Honey bee (Apis mellifera) nurse bee visitation of worker and drone larvae increases Varroa destructor mite cell invasion.

The life cycle of Varroa destructor, the ectoparasitic mite of honey bees (Apis mellifera), includes a dispersal phase, in which mites attach to adult bees for transport and feeding, and a reproductive phase, in which mites invade worker and drone brood cells just prior to pupation to reproduce while their bee hosts complete development. In this study, we wanted to determine whether increased nurse bee visitations of adjacent drone and worker brood cells would increase the likelihood of Varroa mites invading those cells. We also explored whether temporarily restricting the nurses' access to sections of worker brood for 2 or 4 h would subsequently cause higher nurse visitations, and thus, higher Varroa cell invasions. Temporarily precluding larvae from being fed by nurses subsequently led to higher Varroa infestation of those sections in some colonies, but this pattern was not consistent across colonies. Therefore, removing highly infested sections of capped worker brood could be further explored as a potential mechanical/cultural method for mite control. Our results provide more information on how nurse visitations affect the patterns of larval cell invasion by Varroa. Given that the mite's successful reproduction depends on the nurses' ability to visit and feed developing brood, more studies are needed to understand the patterns of Varroa mite invasion of drone and worker cells to better combat this pervasive honey bee parasite.

Social life results in social stress protection: a novel concept to explain individual life-history patterns in social insects.

Resistance to and avoidance of stress slow aging and confer increased longevity in numerous organisms. Honey bees and other superorganismal social insects have two main advantages over solitary species to avoid or resist stress: individuals can directly help each other by resource or information transfer, and they can cooperatively control their environment. These benefits have been recognised in the context of pathogen and parasite stress as the concept of social immunity, which has been extensively studied. However, we argue that social immunity is only a special case of a general concept that we define here as social stress protection to include group-level defences against all biotic and abiotic stressors. We reason that social stress protection may have allowed the evolution of reduced individual-level defences and individual life-history optimization, including the exceptional aging plasticity of many social insects. We describe major categories of stress and how a colonial lifestyle may protect social insects, particularly against temporary peaks of extreme stress. We use the honey bee (Apis mellifera L.) to illustrate how patterns of life expectancy may be explained by social stress protection and how modern beekeeping practices can disrupt social stress protection. We conclude that the broad concept of social stress protection requires rigorous empirical testing because it may have implications for our general understanding of social evolution and specifically for improving honey bee health.

Life-history stage determines the diet of ectoparasitic mites on their honey bee hosts.

Ectoparasitic mites of the genera Varroa and Tropilaelaps have evolved to exclusively exploit honey bees as food sources during alternating dispersal and reproductive life history stages. Here we show that the primary food source utilized by Varroa destructor depends on the host life history stage. While feeding on adult bees, dispersing V. destructor feed on the abdominal membranes to access to the fat body as reported previously. However, when V. destructor feed on honey bee pupae during their reproductive stage, they primarily consume hemolymph, indicated by wound analysis, preferential transfer of biostains, and a proteomic comparison between parasite and host tissues. Biostaining and proteomic results were paralleled by corresponding findings in Tropilaelaps mercedesae, a mite that only feeds on brood and has a strongly reduced dispersal stage. Metabolomic profiling of V. destructor corroborates differences between the diet of the dispersing adults and reproductive foundresses. The proteome and metabolome differences between reproductive and dispersing V. destructor suggest that the hemolymph diet coincides with amino acid metabolism and protein synthesis in the foundresses while the metabolism of non-reproductive adults is tuned to lipid metabolism. Thus, we demonstrate within-host dietary specialization of ectoparasitic mites that coincides with life history of hosts and parasites.

Group size influences maternal provisioning and compensatory larval growth in honeybees.

Environmental variation selects for the adaptive plasticity of maternal provisioning. Even though developing honeybees find themselves in a protected colony environment, their reproductively specialized queens actively adjust their maternal investment, even among worker-destined eggs. However, the potentially adaptive consequences of this flexible provisioning strategy and their mechanistic basis are unknown. Under natural conditions, we find that the body size of larvae hatching from small eggs in large colonies converges with that of initially larger larvae hatching from large eggs typically produced in small colonies. However, large eggs confer a persistent body size advantage when small and large eggs are cross-fostered in small and large colonies, respectively. We substantiate the increased maternal investment by identifying growth-promoting metabolomes and proteomes in large eggs compared to small eggs, which are primarily enriched in amino acid metabolism and cell maturation. Thus, our study provides a comprehensive adaptive explanation for the worker egg size plasticity of honeybees.

