alexander m. vaiserman1, oleh v. lushchak2, alexander k. koliada1 · is the question of whether the...

271Epigenetics of Aging and Longevity. http://dx.doi.org/10.1016/B978-0-12-811060-7.00013-9Copyright © 2018 Elsevier Inc. All rights reserved.

CHAPTER

EPIGENETICS OF LONGEVITY IN SOCIAL INSECTS

Alexander M. Vaiserman1, Oleh V. Lushchak2, Alexander K. Koliada1

1D.F. Chebotarev State Institute of Gerontology NAMS of Ukraine, Kiev, Ukraine; 2Vasyl Stefanyk Precarpathian

National University, Ivano-Frankivsk, Ukraine

13CHAPTER OUTLINE

1. Introduction .....................................................................................................................................271 2. Epigenetics of Caste Differentiation ...................................................................................................273 2.1 DNA Methylation .......................................................................................................... 273 2.2 Alternative Splicing ....................................................................................................... 276 2.3 Histone Modifications ................................................................................................... 276 2.4 Micro-RNAs ................................................................................................................. 277 2.5 Caste-Specific Differences in Gene Expression Patterns .................................................... 278 3. Interplay Between Epigenetic and Endocrine Factors in Regulation of Longevity in Social Insects ........281 4. Conclusions and Future Perspectives ................................................................................................284 Acknowledgment ..................................................................................................................................285 References ...........................................................................................................................................285

1. INTRODUCTIONAging is well-known to be characterized by gradual loss of most physiological functions. A complete understanding of causal mechanisms underlying these processes and determining the lifespan is, how-ever, still far from being reached. One of the most intriguing issues in modern gerontological research is the question of whether the organismal longevity may be originated throughout the early develop-mental stages. An opportunity of the programming of later-life health and life expectancy during the early-life history periods has been repeatedly demonstrated in both animal and human studies [1–3]. Since basic metabolic pathways are largely conserved between mammals and insects [4], various insect models could be helpful for deeply understanding the mechanisms mediating developmental program-ming of aging process. Among these models, social insects are likely the most attractive model system for deeper insight into this topic as they have a considerable plasticity of aging process across different castes [5–7].

The mechanisms determining this plasticity are more fully characterized in honey bee, Apis mel-lifera. A colony of honey bees typically consists of 20–40 thousand worker bees, from zero to few thousand males, depending on the season, and a single queen, the mother of the colony [8]. A. mellifera

CHAPTER 13 EPIGENETICS OF LONGEVITY IN SOCIAL INSECTS272

have haplodiploid system of the sex determination. The unfertilized bee eggs develop into males (drones) while the fertilized eggs develop into female workers or queens [9]. Worker bees are sterile, whereas queens are reproductive. Workers spend the first 2 weeks of their adult life cycle inside the hive and, thereafter, they forage outside the hive to gather pollen and nectar. Queens reach the sexual matu-rity at 5–6 days after emergence and mate usually with 10–17 drones throughout their mating flights in early adult life. Afterward, they store the sperm needed to fertilize eggs throughout their entire life cycle. During the reproductive live, the queen may lay up to 1500–2000 eggs per day. Queens develop from eggs that are genetically not different from those that develop into workers. They are, however, much larger in size, have specialized anatomy, develop significantly faster and live much longer than worker bees. The caste switching is determined by hormonal signals triggered by the quantity and qual-ity of nutrition during the third larval instar stage [10].

The honey bee queens develop from those larvae who fed with a nutritive mixture consisting of essen-tial amino acids, proteins, lipids, vitamins, and other compounds (“royal jelly”) until they enter metamor-phosis, while workers develop from larvae fed with a pure royal jelly until the late-instar stage and then fed a worker jelly (a mixture of glandular secretions, pollen, and honey). Royal jelly contains about 12% sugar while worker jelly contains only 4% sugar, a difference also exists between royal and worker jelly in a protein type and content [11,12]. In the Kamakura [13] study, a specific factor in royal jelly, royalac-tin, has been shown to be a crucial factor driving the queen development. The role of royalactin in queen differentiation remains, however, controversial. In recent study by Buttstedt et al., [14] neither mono-meric major royal jelly protein (MRJP1, royalactin) nor any other MRJPs (MRJP2, 3 and 5) was shown to be single-key driver for queen caste determination. The authors concluded that queen phenotype is driven primarily by well-balanced amount of nutrients ingested, but not by a single deter-mining compound.

After metabolizing in the larval fat body, the nutrients contained in royal jelly activate the insulin signaling pathway, which in turn cause a faster utilization of ingested nutrients and stimulate larval growth [15]. The triggering of varying ontogenetic trajectories with different dietary patterns results in differential morphology and physiology in adult insects [16]. In addition, queens and workers exhibit marked differences in behavioral patterns [17].

In terms of biogerontological research, the most important point is that in many social insect species including ants, bees, wasps, and termites, queens and workers demonstrate up to a 100-fold difference in longevity, with reproductive queens having longer lifespan than nonreproductive workers [18,19]. In the honey bee, worker bees develop in 21 days and their lifespan reaches up to 15–38 days in summer and up to 150–200 days in winter, while queens develop in 16 days and commonly have a lifespan of about 1–2 years [9], although a lifespan of 8 years has been reported by Bozina [20]. In several ant spe-cies, the queen’s longevity may reach up to 30 years [21]. Such differences between reproductive queens and nonreproductive workers are difficult to explain, especially in view of well-known inverse relationship between reproductive activity and longevity across insect species. Remarkably, the lifes-pan can be substantially changeable even within the same castes. In A. mellifera, if the nurse bees responsible for feeding the colony’s larvae in hives are prevented from becoming the forager bees assigned to perform field tasks, they live several times as long as foragers [22]. The worker life expec-tancy may also be substantially enhanced in consequence of removal of the queen from the colony to trigger the worker reproduction [23]. In ant species, the mated queens live significantly longer than the nonmated ones [24]. The differences in life expectancy within the same castes, however, are much smaller than those reported between the castes. Through these features, social insects provide a useful

2732. EPIGENETICS OF CASTE DIFFErENTIATION

model to identify candidate pathways implicated in control of aging and longevity. Genome-wide research of gene expression in markedly differing in lifespan castes of social insect is likely a promising approach for the screening of genes involved in lifespan determination [25].

