Epigenetic Modifications May Regulate the Activation of the Hypopharyngeal Gland of Honeybees (Apis Mellifera) During Winter.
ABSTRACT: DNA methylation is an epigenetic modification primarily responsible for individual phenotypic variation. This modification has been reported to play an important role in caste, brain plasticity, and body development in honeybees (Apis mellifera). Here, we report the DNA methylation profile of honeybee hypopharyngeal glands, from atrophy in winter to arousal in the following spring, through the use of whole-genome bisulfite sequencing. Consistent with previous studies in other Apis species, we found low methylation levels of the hypopharyngeal gland genome that were mostly of the CG type. Notably, we observed a strong preference for CpG methylation, which was localized in promoters and exon regions. This result further indicated that, in honeybees, DNA methylation may regulate gene expression by mediating alternative splicing, in addition to silencing gene in the promoter regions. After assessment by correlation analysis, we identified seven candidate proteins encoded by differentially methylated genes, including aristaless-related homeobox, forkhead box protein O, headcase, alpha-amylase, neural-cadherin, epidermal growth factor receptor, and aquaporin, which are reported to be involved in cell growth, proliferation, and differentiation. Hypomethylation followed by upregulated expression of these candidates suggested that DNA methylation may play significant roles in the activation of hypopharyngeal glands in overwintering honeybees. Overall, this study elucidates epigenetic modification differences in honeybee hypopharyngeal glands by comparing an inactive winter state to an aroused state in the following spring, which could provide further insight into the evolution of insect sociality and regulatory plasticity.
Project description:Global decreases in bee populations emphasize the importance of assessing how environmental stressors affect colony maintenance, especially considering the extreme task specialization observed in honeybee societies. Royal jelly, a protein secretion essential to colony nutrition, is produced by nurse honeybees, and development of bee mandibular glands, which comprise a reservoir surrounded by secretory cells and hypopharyngeal glands that are shaped by acini, is directly associated with production of this secretion. Here, we examined individual and combined effects of the systemic fungicide pyraclostrobin and insecticide fipronil in field-relevant doses (850 and 2.5 ppb, respectively) on mandibular and hypopharyngeal glands in nurse honeybees. Six days of pesticide treatment decreased secretory cell height in mandibular glands. When pyraclostrobin and fipronil were combined, the reservoir volume in mandibular glands also decreased. The total number of acini in hypopharyngeal glands was not affected, but pesticide treatment reduced the number of larger acini while increasing smaller acini. These morphological impairments appeared to reduce royal jelly secretion by nurse honeybees and consequently hampered colony maintenance. Overall, pesticide exposure in doses close to those experienced by bees in the field impaired brood-food glands in nurse honeybees, a change that could negatively influence development, survival, and colony maintenance.
Project description:The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication. The first MRJP was identified in royal jelly, a secretion of the bees' hypopharyngeal glands that is used by young worker bees, called nurses, to feed developing larvae. Thus, MRJPs are frequently assumed to mainly have functions for developing bee larvae and to be expressed in the food glands of nurse bees. In-depth knowledge on caste- and age-specific role and abundance of MRJPs is missing. We here show, using combined quantitative real-time PCR with quantitative mass spectrometry, that expression and protein amount of mrjp1-5 and mrjp7 show an age-dependent pattern in worker's hypopharyngeal glands as well as in brains, albeit lower relative abundance in brains than in glands. Expression increases after hatching until the nurse bee period and is followed by a decrease in older workers that forage for plant products. Mrjp6 expression deviates considerably from the expression profiles of the other mrjps, does not significantly vary in the brain, and shows its highest expression in the hypopharyngeal glands during the forager period. Furthermore, it is the only mrjp of which transcript abundance does not correlate with protein amount. Mrjp8 and mrjp9 show, compared to the other mrjps, a very low expression in both tissues. Albeit mrjp8 mRNA was detected via qPCR, the protein was not quantified in any of the tissues. Due to the occurrence of MRJP8 and MRJP9 in other body parts of the bees, for example, the venom gland, they might not have a hypopharyngeal gland- or brain-specific function but rather functions in other tissues. Thus, mrjp1-7 but not mrjp8 and mrjp9 might be involved in the regulation of phenotypic plasticity and age polyethism in worker honeybees.
Project description:Deformed wing virus (DWV) is capable of infecting honeybees at every stage of development causing symptomatic and asymptomatic infections. To date, very little is known about the histopathological lesions caused by the virus. Therefore, 40 honeybee samples were randomly collected from a naturally DWV infected hive and subjected to anatomopathological examination to discriminate between symptomatic (29) and asymptomatic (11) honeybees. Subsequently, 15 honeybee samples were frozen at -80° and analyzed by PCR and RTqPCR to determinate the presence/absence of the virus and the relative viral load, while 25 honeybee samples were analyzed by histopathological techniques. Biomolecular results showed a fragment of the expected size (69bp) of DWV in all samples and the viral load was higher in symptomatic honeybees compared to the asymptomatic group. Histopathological results showed degenerative alterations of the hypopharyngeal glands (19/25) and flight muscles (6/25) in symptomatic samples while 4/25 asymptomatic samples showed an inflammatory response in the midgut and the hemocele. Results suggest a possible pathogenic action of DWV in both symptomatic and asymptomatic honeybees, and a role of the immune response in keeping under control the virus in asymptomatic individuals.