Quantitative trait loci mapping for survival of virus infection and virus levels in honey bees.

Israeli acute paralysis virus (IAPV) is a highly virulent, Varroa-vectored virus that is of global concern for honey bee health. Little is known about the genetic basis of honey bees to withstand infection with IAPV or other viruses. We set up and analyzed a backcross between preselected honey bee colonies of low and high IAPV susceptibility to identify quantitative trait loci (QTL) associated with IAPV susceptibility. Experimentally inoculated adult worker bees were surveyed for survival and selectively sampled for QTL analysis based on SNPs identified by whole-genome resequencing and composite interval mapping. Additionally, natural titers of other viruses were quantified in the abdomen of these workers via qPCR and also used for QTL mapping. In addition to the full dataset, we analyzed distinct subpopulations of susceptible and non-susceptible workers separately. These subpopulations are distinguished by a single, suggestive QTL on chromosome 6, but we identified numerous other QTL for different abdominal virus titers, particularly in the subpopulation that was not susceptible to IAPV. The pronounced QTL differences between the susceptible and non-susceptible subpopulations indicate either an interaction between IAPV infection and the bees' interaction with other viruses or heterogeneity among workers of a single cohort that manifests itself as IAPV susceptibility and results in distinct subgroups that differ in their interaction with other viruses. Furthermore, our results indicate that low susceptibility of honey bees to viruses can be caused by both, virus tolerance and virus resistance. QTL were partially overlapping among different viruses, indicating a mixture of shared and specific processes that control viruses. Some functional candidate genes are located in the QTL intervals, but their genomic co-localization with numerous genes of unknown function delegates any definite characterization of the underlying molecular mechanisms to future studies.

Major royal jelly proteins influence the neurobiological regulation of the division of labor among honey bee workers.

Age-based division of labor among workers is a fundamental life-history trait of many social insects, including the Western honey bee, Apis mellifera L. Extensive studies of the causation of the most pronounced transition from performing tasks in the nest to outside foraging indicate hormonal regulation of complex physiological changes. However, the proximate neurobiological mechanisms that cause the behavioral repertoire to change are still not understood and require novel approaches to be fully characterized. Thus, we established the first comprehensive monoclonal antibody microarray in honey bees with 16,320 antibodies to directly identify proteins in the brain that regulate the transition to foraging. Major royal jelly protein (MRJP) 1 and MRJP3 were identified as potential protein effectors and further investigated. A series of experimental manipulations of the workers' behavioral transition led to changes in MRJP1 and MRJP3 quantities in accordance with their presumed functional role. Injection of MRJPs into the brain resulted in increased task-reversal from foraging to nursing and decreased task-progression from nursing to foraging, while the latter was increased by injection with MRJP antibodies. Finally, down-regulation of MRJP1 and MRJP3 expression via RNAi injection into the brain increased the transition from in-hive nursing to outside foraging, confirming a causal role of these two proteins in the proximate regulation of behavior and life-history of honey bee workers. Interaction partners of MRJP1 and MRJP3 in the honey bee brain included other regulators of honey bee behavior and life history. Thus, our transformative methodological advancement of proteome analysis in honey bees reveals novel regulators of honey bee behavior, extends our understanding of the functional pleiotropy of MRJPs, and supports a general nutrition-based model of the regulation of the age-based division of labor in honey bees.

The molecular basis of socially induced egg-size plasticity in honey bees.