In this review, we discuss the caste-specific diversity in life span in different social insect species by focusing primarily on mechanisms of epigenetic regulation implicated in caste determination.

2. EPIGENETICS OF CASTE DIFFERENTIATIONMost of data on epigenetic mechanisms involved in caste differentiation have been obtained from stud-ies performed on A. mellifera. From these studies, it became evident that queen phenotype is driven by epigenetic reprogramming in developing larvae by particular nutritional components contained in royal jelly [26,27]. Epigenetics, that deals with heritable changes in gene function occurring without a change in the DNA sequence [28], seems an attractive system for the developmental caste-biasing, since, as it was stressed by Maleszka et al. [29], “the epigenetic system is a self-organizing regulatory level of organization that operates above the genome and provides the high level of flexibility needed for coor-dinated and context-dependent expression of multitudes of genes.” Epigenetic changes induced by specific environmental cues during the early development have been demonstrated to be persistent throughout entire life cycle, thereby causing a process known as “developmental programming” [30]. Epigenetic modifications, including DNA methylation, histone modifications, and changes in noncod-ing RNAs, can be induced in a context-dependent manner in response to both external and internal stimuli and can lead to context-dependent programming effects through the persistent effects on gene regulatory cascades [31]. In A. mellifera, feeding the female larvae with royal jelly in general leads to a reduced level of global DNA methylation and correlated alteration in gene expression along with elevated juvenile hormone (JH) levels. Such changes collectively cause formation of highly reproduc-tive and long-lived queen phenotype, while larvae fed with less-nutritious worker jelly develop into short-lived and functionally sterile worker bees. Initially, the developing larva has a certain degree of plasticity. By the fourth day of growth, however, the larval commitment to a particular ontogenetic trajectory is becoming irreversible, suggesting that caste determination is a multistep, threshold-based process [26]. This process proceeds via discrete switches inducing the expression of many genes and hormonal signaling pathways [15]. In the following subchapters, we summarize the evidence for the role of epigenetic mechanisms in developmental determination of the caste fate in various social insect species.

2.1 DNA METHYLATIONDNA methylation has been widely identified as a key mechanism implicated in shifting the caste devel-opmental trajectories in A. mellifera and other species of social insects [32,33]. It consists of the addi-tion by DNA methyltransferase (DNMT) of a methyl group at the 5-carbon of the cytosine pyrimidine ring, resulting in 5-methylcytosine [34]. In vertebrate species, these covalent modifications of DNA occur mostly in the context of cytosine-guanine (CpG) dinucleotides. DNA methylation can inhibit binding of nuclear factors, and may also be recognized by methyl-CpG–binding proteins, that can recruit additional regulatory complexes to influence the transcriptional activity. The methylation of CpG islands in promoter gene regions is associated across various taxa with transcriptional silencing


[35]. In different species, aging has been repeatedly demonstrated to be associated with global hypo-methylation; in particular CpG islands or promoters, an increase of the methylation level can also occur [36]. The presence of the methylation machinery has been, however, shown to be not universal across insect species. In contrast to vertebrates, low to zero DNA methylation levels have been detected in insect models such as Tribolium and Drosophila [37]. Recent research, however, highlighted the role of DNA methylation in different insect species including the honey bee [34]. In A. mellifera, similarly to mammals, all three main subfamilies of Dnmt enzymes, namely Dnmt1, Dnmt2, and Dnmt3, are presented [6]. In various insect species, the methylation of DNA appears to occur exclusively in the CpG context, and the levels of methylation are markedly lower than in mammals, with about 5% of cytosines methylated in differentiated human cells versus 0.7% in the honey bee brain only [38]. Furthermore, in insects the DNA methylation was found to be predominantly restricted to coding exons, and it is absent in the promoter regions [34,38].

Remarkably, in the honey bee, similarly to mammals, genes that are dynamically methylated with respect to the caste differentiation vary with age [39]. This is important since there are numerous lines of evidence indicating that epigenetically regulated developmental mechanisms can be potentially involved in aging processes [30]. For example, Kananen et al. [40] recently found in human longitudi-nal studies that trajectories of the blood DNA methylome aging rate were mostly fixed before adult-hood and remained almost unchanged afterward, even in the oldest-old ages.

Based on the evident analogy between the processes of reprogramming in mammalian cells and specialization in social insects, highlighting the possible similarity of epigenetic mechanisms regulating these processes, Patalano et al. [38] proposed a model for identifying four stages of phenotypic plasticity in social insects, which are associated with dynamic changes in their methylome: 1. “Totipotency”—where castes are nondefined, thus there is no consistent transcriptional differen-

tiation among individuals; 2. “The point of divergence”—where insects differentiate into their caste-specific lineage; 3. “Reprogramming window”—a period of metastability accompanied by stochastic changes in the

epigenetic profile and resulting gene expression, where differentiation is controlled but still allows plasticity;

4. “Differentiation”—where the caste-specific methylation patterns becoming fully established and maintained. After this point, it is difficult for worker and queen to change their caste fate.