Project description:In honeybees (Apis mellifera), the rate of aging is modulated through social interactions and according to caste differentiation and the seasonal (winter/summer) generation of workers. Winter generation workers, which hatch at the end of summer, have remarkably extended lifespans as an adaptation to the cold season when the resources required for the growth and reproduction of colonies are limited and the bees need to maintain the colony until the next spring. In contrast, the summer bees only live for several weeks. To better understand the lifespan differences between summer and winter bees, we studied the fat bodies of honeybee workers and identified several parameters that fluctuate in a season-dependent manner. In agreement with the assumption that winter workers possess greater fat body mass, our data showed gradual increases in fat body mass, the size of the fat body cells, and Vg production as the winter season proceeded, as well as contrasting gradual decreases in these parameters in the summer season. The differences in the fat bodies between winter and summer bees are accompanied by respective increases and decreases in telomerase activity and DNA replication in the fat bodies. These data show that although the fat bodies of winter bees differ significantly from those of summer bees, these differences are not a priori set when bees hatch at the end of summer or in early autumn but instead gradually evolve over the course of the season, depending on environmental factors.
Project description:The honeybee model organism, Apis mellifera carnica, is a hymenopteran with a remarkable ability to withstand cold winters by instinctively entering into hibernation in the hive. Hibernation is characterized by a significant reduction in the metabolic rate, triggered by a drop in the ambient temperature and a shortage of reserves. It provides a token cue that initiates an alternative developmental program that leads to dormancy and switches social behaviors. The age-dependent changes in the generic proteins and substance composition of royal jelly (RJ) have been well documented. However, the molecular mechanism of RJ secretion and the biological consequences of regulation by the hypopharyngeal gland (HG) according to the season remain to be addressed. In this study, large-scale transcriptomic and proteomic approaches were used to detect statistically significant changes in the honeybee proteome and to explore the mechanistic basis for the HG regulation that accompanies hibernation in the winter and arousal in the spring. HG activity was associated with hundreds of gene and protein expression changes in the winter-spring cycle. A systematic study of HG activity and secretion identified 21,441 genes and 2,156 proteins at three time points (i.e., A, winter bees; B, spring bees; and C, retired bees). Of these, 737 genes and 698 proteins were qualified and compared to reveal differential expression patterns, including 197 up-regulated and 291 down-regulated DEGs in group A vs. B, 118 up-regulated and 259 down-regulated DEGs in group A vs. C, and 106 up-regulated and 53 down-regulated DEGs in group B vs. C, respectively. The results were validated by qPCR.Honeybee HG activity was associated with multiple genes regulation.The results present the first global pictures of the regulation of HG activity in winter bee. Overall design: The 3 samples at given time (A,winter bee; B,spring bee; and C, retired bee) are collected to reveal the mechanism of the regulation of HG activity and RJ secretion, the differentially expressed genes involved in the Metabolic process and protein processing in endoplasmic reticulum related DEGs were identified and the shifts appeared to show dormancy-induced transcriptional changes across the winter. 30 pooled heads of each samples were pooled as one biologiacl repeat.
Project description:The honeybee is a model organism for studying learning and memory formation and its underlying molecular mechanisms. While DNA methylation is well studied in caste differentiation, its role in learning and memory is not clear in honeybees. Here, we analyzed genome-wide DNA methylation changes during olfactory learning and memory process in A. mellifera using whole genome bisulfite sequencing (WGBS) method. A total of 853 significantly differentially methylated regions (DMRs) and 963 differentially methylated genes (DMGs) were identified. We discovered that 440 DMRs of 648 genes were hypermethylated and 274 DMRs of 336 genes were hypomethylated in trained group compared to untrained group. Of these DMGs, many are critical genes involved in learning and memory, such as Creb, GABA B R and Ip3k, indicating extensive involvement of DNA methylation in honeybee olfactory learning and memory process. Furthermore, key enzymes for histone methylation, RNA editing and miRNA processing also showed methylation changes during this process, implying that DNA methylation can affect learning and memory of honeybees by regulating other epigenetic modification processes.
Project description:There is increasing evidence that honeybees (Apis mellifera L.) can adapt naturally to survive Varroa destructor, the primary cause of colony mortality world-wide. Most of the adaptive traits of naturally varroa-surviving honeybees concern varroa reproduction. Here we investigate whether factors in the honeybee metagenome also contribute to this survival. The quantitative and qualitative composition of the bacterial and viral metagenome fluctuated greatly during the active season, but with little overall difference between varroa-surviving and varroa-susceptible colonies. The main exceptions were Bartonella apis and sacbrood virus, particularly during early spring and autumn. Bombella apis was also strongly associated with early and late season, though equally for all colonies. All three affect colony protein management and metabolism. Lake Sinai virus was more abundant in varroa-surviving colonies during the summer. Lake Sinai virus and deformed wing virus also showed a tendency towards seasonal genetic change, but without any distinction between varroa-surviving and varroa-susceptible colonies. Whether the changes in these taxa contribute to survival or reflect demographic differences between the colonies (or both) remains unclear.
Project description:Honeybees represent one of the most important insect species we have, particularly due to their pollinating services. Several emerging fungal and bacterial diseases, however, are currently threatening honeybees without known mechanisms of pathogenicity. Therefore, the aim of the current work was to investigate the seasonal (winter, spring, summer, and autumn) fungal and bacterial distribution through different gut segments (crop, midgut, ileum, and rectum). This was done from two hives in Norway. Our main finding was that bacteria clustered by gut segments, while fungi were clustered by season. This knowledge can therefore be important in studying the epidemiology and potential mechanisms of emerging diseases in honeybees, and also serve as a baseline for understanding honeybee health.