Reproduction involves the investment of resources into offspring. Although variation in reproductive effort often affects the number of offspring, adjustments of propagule size are also found in numerous species, including the Western honey bee, Apis mellifera. However, the proximate causes of these adjustments are insufficiently understood, especially in oviparous species with complex social organization in which adaptive evolution is shaped by kin selection. Here, we show in a series of experiments that queens predictably and reversibly increase egg size in small colonies and decrease egg size in large colonies, while their ovary size changes in the opposite direction. Additional results suggest that these effects cannot be solely explained by egg-laying rate and are due to the queens' perception of colony size. Egg-size plasticity is associated with quantitative changes of 290 ovarian proteins, most of which relate to energy metabolism, protein transport, and cytoskeleton. Based on functional and network analyses, we further study the small GTPase Rho1 as a candidate regulator of egg size. Spatio-temporal expression analysis via RNAscope and qPCR supports an important role of Rho1 in egg-size determination, and subsequent RNAi-mediated gene knockdown confirmed that Rho1 has a major effect on egg size in honey bees. These results elucidate how the social environment of the honey bee colony may be translated into a specific cellular process to adjust maternal investment into eggs. It remains to be studied how widespread this mechanism is and whether it has consequences for population dynamics and epigenetic influences on offspring phenotype in honey bees and other species.

Honey bees are social insects that live in large colonies containing tens of thousands of individuals. The vast majority of bees are sterile females known as worker bees. They perform most of the activities essential for the survival of the colony, including foraging for pollen and nectar and taking care of eggs and larvae. An individual known as the queen bee is the mother of the colony and is normally the only female who reproduces. She has two massive ovaries and can produce up to two thousand eggs per day. Previous studies indicate that the number and size of the eggs vary according to the conditions inside the colony and in the surrounding environment. Larger eggs contain more nutrients so the resulting embryos may have a better chance of survival. However, producing bigger eggs requires the queen to invest more resources, which is costly to the colony as a whole. It remains unclear which mechanisms regulate the size of honey bee eggs. To address this question, Han, Wei, Amiri et al. carried out a series of experiments on the Western honey bee, Apis mellifera. The experiments showed that queen bees in small colonies had smaller ovaries and produced bigger eggs than those in large colonies. The difference in egg size appeared to be due to the queen bee's perception of the size of the colony, rather than its actual size. An approach called proteomics revealed that 290 ovarian proteins were produced at different levels in big-egg producing ovaries compared to small-egg producing ovaries. Further experiments suggested that a protein known as Rho1 regulates the size of the eggs the queen bees produce. These findings provide an explanation for how the social environment of the Western honey bee colony may influence the queen bee's reproductive investment at the molecular level. Further studies to confirm and expand on this work may help to improve honey bee health and also contribute to our general understanding of this life stage in bees and other insects.

Fertility costs of cryptic viral infections in a model social insect.

Declining insect populations emphasize the importance of understanding the drivers underlying reductions in insect fitness. Here, we investigated viruses as a threat to social insect reproduction, using honey bees as a model species. We report that in two independent surveys (N = 93 and N = 54, respectively) of honey bee (Apis mellifera) queens taken from a total of ten beekeeping operations across British Columbia, high levels of natural viral infection are associated with decreased ovary mass. Failed (poor quality) queens displayed higher levels of viral infection, reduced sperm viability, smaller ovaries, and altered ovary protein composition compared to healthy queens. We experimentally infected queens with Israeli acute paralysis virus (IAPV) and found that the ovary masses of IAPV-injected queens were significantly smaller than control queens, demonstrating a causal relationship between viral infection and ovary size. Queens injected with IAPV also had significantly lower expression of vitellogenin, the main source of nutrition deposited into developing oocytes, and higher levels of heat-shock proteins, which are part of the honey bee's antiviral response. This work together shows that viral infections occurring naturally in the field are compromising queen reproductive success.

Multiple benefits of breeding honey bees for hygienic behavior.