The queen–worker developmental dichotomy triggered by larval nutritional conditions is the

only biological process by now in which the causal role of gene body methylation has been con-firmed experimentally [41]. In A. mellifera, the downregulation of Dnmt3, which is a key driver of global epigenetic remodeling, was found to cause profound shift in the caste fate of developing larvae. Kucharski et al. [41] found that silencing the expression of Dnmt3 by specific small inter-fering RNAs in newly hatched larvae led to changes in the larval developmental trajectory similar to those induced by royal jelly: the majority (72%) of Dnmt3 siRNA-treated females exhibited queen-like morphological features while only 28% bees emerged as workers with rudimentary ovaries. Remarkably, the similarly decreased levels of DNA methylation at CpG dinucleotides have been found in particular genes in the heads of queen-destined larvae regardless of whether they were hive-reared or generated via siRNA silencing of Dnmt3 [6,41]. Fully developed ovaries and decreased level of CpG methylation in the Dynactin p62 gene have been also observed in such


siRNA-induced queen phenotypes. A role of DNA methylation in the development of castes is also evident from the Lyko et al. [17] study, where more than 550 genes exhibited differential patterns of DNA methylation between the worker and queen brains. By characterizing the dynamics of the context-dependent methylation patterns throughout the caste determination in A. mellifera, Foret et al. [26] show that 2399 of 6086 methylated genes demonstrated differential patterns of methyla-tion in the queen and worker larval heads. Among these differentially methylated genes, several evolutionarily conserved signaling pathways were found to be enriched, including those genes related to hormones which have been shown previously to regulate caste determination, in particu-lar, insulin and JH. A distinctive role of the anaplastic lymphoma kinase (ALK) gene known to be an important regulator of metabolism has been also demonstrated in this study. The alk gene was found to be both differentially methylated and alternatively spliced in different castes. The extraor-dinarily complex regulation of ALK, according to the authors, can suggest that this protein might serve as an important factor in a process of activating the downstream signaling in honey bee in accordance with a nutritional context.

The crucial role of Dnmt3 in caste differentiation has been also confirmed in some recent studies, where substantially decreased CpG methylation of several genes involved in a variety of biologically important pathways was reported in A. mellifera queens. In the study by Ikeda et al. [42], some CpG sites in the hexamerin 110 gene encoding a storage protein have been shown to be differentially methyl-ated between the worker and queen larvae. Research by Shi et al. [43] demonstrated that larval dietary conditions can affect various methylation sites inside the dynactin p62, a conserved gene responding to feeding. In this study, it has been also indicated that caste determination may be influenced by cell size as well. In a more recent work by Shi et al. [44], where profiles of the DNA methylome in both worker and queen larvae of different ages (2, 4, and 6 days old) have been examined, the global DNA methyla-tion levels differed significantly between the castes. In the queen-destined larvae, levels of DNA meth-ylation increased from 2-day- to 4-day-old larvae and thereafter decreased in 6-day-old larvae, while in worker-destined larvae the methylation levels have been gradually increased with age. The total num-bers of differentially methylated genes between the 2-, 4-, and 6-day-old worker and queen larvae were 725, 3,013, and 5,049, respectively. In the queen larvae, a large number of genes have been found to be down-methylated compared to 4- and 6-day-old worker larvae. Most of these genes are known to be involved in the biologically important processes linked to development, reproduction, and metabolic regulation. Several among these genes have been previously reported to play an important role in caste differentiation. In the study by Lyko et al. [17], differential methylation levels have been observed in the queen and worker brains, including those such as GB18602 (a gene coding a putative transmem-brane protein) where the level of DNA methylation was reduced to almost zero level in the queen brains.

An important role of DNA methylation in the regulation of behavioral and physiological differ-ences in primitively social species having more flexible distinctions among castes than those observed in A. mellifera has been revealed in the study by Weiner et al. [45]. By investigating the caste- and life stage–associated DNA methylation patterns in some species of honey bees and vespid wasps which have different levels of social organization, the moderate levels of DNA methylation in most bees and wasps were obtained, with no clear link to the levels of sociality. The primitive social Polistes dominula paper wasps have demonstrated surprisingly high overall DNA methylation and substantial differences between castes in site-specific methylation levels. An importance of DNA methylation to the process of caste differentiation has been also reported in several ant species.


Similarly to other social insect species, ants have a full set of DNMTs and their genomes contain methylcytosine [46]. Furthermore, like in A. mellifera, the levels of methylation of the CpG sites are highest in the exons of the constitutively expressed housekeeping genes. By studying the caste-related methylome differences in various ant species, such as the Florida carpenter ant (Camponotus floridanus) and the Jerdon’s jumping ant (Harpegnathos saltator), Bonasio et al. [46] have found that the caste- and allele-specific methylation was significantly correlated with the allele-specific expres-sion in these species. In another study for the same ant species, Bonasio et al. [47] revealed that sir-tuin deacetylases and telomerase are upregulated in longer-lived H. saltator reproductives. Furthermore, the caste-specific expression of microRNAs and SET and MYND Domain (SMYD) histone methyltransferases, as well as differential regulation of genes implicated in chemical com-munication and neuronal function, have been observed.

Lyko et al. [17] noted that the fact that honey bee queens continue to feed on royal jelly during adulthood indicates that this specialized diet may play an important role for maintaining the behavioral and reproductive patterns characteristic to the queen phenotype. Thus, queens can adjust their brain methylomes throughout adult life in accordance with the specific nutritional cues. One of the royal jelly ingredients, phenyl butyrate, which is a histone deacetylase inhibitor and growth regulator known to extend longevity in fruit fly [48] may likely be among these nutritional factors.

