would be beneficial for patients. The wealth of the presented data was an impressive showcase of the enormous challenges faced by researchers in their quest for understanding the benefits and limitations of the available information in the medical context. A shared view among the participants was that merging the current state of knowledge with clinical applications will be promptly achieved.

Epigenomic communication systems in humans and honey bees: from molecules to behavior.

A 2010 Nature editorial entitled "Time for the Epigenome" trumpets the appearance of the International Human Epigenome Consortium and likens it to Biology's equivalent of the Large Hadron Collider. It strongly endorses the viewpoint that selective modifications of "marks" on DNA and histones constitute the crucial codes of life, a proposition which is hotly contested (Ptashne et al., in 2010). This proposition reflects the current mindset that DNA and histone modifications are the prime movers in gene regulation during evolution. This claim is perplexing, since the well characterized organisms, Drosophila melanogaster and Caenorhabditis elegans, lack methylated DNA "marks" and the DNA methytransferase enzymology. Despite their complete absence, D. melanogaster nevertheless has extensive gene regulatory networks which drive sophisticated development, gastrulation, migration of germ cells and yield a nervous system with significant neural attributes. In stark contrast, the honey bee Apis mellifera deploys its human-type DNA methyltransferase enzymology to "mark" its DNA and it too has sophisticated development. What roles therefore is DNA methylation playing in different animals? The honey bee brings a fresh perspective to this question. Its combinatorial chemistry of pheromones, tergal and cuticular exudates provide an exquisite communication system between thousands of individuals. The development of queen and worker is strictly controlled by differential feeding of royal jelly and their adult behaviors are accompanied by epigenomic changes. Their interfaces with different "environments" are extensive, allowing an evaluation of the roles of epigenomes in behavior in a natural environment, in the space of a few weeks, and at requisite levels of experimental rigor.

Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining.

In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 µm long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (~ 2.1 µm). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain.

Exploring DNA Methylation Diversity in the Honey Bee Brain by Ultra-Deep Amplicon Sequencing.

Understanding methylation dynamics in organs or tissues containing many different cell types is a challenging task that cannot be efficiently addressed by the low-depth bisulphite sequencing of DNA extracted from such sources. Here we explored the feasibility of ultra-deep bisulphite sequencing of long amplicons to reveal the brain methylation patterns in three selected honey bee genes analysed across five distinct conditions on the Illumina MiSeq platform. By combing 15 libraries in one run we achieved a very high sequencing depth of 240,000-340,000 reads per amplicon, suggesting that most of the cell types in the honey bee brain, containing approximately 1 million neurons, are represented in this dataset. We found a small number of gene-specific patterns for each condition in individuals of different ages and performing distinct tasks with 80-90% of those were represented by no more than a dozen patterns. One possibility is that such a small number of frequent patterns is the result of differentially methylated epialleles, whereas the rare and less frequent patterns reflect activity-dependent modifications. The condition-specific methylation differences within each gene appear to be position-dependent with some CpGs showing significant changes and others remaining stable in a methylated or non-methylated state. Interestingly, no significant loss of methylation was detected in very old individuals. Our findings imply that these diverse patterns represent a special challenge in the analyses of DNA methylation in complex tissues and organs that cannot be investigated by low-depth genome-wide bisulphite sequencing. We conclude that ultra-deep sequencing of gene-specific amplicons combined with genotyping of differentially methylated epialleles is an effective way to facilitate more advanced neuro-epigenomic studies in honey bees and other insects.

Epigenetic regulation of the honey bee transcriptome: unravelling the nature of methylated genes.

BACKGROUND: Epigenetic modification of DNA via methylation is one of the key inventions in eukaryotic evolution. It provides a source for the switching of gene activities, the maintenance of stable phenotypes and the integration of environmental and genomic signals. Although this process is widespread among eukaryotes, both the patterns of methylation and their relevant biological roles not only vary noticeably in different lineages, but often are poorly understood. In addition, the evolutionary origins of DNA methylation in multicellular organisms remain enigmatic. Here we used a new 'epigenetic' model, the social honey bee Apis mellifera, to gain insights into the significance of methylated genes. RESULTS: We combined microarray profiling of several tissues with genome-scale bioinformatics and bisulfite sequencing of selected genes to study the honey bee methylome. We find that around 35% of the annotated honey bee genes are expected to be methylated at the CpG dinucleotides by a highly conserved DNA methylation system. We show that one unifying feature of the methylated genes in this species is their broad pattern of expression and the associated 'housekeeping' roles. In contrast, genes involved in more stringently regulated spatial or temporal functions are predicted to be un-methylated. CONCLUSION: Our data suggest that honey bees use CpG methylation of intragenic regions as an epigenetic mechanism to control the levels of activity of the genes that are broadly expressed and might be needed for conserved core biological processes in virtually every type of cell. We discuss the implications of our findings for genome-scale regulatory network structures and the evolution of the role(s) of DNA methylation in eukaryotes. Our findings are particularly important in the context of the emerging evidence that environmental factors can influence the epigenetic settings of some genes and lead to serious metabolic and behavioural disorders.