Honey bee colonies are prone to invasion by pests and pathogens. The combination of the parasitic mite Varroa destructor (Varroa) and the multiple viruses it vectors, is a major driver of colony losses. Breeding for hygienic behavior to reduce Varroa populations is considered a sustainable way to reduce the impact of Varroa on honey bee health. However, hygienic behavior may have a cost to the health of individual bees, both in terms of viral infection risk and immune function. To determine whether selection for hygienic behavior at the colony level is associated with trade-offs in honey bee viral infection and immune function, we compared Varroa populations, viral loads, and individual immune function between honey bee colonies that were bred for high and low hygienic behavior. Specifically, we measured Varroa infestation, Deformed wing virus DWV-A, DWV-B, Acute bee paralysis virus (ABPV), and Israeli acute paralysis virus IAPV viral genome levels in bee samples from artificially inseminated queens in our bi-directional selection program for hygienic behavior in Israel. In addition, we evaluated the expression of 12 genes from the Jak-STAT, Toll, IMD and RNAi immune pathways. We found significantly lower Varroa infestation and DWV loads in highly hygienic colonies than in colonies exhibiting low hygienic behavior. In addition, workers of the hygienic colonies had significantly higher expression of the immune genes PGRP-S2 and hymenoptaecin compared to workers from low hygienic colonies. These results indicate no trade-offs in breeding for hygienic behavior. Hygienic honey bees were associated with reduced Varroa populations and reduced DWV prevalence or load at the colony level. Individual immunity of hygienic bees was increased, which could also contribute to lower virus levels, although lower Varroa levels due to social immunity presumably contributed as well. In sum, we demonstrate multiple health benefits of breeding for honey bee hygiene.

Impact of Honey Bee Migratory Management on Pathogen Loads and Immune Gene Expression is Affected by Complex Interactions With Environment, Worker Life History, and Season.

The effects of honey bee management, such as intensive migratory beekeeping, are part of the ongoing debate concerning causes of colony health problems. Even though comparisons of disease and pathogen loads among differently managed colonies indicate some effects, the direct impact of migratory practices on honey bee pathogens is poorly understood. To test long- and short-term impacts of managed migration on pathogen loads and immunity, experimental honey bee colonies were maintained with or without migratory movement. Individuals that experienced migration as juveniles (e.g., larval and pupal development), as adults, or both were compared to control colonies that remained stationary and therefore did not experience migratory relocation. Samples at different ages and life-history stages (hive bees or foragers), taken at the beginning and end of the active season, were analyzed for pathogen loads and physiological markers of health. Bees exposed to migratory management during adulthood had increased levels of the AKI virus complex (Acute bee paralysis, Kashmir bee, and Israeli acute bee paralysis viruses) and decreased levels of antiviral gene expression (dicer-like). However, those in stationary management as adults had elevated gut parasites (i.e. trypanosomes). Effects of environment during juvenile development were more complex and interacted with life-history stage and season. Age at collection, life-history stage, and season all influenced numerous factors from viral load to immune gene expression. Although the factors that we examined are not independent, the results illuminate potential factors in both migratory and nonmigratory beekeeping that are likely to contribute to colony stress, and also indicate potential mitigation measures.

High royal jelly production does not impact the gut microbiome of honey bees.

BACKGROUND: Honey bees are not only essential for pollination services, but are also economically important as a source of hive products (e.g., honey, royal jelly, pollen, wax, and propolis) that are used as foods, cosmetics, and alternative medicines. Royal jelly is a popular honey bee product with multiple potential medicinal properties. To boost royal jelly production, a long-term genetic selection program of Italian honey bees (ITBs) in China has been performed, resulting in honey bee stocks (here referred to as RJBs) that produce an order of magnitude more royal jelly than ITBs. Although multiple studies have investigated the molecular basis of increased royal jelly yields, one factor that has not been considered is the role of honey bee-associated gut microbes. RESULTS: Based on the behavioral, morphological, physiological, and neurological differences between RJBs and ITBs, we predicted that the gut microbiome composition of RJBs bees would differ from ITBs. To test this hypothesis, we investigated the bacterial composition of RJB and ITB workers from an urban location and RJBs from a rural location in China. Based on 16S rRNA gene profiling, we did not find any evidence that RJBs possess a unique bacterial gut community when compared to ITBs. However, we observed differences between honey bees from the urban versus rural sites. CONCLUSIONS: Our results suggest that the environmental factors rather than stock differences are more important in shaping the bacterial composition in honey bee guts. Further studies are needed to investigate if the observed differences in relative abundance of taxa between the urban and rural bees correspond to distinct functional capabilities that impact honey bee health. Because the lifestyle, diet, and other environmental variables are different in rural and urban areas, controlled studies are needed to determine which of these factors are responsible for the observed differences in gut bacterial composition between urban and rural honeybees.

Recombination mapping of the Brazilian stingless bee Frieseomelitta varia confirms high recombination rates in social hymenoptera.