2.2 ALTERNATIVE SPLICINGAlternative splicing (a process through which various combinations of exons from the same gene are joined together to form different mature mRNA isoforms and protein products) was also proposed as a potential mechanism mediating the link between DNA methylation and caste-specific gene regulation in social insects, including A. mellifera [26]. Some caste-associated CpG methylation patterns have been demonstrated to be enriched in exon regions, in particular in the alternative splicing sites [17]. In the conceptual framework, Weiner and Toth assumed that differential methylation triggered by different nutritional conditions throughout the larval development could result in alternative splicing and caste-biased expression, which, in turn, may cause caste-biased phenotypes [33].

A strong association between the patterns of DNA methylation and sites demonstrating the potential to generate the alternative splicing isoforms was revealed in the study by Lyko et al. [17]. In a genome-wide analysis of DNA methylation in the adult worker and queen brains, it was found that methylated CpGs are predominantly colocalized in the exons that are alternatively spliced. Such association between DNA methylation and splicing sites was evident, in particular, for the genes belonging to the histone gene family. Furthermore, a possible role of relationship between the DNA methylation and splicing in determining the caste-specific developmental pathways has been confirmed in two ant spe-cies in the study by Bonasio et al. [46]. On the basis of these findings, several authors assume that behavioral and morphological differences between castes across social insect species could rather be attributed to the production of caste-specific protein isoforms than to alterations in expression levels of particular genes per se [34].

2.3 HISTONE MODIFICATIONSHistone modification is another key mechanism of epigenetic regulation implicated in phenotypic plas-ticity in social insects. N-terminal tails of histones are known to be subjected to a many posttransla-tional modifications, such as acetylation, phosphorylation, methylation, and ubiquitylation. Such


modifications, in turn, may change nucleosome stability and thereby affect various important biological processes. The lysine acetylation of the N-terminal tails catalyzed by histone acetyltransferases (HATs) is the best-studied modification of the core histones, commonly linked to gene expression. On the con-trary, histone deacetylation by histone deacetylases (HDACs) promotes the formation of compact nucleosomes, leading to repression of transcription. The epigenetic mechanisms act in a coordinated manner to control gene expression. Generally, DNA methylation represses transcription, while acetyla-tion of histones is linked to the activation of transcription [49].

The role of the “histone code” in regulation of epigenetic processes in A. mellifera was clearly indi-cated by findings from the study by Dickman et al.[31], who identified 23 posttranslational modifica-tions of histones H3.1, H3.3, and H4 in 96-h-old larvae, an important time point when the key ontogenetic modifications occur due to epigenetic reprogramming triggered by royal jelly. The caste-specific differences at the level of histone modification have been also revealed in the ant C. floridanus [50]. By profiling genome-wide localization of modifications of the histone H3, a strong variability between two female worker and male castes were found for most of the histone modifications investi-gated. The level of acetylation of lysine 27 of histone H3 (H3K27ac) was demonstrated to be a power-ful predictor for caste identity. Most genes demonstrating difference in H3K27ac levels between castes have been shown to be linked to muscle development, sensory responses, and neuronal regulation. The importance of this mechanism of epigenetic regulation in determining ontogenetic pathways in social insects is clearly evident from the study by Spannhoff et al. [51] where it has been demonstrated that epigenetic control of queen-bee development can be driven by the activity of HDAC inhibitors in royal jelly. Therefore the royal jelly seems to have a potential to directly influence the level of histone lysine acetylation and to control the expression of genes implicated in honey bee caste differentiation. Foret et al. [26] by discussing these findings, stressed that “the presence of compounds with histone deacety-lase inhibitor activities in royal jelly suggests that the queen/worker developmental dichotomy is driven by a combinatorial action of DNA methylation, histone modification, and possibly other elements that respond to changes in the in vivo microenvironment brought about by fluctuations of metabolite levels.”

2.4 MICRO-RNASEpigenetic regulation by microRNAs is suggested to be another candidate mechanisms underlying caste differentiation. MicroRNAs are a class of small (17–27 nucleotides) noncoding RNA molecules playing a crucial role in the regulation of levels of proteins implicated in development, metabolism, and also other life history traits across all species including social insects [52]. A key role of microRNAs in determining many aging pathways has been also demonstrated [53]. Substantial age- and caste-associated differences in the transcriptional patterns of miRNAs have been revealed in the Weaver et al. study [52]. In this research, genes in proximity to miRNAs have been found to be associated with gene ontology terms, such as “physiological process,” “nucleus”, and “response to stress”.

Remarkably, by studying regulatory properties of microRNAs contained in the larval food of A. mellifera, Guo et al. demonstrated that worker jelly is significantly enriched in complexity and abun-dance of miRNAs in comparison with royal jelly [54]. Specifically, 7- to 215-fold higher levels of particular miRNAs have been found in worker jelly compared to royal jelly. Most of these miRNAs are known to have the potential to regulate mRNAs functionally linked to the development of the insects’ central nervous system. The worker and queen larvae also were shown to contain a common set of highly, but differentially, expressed miRNAs. These miRNAs have been demonstrated to coincide in


both composition and relative expression levels with the miRNAs identified in worker jelly, but they have been expressed at 2- to 4-fold higher levels in worker larvae compared to queen larvae. The addi-tion of certain miRNAs to royal jelly caused significant alterations in the levels of mRNA expression in queen-destined larvae and in morphological traits of the emerging insects. Supplementation of the lar-val food of prospective queens with particular miRNAs has been demonstrated to be able to alter the morphology of adult bees in the direction of the worker phenotype. Especially significant changes in the patterns of expression of larval mRNAs and in many adult characteristics were detected for miR-184.