Effect of age, behaviour and social environment on honey bee brain plasticity.

We examined the effects of behaviour, age and social environment on mushroom body volume in adult bees. The mushroom bodies are regions of the central brain important for sensory integration and learning. Their volume was influenced by behaviour throughout life: always larger in forager bees than age-matched nurse bees, even in old bees up to 93 days of age as adults. Mushroom body development was influenced by the social environment in the first 8 days of adult life, with different environments having markedly different effects on mushroom body size. Compared to hive-reared bees, isolation slowed mushroom body growth, but bees reared in isolation confined with a single dead bee showed a dramatic increase in mushroom body volume comparable to that seen in active foragers. Despite their precocious mushroom body development, these bees did not show improved performance in an olfactory learning test. Since simple environmental manipulations can both accelerate and delay mushroom body growth in young bees, and since mushroom body volume is sensitive to behaviour throughout life, the honey bee has great potential as a model for exploring the interactions between environment, behaviour and brain structure.

Characterization of a Dopamine Transporter and Its Splice Variant Reveals Novel Features of Dopaminergic Regulation in the Honey Bee.

Dopamine is an important neuromodulator involved in reward-processing, movement control, motivational responses, and other aspects of behavior in most animals. In honey bees (Apis mellifera), the dopaminergic system has been implicated in an elaborate pheromonal communication network between individuals and in the differentiation of females into reproductive (queen) and sterile (worker) castes. Here we have identified and characterized a honey bee dopamine transporter (AmDAT) and a splice variant lacking exon 3 (AmDATΔex3). Both transcripts are present in the adult brain and antennae as well as at lower levels within larvae and ovaries. When expressed separately in the Xenopus oocyte system, AmDAT localizes to the oocyte surface whereas the splice variant is retained at an internal membrane. Oocytes expressing AmDAT exhibit a 12-fold increase in the uptake of [3H]dopamine relative to non-injected oocytes, whereas the AmDATΔex3-expressing oocytes show no change in [3H]dopamine transport. Electrophysiological measurements of AmDAT activity revealed it to be a high-affinity, low-capacity transporter of dopamine. The transporter also recognizes noradrenaline as a major substrate and tyramine as a minor substrate, but does not transport octopamine, L-Dopa, or serotonin. Dopamine transport via AmDAT is inhibited by cocaine in a reversible manner, but is unaffected by octopamine. Co-expression of AmDAT and AmDATΔex3 in oocytes results in a substantial reduction in AmDAT-mediated transport, which was also detected as a significant decrease in the level of AmDAT protein. This down-regulatory effect is not attributable to competition with AmDATΔex3 for ER ribosomes, nor to a general inhibition of the oocyte's translational machinery. In vivo, the expression of both transcripts shows a high level of inter-individual variability. Gene-focused, ultra-deep amplicon sequencing detected methylation of the amdat locus at ten 5'-C-phosphate-G-3' dinucleotides (CpGs), but only in 5-10% of all reads in whole brains or antennae. These observations, together with the localization of the amdat transcript to a few clusters of dopaminergic neurons, imply that amdat methylation is positively linked to its transcription. Our findings suggest that multiple cellular mechanisms, including gene splicing and epigenomic communication systems, may be adopted to increase the potential of a conserved gene to contribute to lineage-specific behavioral outcomes.

Beyond Royalactin and a master inducer explanation of phenotypic plasticity in honey bees.

Distinct female castes produced from one genotype are the trademark of a successful evolutionary invention in eusocial insects known as reproductive division of labour. In honey bees, fertile queens develop from larvae fed a complex diet called royal jelly. Recently, one protein in royal jelly, dubbed Royalactin, was deemed to be the exclusive driver of queen bee determination. However, this notion has not been universally accepted. Here I critically evaluate this line of research and argue that the sheer complexity of creating alternate phenotypes from one genotype cannot be reduced to a single dietary component. An acceptable model of environmentally driven caste differentiation should include the facets of dynamic thinking, such as the concepts of attractor states and genetic hierarchical networks.

Cocaine Directly Impairs Memory Extinction and Alters Brain DNA Methylation Dynamics in Honey Bees.

Drug addiction is a chronic relapsing behavioral disorder. The high relapse rate has often been attributed to the perseverance of drug-associated memories due to high incentive salience of stimuli learnt under the influence of drugs. Drug addiction has also been interpreted as a memory disorder since drug associated memories are unusually enduring and some drugs, such as cocaine, interfere with neuroepigenetic machinery known to be involved in memory processing. Here we used the honey bee (an established invertebrate model for epigenomics and behavioral studies) to examine whether or not cocaine affects memory processing independently of its effect on incentive salience. Using the proboscis extension reflex training paradigm we found that cocaine strongly impairs consolidation of extinction memory. Based on correlation between the observed effect of cocaine on learning and expression of epigenetic processes, we propose that cocaine interferes with memory processing independently of incentive salience by directly altering DNA methylation dynamics. Our findings emphasize the impact of cocaine on memory systems, with relevance for understanding how cocaine can have such an enduring impact on behavior.