BACKGROUND: Meiotic recombination is a fundamental genetic process that shuffles allele combinations and promotes accurate segregation of chromosomes. Analyses of the ubiquitous variation of recombination rates within and across species suggest that recombination is evolving adaptively. All studied insects with advanced eusociality have shown exceptionally high recombination rates, which may represent a prominent case of adaptive evolution of recombination. However, our understanding of the relationship between social evolution and recombination rates is incomplete, partly due to lacking empirical data. Here, we present a linkage map of the monandrous, advanced eusocial Brazilian stingless bee, Frieseomelitta varia, providing the first recombination analysis in the diverse Meliponini (Hymenoptera, Apidae). RESULTS: Our linkage map includes 1417 markers in 19 linkage groups. This map spans approximately 2580 centimorgans, and comparisons to the physical genome assembly indicate that it covers more than 75 % of the 275 Megabasepairs (Mbp) F. varia genome. Thus, our study results in a genome-wide recombination rate estimate of 9.3-12.5 centimorgan per Mbp. This value is higher than estimates from nonsocial insects and comparable to other highly social species, although it does not support our prediction that monandry and strong queen-worker caste divergence of F. varia lead to even higher recombination rates than other advanced eusocial species. CONCLUSIONS: Our study expands the association between elevated recombination and sociality in the order Hymenoptera and strengthens the support for the hypothesis that advanced social evolution in hymenopteran insects invariably selects for high genomic recombination rates.

The genomic basis of evolutionary differentiation among honey bees.

In contrast to the western honey bee, Apis mellifera, other honey bee species have been largely neglected despite their importance and diversity. The genetic basis of the evolutionary diversification of honey bees remains largely unknown. Here, we provide a genome-wide comparison of three honey bee species, each representing one of the three subgenera of honey bees, namely the dwarf (Apis florea), giant (A. dorsata), and cavity-nesting (A. mellifera) honey bees with bumblebees as an outgroup. Our analyses resolve the phylogeny of honey bees with the dwarf honey bees diverging first. We find that evolution of increased eusocial complexity in Apis proceeds via increases in the complexity of gene regulation, which is in agreement with previous studies. However, this process seems to be related to pathways other than transcriptional control. Positive selection patterns across Apis reveal a trade-off between maintaining genome stability and generating genetic diversity, with a rapidly evolving piRNA pathway leading to genomes depleted of transposable elements, and a rapidly evolving DNA repair pathway associated with high recombination rates in all Apis species. Diversification within Apis is accompanied by positive selection in several genes whose putative functions present candidate mechanisms for lineage-specific adaptations, such as migration, immunity, and nesting behavior.

Reproductive activation in honeybee (Apis mellifera) workers protects against abiotic and biotic stress.

Social insect reproductives exhibit exceptional longevity instead of the classic trade-off between somatic maintenance and reproduction. Even normally sterile workers experience a significant increase in life expectancy when they assume a reproductive role. The mechanisms that enable the positive relation between the antagonistic demands of reproduction and somatic maintenance are unclear. To isolate the effect of reproductive activation, honeybee workers were induced to activate their ovaries. These reproductively activated workers were compared to controls for survival and gene expression patterns after exposure to Israeli Acute Paralysis Virus or the oxidative stressor paraquat. Reproductive activation increased survival, indicating better immunity and oxidative stress resistance. After qPCR analysis confirmed our experimental treatments at the physiological level, whole transcriptome analysis revealed that paraquat treatment significantly changed the expression of 1277 genes in the control workers but only two genes in reproductively activated workers, indicating that reproductive activation preemptively protects against oxidative stress. Significant overlap between genes that were upregulated by reproductive activation and in response to paraquat included prominent members of signalling pathways and anti-oxidants known to affect ageing. Thus, while our results confirm a central role of vitellogenin, they also point to other mechanisms to explain the molecular basis of the lack of a cost of reproduction and the exceptional longevity of social insect reproductives. Thus, socially induced reproductive activation preemptively protects honeybee workers against stressors, explaining their longevity. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Tachykinin signaling inhibits task-specific behavioral responsiveness in honeybee workers.