2.5 CASTE-SPECIFIC DIFFERENCES IN GENE EXPRESSION PATTERNSCollectively, developmentally triggered histone modifications and changes in DNA methylation and microRNA regulation lead to highly coordinated modulation of gene expression, which in turn control many aspects of the life cycle of social insects, including aging. These processes eventually result in caste-biased phenotypes, such as long-lived queens and short-lived workers [55–57]. Significant differ-ences among castes in transcriptional profiles were shown in numerous studies. First, the caste-specific differences in transcriptional patterns throughout both larval and prepupal stages have been demon-strated by Severson et al. [58] in 1989. The majority of genes exhibiting differential expression patterns between queen- and worker-destined larvae were found to be involved in metabolic pathways [55,56]. Most of metabolic enzymes have been shown to be upregulated in queen-destined larvae apparently reflecting the increased growth rate of queens throughout the late larval development. Moreover, the worker-destined larvae had enhanced levels of expression of a member of the cytochrome P450 family, dihydrodiol dehydrogenase, and hexameric storage proteins, as well as two heat-shock proteins, HSP-70 and -90. Several proteins related to RNA processing and translation had also been observed to be upregulated in young larvae [55,56]. A higher level of expression of the AmIF-2mt gene encoding the mitochondrial translation initiation factor has been also revealed in queen larvae relative to the worker ones [12]. Furthermore, differential expression levels of some genes encoding mitochondrial proteins, such as mitochondrial-encoded gene cytochrome oxidase subunit 1, were found among the queen and worker bees. Significant quantitative and qualitative differences in protein expression among the mito-chondrial proteomes in worker- and queen-destined larvae have been also revealed during three early developmental stages [59]. Specifically, significant portion of proteins related to the metabolism of carbohydrate and energy, as well as fatty acids and amino acids, and also implicated in protein folding have been shown to be upregulated in the queen larvae. In the research by Cristino et al.[60], it has been also confirmed that most genes that are differentially expressed between castes are linked to metabolic processes. Among these genes, those encoding enzymes which demonstrate hydrolase activity have been found to be upregulated in worker larvae whereas those with oxidoreductase activity were shown to be upregulated in the queen larvae. By identifying genes responsible for worker–queen caste deter-mination in honey bee, Barchuk et al. [15] identified 240 genes differentially expressed among the worker and queen larvae. The worker larvae have been found to upregulate more developmental genes than the queen ones. Among them, there were genes responsible for development of organs unique to worker bees such as, for example, pollen collecting baskets. The queen-destined larvae were demon-strated to mostly upregulate a considerable amount of physio-metabolic genes, including those encod-ing the metabolic enzymes, and also those regulating both mass-transforming processes and the organism’s growth rate. Among these differentially expressed genes, there were those involved in


processes linked to development of caste-biased structures, including the legs, brain, and ovaries, and also in the coding for components of cytoskeleton. In addition, treatment of worker larvae throughout the critical periods of caste determination with JH allowed to reveal 52 JH-responsive genes, suggest-ing important role of this pathway in caste determination. The caste-specific DNA methylome patterns were identified in the genome-wide expressional profiling of the Dnmt3-silenced larvae conducted by Kucharski et al. [41]. Most of genes that have been differentially expressed in the worker and queen larvae were shown to be associated with posttranslational modification, protein turnover, hormonal regulation, lipid transport, ribosomal biogenesis, energy transfer, and other physio-metabolic processes. Among the genes responsive to Dnmt3 silencing, there were some genes involved in the chromatin integrity and remodeling, including the ATPase of the type implicated in the maintenance of chromo-somal stability and also the subunit of the INO80 nucleosome remodeling complex. Those findings suggest that either feeding with royal jelly or Dnmt3 silencing caused epigenetic reprogramming that is characterized by the shift of larval transcriptional profile toward higher expression of physio-meta-bolic genes, including the genes implicated in growth and metabolic regulation. In another transcrip-tome comparison among the castes of honey bee, more than 4500 genes have been revealed to be differentially expressed between the worker- and queen-destined larvae [57]. In each larval instar, over 70% across the differentially expressed genes have been demonstrated to be more highly expressed in queen than in worker larvae suggesting that overall levels of transcriptional activity throughout the dif-ferentiation are higher in queen larvae than in worker ones. In the genome-wide expression analysis conducted on brains of the same-aged virgin honey bee queens, sterile workers, and reproductive work-ers, substantial differences in the expression levels for ∼2000 genes between either queens or both worker groups, and much smaller differences (221 genes only) among the sterile and reproductive worker bees have been revealed [61]. Remarkably, some functional groups of genes linked to longevity in other species have been found to be significantly upregulated in queens. In some research, the iden-tification of subsets of differentially expressed genes specifically involved in longevity-associated path-ways in honey bee has been carried out. For example, in the study by Azevedo et al.[62], the caste-specific differential expression of genes related to the regulation of the hypoxia pathway has been investigated. In this study, enhanced levels of expression of the honey bee homologs of the hypoxia signaling factors, HIFα/Sima, HIFβ/Tango, and PHD/Fatig, during the critical stages of larval development were found in worker larvae in comparison with those in queen larvae. By studying adult queens and aging work-ers, the elevated expression levels of two genes potentially implicated in repair and prevention of oxida-tive damage has been revealed in queens as compared with workers; these genes have tend to be downregulated throughout the aging trajectory in worker bees [63].