Behavioral specialization is key to the success of social insects and leads to division of labor among colony members. Response thresholds to task-specific stimuli are thought to proximally regulate behavioral specialization, but their neurobiological regulation is complex and not well understood. Here, we show that response thresholds to task-relevant stimuli correspond to the specialization of three behavioral phenotypes of honeybee workers in the well-studied and important Apis mellifera and Apis cerana. Quantitative neuropeptidome comparisons suggest two tachykinin-related peptides (TRP2 and TRP3) as candidates for the modification of these response thresholds. Based on our characterization of their receptor binding and downstream signaling, we confirm a functional role of tachykinin signaling in regulating specific responsiveness of honeybee workers: TRP2 injection and RNAi-mediated downregulation cause consistent, opposite effects on responsiveness to task-specific stimuli of each behaviorally specialized phenotype but not to stimuli that are unrelated to their tasks. Thus, our study demonstrates that TRP signaling regulates the degree of task-specific responsiveness of specialized honeybee workers and may control the context specificity of behavior in animals more generally.

Comparing Survival of Israeli Acute Paralysis Virus Infection among Stocks of U.S. Honey Bees.

Among numerous viruses that infect honey bees (Apis mellifera), Israeli acute paralysis virus (IAPV) can be linked to severe honey bee health problems. Breeding for virus resistance may improve honey bee health. To evaluate the potential for this approach, we compared the survival of IAPV infection among stocks from the U.S. We complemented the survival analysis with a survey of existing viruses in these stocks and assessing constitutive and induced expression of immune genes. Worker offspring from selected queens in a common apiary were inoculated with IAPV by topical applications after emergence to assess subsequent survival. Differences among stocks were small compared to variation within stocks, indicating the potential for improving honey bee survival of virus infections in all stocks. A positive relation between worker survival and virus load among stocks further suggested that honey bees may be able to adapt to better cope with viruses, while our molecular studies indicate that toll-6 may be related to survival differences among virus-infected worker bees. Together, these findings highlight the importance of viruses in queen breeding operations and provide a promising starting point for the quest to improve honey bee health by selectively breeding stock to be better able to survive virus infections.

Local Variation in Recombination Rates of the Honey Bee (Apis mellifera) Genome among Samples from Six Disparate Populations.

Meiotic recombination is an essential component of eukaryotic sexual reproduction but its frequency varies within and between genomes. Although it is well-established that honey bees have a high recombination rate with about 20 cM/Mbp, the proximate and ultimate causes of this exceptional rate are poorly understood. Here, we describe six linkage maps of the Western Honey Bee Apis mellifera that were produced with consistent methodology from samples from distinct parts of the species' near global distribution. We compared the genome-wide rates and distribution of meiotic crossovers among the six maps and found considerable differences. Overall similarity of local recombination rates among our samples was unrelated to geographic or phylogenetic distance of the populations that our samples were derived from. However, the limited sampling constrains the interpretation of our results because it is unclear how representative these samples are. In contrast to previous studies, we found only in two datasets a significant relation between local recombination rate and GC content. Focusing on regions of particularly increased or decreased recombination in specific maps, we identified several enriched gene ontologies in these regions and speculate about their local adaptive relevance. These data are contributing to an increasing comparative effort to gain an understanding of the intra-specific variability of recombination rates and their evolutionary role in honey bees and other social insects.

Transcriptomic and Epigenomic Dynamics of Honey Bees in Response to Lethal Viral Infection.

Honey bees (Apis mellifera L.) suffer from many brood pathogens, including viruses. Despite considerable research, the molecular responses and dynamics of honey bee pupae to viral pathogens remain poorly understood. Israeli Acute Paralysis Virus (IAPV) is emerging as a model virus since its association with severe colony losses. Using worker pupae, we studied the transcriptomic and methylomic consequences of IAPV infection over three distinct time points after inoculation. Contrasts of gene expression and 5 mC DNA methylation profiles between IAPV-infected and control individuals at these time points - corresponding to the pre-replicative (5 h), replicative (20 h), and terminal (48 h) phase of infection - indicate that profound immune responses and distinct manipulation of host molecular processes accompany the lethal progression of this virus. We identify the temporal dynamics of the transcriptomic response to with more genes differentially expressed in the replicative and terminal phases than in the pre-replicative phase. However, the number of differentially methylated regions decreased dramatically from the pre-replicative to the replicative and terminal phase. Several cellular pathways experienced hyper- and hypo-methylation in the pre-replicative phase and later dramatically increased in gene expression at the terminal phase, including the MAPK, Jak-STAT, Hippo, mTOR, TGF-beta signaling pathways, ubiquitin mediated proteolysis, and spliceosome. These affected biological functions suggest that adaptive host responses to combat the virus are mixed with viral manipulations of the host to increase its own reproduction, all of which are involved in anti-viral immune response, cell growth, and proliferation. Comparative genomic analyses with other studies of viral infections of honey bees and fruit flies indicated that similar immune pathways are shared. Our results further suggest that dynamic DNA methylation responds to viral infections quickly, regulating subsequent gene activities. Our study provides new insights of molecular mechanisms involved in epigenetic that can serve as foundation for the long-term goal to develop anti-viral strategies for honey bees, the most important commercial pollinator.

Egg transcriptome profile responds to maternal virus infection in honey bees, Apis mellifera.

Trans-generational disease effects include vertical pathogen transmission but also immune priming to enhance offspring immunity. Accordingly, the survival consequences of maternal virus infection can vary and its molecular consequences during early development are poorly understood. The honey bee queen is long-lived and represents the central hub for vertical virus transmission as the sole reproductive individual in her colony. Even though virus symptoms in queens are mild, viral infection may have severe consequences for the offspring. Thus, transcriptome patterns during early developmental are predicted to respond to maternal virus infection. To test this hypothesis, gene expression patterns were compared among pooled honey bee eggs laid by queens that were either infected with Deformed wing virus (DWV1), Sacbrood virus (SBV2), both viruses (DWV and SBV), or no virus. Whole transcriptome analyses revealed significant expression differences of a few genes, some of which have hitherto no known function. Despite the paucity of single gene effects, functional enrichment analyses revealed numerous biological processes in the embryos to be affected by virus infection. Effects on several regulatory pathways were consistent with maternal responses to virus infection and correlated with responses to DWV and SBV in honey bee larvae and pupae. Overall, effects on egg transcriptome patterns were specific to each virus and the results of dual-infection samples suggested synergistic effects of DWV and SBV. We interpret our results as consequences of maternal infections. Thus, this first study to document and characterize virus-associated changes in the transcriptome of honey bee eggs represents an important contribution to understanding trans-generational virus effects, although more in-depth studies are needed to understand the detailed mechanisms of how viruses affect honey bee embryos.

The Neuroproteomic Basis of Enhanced Perception and Processing of Brood Signals That Trigger Increased Reproductive Investment in Honeybee (Apis mellifera) Workers.

The neuronal basis of complex social behavior is still poorly understood. In honeybees, reproductive investment decisions are made at the colony-level. Queens develop from female-destined larvae that receive alloparental care from nurse bees in the form of ad-libitum royal jelly (RJ) secretions. Typically, the number of raised new queens is limited but genetic breeding of "royal jelly bees" (RJBs) for enhanced RJ production over decades has led to a dramatic increase of reproductive investment in queens. Here, we compare RJBs to unselected Italian bees (ITBs) to investigate how their cognitive processing of larval signals in the mushroom bodies (MBs) and antennal lobes (ALs) may contribute to their behavioral differences. A cross-fostering experiment confirms that the RJB syndrome is mainly due to a shift in nurse bee alloparental care behavior. Using olfactory conditioning of the proboscis extension reflex, we show that the RJB nurses spontaneously respond more often to larval odors compared with ITB nurses but their subsequent learning occurs at similar rates. These phenotypic findings are corroborated by our demonstration that the proteome of the brain, particularly of the ALs differs between RJBs and ITBs. Notably, in the ALs of RJB newly emerged bees and nurses compared with ITBs, processes of energy and nutrient metabolism, signal transduction are up-regulated, priming the ALs for receiving and processing the brood signals from the antennae. Moreover, highly abundant major royal jelly proteins and hexamerins in RJBs compared with ITBs during early life when the nervous system still develops suggest crucial new neurobiological roles for these well-characterized proteins. Altogether, our findings reveal that RJBs have evolved a strong olfactory response to larvae, enabled by numerous neurophysiological adaptations that increase the nurse bees' alloparental care behavior.