Differential patterns of gene expression between castes have been also identified in species of social insects other than honey bee. Pereboom et al.[64] demonstrated that genes whose differential expres-sion is associated with caste determination are substantially similar in the bumble-bee, Bombus terres-tris, and in A. mellifera. Most of these differentially expressed genes have been found to be upregulated in queen-destined larvae early in development. The same genes have been revealed to be upregulated in worker-destined larvae late in development, suggesting that caste determination in B. terrestris arises not from differences in the identity of genes expressed in queen- and worker-destined larvae, but rather from the relative timing of their expression. In the Lockett et al. study [65], age- and caste-related changes in the expression levels of four candidate genes associated with taxonomically widespread aging-associated pathways, namely, Dnmt3, foraging, vitellogenin, and coenzyme Q biosynthesis pro-tein 7 have been observed in B. terrestris.


Caste-specific patterns of gene expression have been also identified in ants. In the ant Temnothorax longispinosus, ∼2500 genes were shown to be differentially expressed between worker and queen castes [66]. Differences among castes in the expression levels of immune response-linked and chemosensory genes have been observed as well in another ant species such as Atta vollenweideri [67]. The greater levels of expression of somatic repair genes have been revealed in long-lived queens than in workers in ant Lasius niger [68]. Specifically, no difference in the expression of these genes was observed in both legs and brains among queens and workers in 1-day-old individuals. With age, the enhancement of the expression levels of these genes was observed, and such upregulation has been demonstrated to be higher in queens than in workers, causing significantly queen-biased expression in 2-month-old insects. Sixteen genes have been demonstrated to be differentially expressed across adult queen and worker ants L. niger in the research by Gräff et al. [69]. Among these genes, three being upregulated in queens are likely related to both repair and maintenance of the soma, considered as the candidate mechanisms for longevity determination. Soldier caste-specific gene expression has been also revealed in the Japanese damp-wood termite Hodotermopsis japonica [70].

Only one research has been conducted to date exclusively focused on the genetic pathways poten-tially implicated in determining longevity in social insect [25]. In this study, the predictions of oxida-tive stress theory of aging, which postulate that the production of highly reactive free radicals and other reactive oxygen species (ROS) cause progressive oxidative damage of different cellular components, and that the accumulation of oxidative damage is the proximate cause of aging [71], were tested in the honey bee model. For this purpose, the expression levels of eight genes encoding antioxidant defense system enzymes and also five mitochondrial proteins involved in respiration in thorax, head, and abdo-men were determined [25]. The expression of antioxidant genes was shown to be commonly decreased with age in queens, but not in workers. Consequently, queens did not have higher mRNA levels of antioxidant genes involved in ROS degradation in their older age than worker bees, thereby suggesting that their extraordinary longevity can unlikely be explained by elevated antioxidant capacity per se. The remarkable differences in the respiration-related gene expression among the worker and queen bees have been indicated as well, demonstrating that obtained differences display, at least partly, the caste-specific difference in metabolic rate. The expression of the mitochondrial genes investigated has been revealed to be higher in young queens, but these genes showed a faster age-associated decline in com-parison with workers. These results indicate that differences in longevity between castes could result from the caste-specific alteration in regulation of mitochondrial genes that, in turn, affect ROS produc-tion. On the basis of these findings, the authors speculated that “if queens produce less ROS than do workers due to increased respiration efficiency, they might not need high antioxidant expression.” Similar data indicating that enhanced levels of antioxidant enzyme gene expression are most likely not necessary for the evolution of the extended life expectancy in social insects, were also obtained in the ant L. niger [72]. In this research, adult queens had equal or lower levels of expression of the Cu–Zn superoxide dismutase 1 (CuZnSOD) gene and CuZnSOD activity in thorax, head, and abdomen rela-tive to those in short-lived workers and male ants. Cellular membrane composition has been suggested by several authors to be another important factor mediating differential tolerance to oxidative stress across the female castes. The honey bee queens have been indeed demonstrated to have more peroxi-dation-resistant membranes in consequence of modified fatty acid composition of phospholipids in comparison with worker bees [73,74].

2813. INTErPLAY BETWEEN EPIGENETIC AND ENDOCrINE FACTOrS

3. INTERPLAY BETWEEN EPIGENETIC AND ENDOCRINE FACTORS IN REGULATION OF LONGEVITY IN SOCIAL INSECTS

Caste-specific differences in the expression of genes that are triggered by nutritional factors throughout larval development are demonstrated to affect various endocrine aspects of both larval and adult stages in different social insect species. In some studies, endocrine pathways involved in the regulation of fertility and life expectancy have been identified. The crucial roles of signaling pathways such as the insulin/insulin-like growth factor 1 (IGF-1) pathway and its associated branch, target of rapamycin (TOR) pathway, along with JH and a female-specific egg yolk precur-sor, vitellogenin, in the regulation of the longevity in A. mellifera and other social insect species have been indicated. The key role of JH known to regulate larval development and inhibiting meta-morphosis in determining the caste fate is well known for decades. Findings from many studies demonstrate that nutritional stimuli throughout larval development induce endocrine responses manifested by enhanced JH titers in queen larvae relative to the worker ones [75]. The queen-inducing capacity of JH was shown as early as in 1972 in the research by Wirtz and Beetsma, [76] who applied JH to the worker larvae, and these data have been subsequently confirmed in many studies [77,78].

The insulin signaling pathway is another crucial pathway causally linked to caste determination. A specific insulin-like peptide highly expressed in queen but not in worker larvae has been identified in the Wheeler et al. study [79]. Another gene, namely that encoding for insulin receptor, has been found to be expressed at higher levels in queen larvae than in worker larvae throughout second larval instar. By investigating the developmental profiles of expression of several genes encoding insulin receptors and insulin-like peptides in A. mellifera, de Azevedo et al. [80] contrary to these findings revealed that expression of gene encoding for the insulin-like peptide, AmILP-2, has been higher in workers than in queens. The expression levels of both studied genes encoding for insulin receptors (AmInR-1 and AmInR-2) has been demonstrated to be sharply declined in fourth instar queen larvae but exhibited little modulation in workers. The authors stressed that their findings are certainly not intuitive, considering the high rate of growth in queen honey bees, but they could be interpreted as probable antagonistic crosstalk between JH and insulin/insulin-like signaling pathway. By using a double-stranded RNA (dsRNA)–mediated gene knockdown approach, Wang et al. [81] also demon-strated that queen-worker dimorphism can be determined by level of expression of genes encoding for insulin-like peptides such as AmILP1 and AmILP2. Indeed, the treatment with AmILP1 dsRNA led to reduced JH levels, while knockdown of AmILP2 resulted in a reduction in ovary size. The authors concluded that the level of expression of AmILP2 may affect the insect body size and devel-opment of particular organs, such as the ovary, whereas the brain-expressed AmILP1 may regulate the JH production. A role of the insulin/insulin-like signaling network in determination of the caste fate has been also confirmed in the study by Wolschin et al. [82], where knockdown of the gene encoding for insulin receptor substrate by RNA interference resulted in the development of worker morphology. It should be, however, noted that findings on the reduced expression levels of insulin receptors in queen-destined larvae obtained by de Azevedo et al. [80] and Wang et al. [81] contradict the commonly accepted view on the growth-promoting roles of the insulin/IGF-1 signaling pathway. Indeed, mutations in this pathway are well known to reduce body size and to extend longevity [83], and decreased signaling of insulin-like peptides has been repeatedly revealed to increase the lifespan


in worms, fruit flies, and rodents [84]. A. mellifera queens, however, exhibited both enhanced body size and longevity. Thus, the roles of insulin signaling in the honey bee queen longevity are largely controversial.

The epidermal growth factor receptor (EGFR) signaling pathway has been proposed as another important factor contributing to the regulation of longevity in honey bee. This pathway has been demonstrated to be greatly implicated in the size control across insect species [85]. In some studies, it has been found that the EGFR pathway, rather than the insulin signaling pathway, may play a potential role in promoting health span and longevity in Caenorhabditis elegans [86,87]. In the research by Kamakura, activating the EGFR pathway by royalactin (a specific 57-kDa protein con-tained in the royal jelly) lead to increased body size and longevity in Drosophila, thereby demon-strating that this factor can play a crucial role in the development of long-lived queen phenotype in honey bee [14]. To determine the pathways mediating the life-extending effect of royalactin, the author fed that compound to the nonsocial insect such as Drosophila melanogaster and found that flies fed with royalactin exhibited enhanced body size, longevity, and fecundity, all characteristics typical for a honey bee queen phenotype. Both by using the mutant fruit flies or by disrupting differ-ent signaling cascades by RNA interference (RNAi), Kamakura showed that the effects of royalactin are mediated via EGFR signaling in the honey bee fat body and via the release of secreted factors, such as the phosphatidylinositol 3-kinase to target of rapamycin to S6 kinase (PI3K-TOR-S6K) path-way known to stimulate growth and protein synthesis. Activating the EGFR signaling has been also shown to lead to enhanced synthesis of JH, to elevated levels of expression of the yolk protein–encoding gene, and also to the high fecundity rate. In the honey bee model, the author also demon-strated that RNAi-triggered inhibition of selective signaling pathway that EGFR is involved in the increase of body weight and shortening of developmental duration in the honey bee queen in response to royalactin.

Target of rapamycin (TOR) pathway, which is known to closely interact with the insulin/IGF-1 signaling pathway, is also revealed to play a key role in the regulation of caste polymorphism in social insects [88]. TOR is a serine/threonine kinase controlling growth and metabolism in response to dietary and growth factor cues [89]. In research by Shao et al., the enhanced levels of TOR expression were observed in the third-instar queen larvae relative to the worker larvae [13]. The third-instar queen lar-vae also had about twofold higher levels of TOR mRNA than third-instar worker larvae in the study by Patel et al. [89]. In this study, the role of TOR in the caste determination was examined by using the TOR inhibitor, rapamycin, and its competitive antagonists, FK506, as well as by RNAi gene knock-down. In the queen-destined larvae, rapamycin induced the development of worker characteristics, and this effect was blocked by FK506. The queen fate has been shown to be associated with enhanced activity of the TOR gene, and knockdown of this gene shifted larval fate from the queen destined to the worker one.

To address the issue how JH signaling and dietary factors determine the honey bee caste fate, Mutti et al. [88] suppressed the nutrient sensing by the RNAi-mediated knockdown of genes encoding the insulin receptor substrate and TOR in larvae fed on queen diet. This treatment influ-enced many levels of epigenetic regulation such as DNA methylation and gene expression, and also proteomic profiles, lipid levels, and some morphological features. The knockdown of these genes resulted in abolishing the JH peak that is required for the queen development, thereby lead-ing to a worker phenotype. In these knockdowns, the JH application rescued the queen phenotype,

2833. INTErPLAY BETWEEN EPIGENETIC AND ENDOCrINE FACTOrS

suggesting that larval response to JH could drive the normal developmental plasticity even when levels of transcripts of insulin receptor substrate or TOR are reduced. In monitoring the expression levels of 14 genes for components of the TOR and insulin/insulin-like signaling pathways in honey bee larvae, Wheeler et al. [90] found that, among these genes, three which is suggested to play an important role in the transduction of nutritional signals into the hormone production, demonstrated substantial differences in the expression levels in the 40-hour-old larvae. Following a shift diet to the worker nutritional state, the expression levels of nine genes have been found to be upregulated relative to queens, whereas a shift to the queen diet caused changes in expression level of one gene only.

Among the potential main candidates in regulating the honey bee longevity, the egg-yolk precur-sor vitellogenin, which is known to affect many aspects of insect life histories, is one of the best studied [91]. Alternative vitellogenin utilization in nursing and somatic maintenance has been pro-posed to be a plausible mechanism mediating both behavior and aging plasticity in worker bees differing in respect to their reproductive status. Beneficial effects of vitellogenin are assumed to be associated with its inhibitory action on JH (an aging-promoting and life-shortening insect hormone) as well as on the insulin-like signaling. Worker division of labor in insect colonies and resulting longevity variation, thereby, can be affected by a mutual negative feedback between the vitellogenin and JH [92]. It is also suggested that vitellogenin can promote longevity via its antioxidant effect [93]. The activity of the vitellogenin gene has been shown to protect the workers from paraquat-induced oxidative stress throughout its antioxidant properties [94]. Moreover, RNAi-induced knock-down of this gene was demonstrated to cause significant life shortening. Basing on these findings, the authors suppose that vitellogenin can play a key role in determining the extraordinary queen longevity. The presumed roles of vitellogenin, as well as the associated roles of JH, insulin, and IGF-1 in determining the differences in lifespan among the honey bee castes have been thoroughly investigated by Corona et al. [93]. The insulin/IGF-1 signaling pathway has been selected for inves-tigation since that pathway is known for its key role in regulating many important biological pro-cesses linked to aging. Downregulating the insulin/IGF-1 signaling pathway has been repeatedly demonstrated to be related to decreased fertility and enhanced life expectancy in different experi-mental models, including fruit flies, worms, and mice [95]. Moreover, the relationship between JH and vitellogenin synthesis was observed [93,96]. In the study by Corona et al. [93], vitellogenin has been observed to be expressed in the head fat body and thorax in an age-dependent manner. The old queen bees exhibited much larger levels of expression of vitellogenin and also much lower levels of expression of insulin-like peptide and its putative receptors than the worker ones. JH influenced the levels of expression of vitellogenin and some genes related to the insulin/IGF-1 signaling in oppo-site ways, suggesting a feedback loop between these pathways. These findings assume that caste-specific differences in the level of vitellogenin could be involved in the plasticity of longevity in honey bee. The authors suggested that vitellogenin can act as a free radical scavenger to protect against oxidative stress, in addition to the endogenous antioxidant defense systems; therefore, queen bees have additional mechanisms to minimize oxidative damage. Moreover, they speculated that negative association between vitellogenin and JH can contribute to changes in insulin/IGF-1 signal-ing to allow for both high fecundity and extended lifespan in queens. A schematic representation of hypothetical regulatory pathways responsible for determining caste fate in Apis mellifera is pre-sented in Fig. 13.1.


4. CONCLUSIONS AND FUTURE PERSPECTIVESEvidence of the extraordinarily life expectancy of queens in different social insect species and amaz-ingly great differences in longevity among the genetically identical worker and queen females consid-erably challenges our understanding of the processes underlying aging and provides unique opportunity for investigating the factors underlying variation in lifespan within and among species [97]. Among various social insect models aimed at investigating the basic mechanisms of aging, A. mellifera seems

FIGURE 13.1

A schematic representation of hypothetical regulatory pathways responsible for determining the caste fate in Apis mellifera. Under this hypothetical mechanistic model, the larval nutrition with royal jelly leads to a reduced level of global DNA methylation and associated changes in gene expression and splicing, in turn triggering the endocrine response manifested in the activation of JH, TOr, and EGFr signaling and also in the alteration of insulin/IGF-1 pathway in the queen-destined larvae in comparison with worker-destined ones. In total, these processes are likely contributing to the development of long-lived queen phenotype. EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor 1; JH, juvenile hormone; TOR, target of rapamycin.

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especially promising. Previous studies conducted with this model were mostly focused on highlighting the roles of factors contributing to longevity in worker bees, such as behavioral factors including forag-ing and nursing and environmental factors influencing extrinsic mortality including predation or weather. Over the last years, the emphasis of such research has been shifted to elucidating mechanisms underlying caste-specific differences in longevity. It draws particular attention to the nutritional epi-genetics, a novel research field that has been emerged via the development of highly effective technolo-gies which allow illuminate the molecular mechanisms contributing to aging and longevity [18,98]. This research direction seems especially promising in the context of modern conceptual frameworks which relate aging process to development and focus on epigenetic pathways responsible for the regu-lation of ontogenesis [99].

The epigenome-wide association study is likely the most promising approach now to address the gaps in our knowledge in this emerging research field. The application of such research strategy will allow us to identify candidate genes that can be subsequently examined more carefully with methods such as RNAi. In the context of research on social insect models, further development of such research methodology can help identify particular genes which make queens so much longer-lived than worker bees. Investigating the molecular, biochemical, and physiological aspects of honey bee biology would likely be a promising avenue for identifying the pathways that contribute to the regulation of longevity and for developing the novel treatment options designed to affect such pathways. Highlighting these pathways could help to identify the potential targets for future therapeutic interventions aimed at preven-tion and treatment of age-associated disorders [100] and also at slowing down the aging process per se.

ACKNOWLEDGMENTThe author would like to thank Oksana Zabuga for the helpful assistance in preparing the manuscript.

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alexander m. vaiserman1, oleh v. lushchak2, alexander k. koliada1 · is the question of whether the...

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