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Background: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses., Results: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses., Conclusions: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

Honeybee colonies (Apis mellifera) serve as attractive hosts for a variety of pathogens providing optimal temperatures, humidity, and an abundance of food. Thus, honeybees have to deal with pathogens throughout their lives and, even as larvae they are affected by severe brood diseases like the European Foulbrood caused by Melissococcus plutonius. Accordingly, it is highly adaptive that larval food jelly contains antibiotic compounds. However, although food jelly is primarily consumed by bee larvae, studies investigating the antibiotic effects of this jelly have largely concentrated on bacterial human diseases. In this study, we show that royal jelly fed to queen larvae and added to the jelly of drone and worker larvae, inhibits not only the growth of European Foulbrood-associated bacteria but also its causative agent M. plutonius. This effect is shown to be caused by the main protein (major royal jelly protein 1) of royal jelly., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Regulation of gene expression in the brain plays an important role in behavioral plasticity and decision making in response to external stimuli. However, both can be severely affected by environmental factors, such as parasites and pathogens. In honey bees, the emergence and re-emergence of pathogens and potential for pathogen co-infection and interaction have been suggested as major components that significantly impaired social behavior and survival. To understand how the honey bee is affected and responds to interacting pathogens, we co-infected workers with two prevalent pathogens of different nature, the positive single strand RNA virus Black queen cell virus (BQCV), and the Microsporidia Nosema ceranae , and explored gene expression changes in brains upon single infections and co-infections. Our data provide an important resource for research on honey bee diseases, and more generally on insect host-pathogen and pathogen-pathogen interactions. Raw and processed data are publicly available in the NCBI/GEO database: (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE81664.

Male-haploidy has independently evolved several times in different phylogenetic groups and has led to various extant lineages in the insects, Arachnida and Rotifera. Although the stability of male-haploidy as an evolutionary strategy is not well understood, various theories address the invasion of male-haploidy in diploid populations. Here two of these theories: (i) the maternal transmission hypothesis (MTH) and (ii) the deleterious mutation hypothesis (DMH), are re-investigated with an agent-based model to understand the role of genetic drift as a mechanism facilitating the spread of male-haploidy. These two hypotheses are analysed separately and comparatively, and the results suggest dominance of the MTH. In addition, comparison of the stochastic results to deterministic results using the same model structure shows how genetic drift can enhance the parameter space where male-haploidy can be expected to invade., (© 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.)

Organisms living in large groups, such as social insects, are particularly vulnerable to parasite transmission. However, they have evolved diverse defence mechanisms which are not only restricted to the individual's immune response, but also include social defences. Here, we review cases of adaptations at the individual and social level in the honeybee Apis mellifera against the ectoparasitic mite Varroa destructor and the endoparasitic microsporidians Nosema ceranae and Nosema apis. They are considered important threats to honeybee health worldwide. We highlight how individual resistance may result in tolerance at the colony level and vice versa., (Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Honey bees (Apis mellifera) are the best studied model of ageing among the social insects. As in other social insects, the reproductive queen far outlives her non-reproductive workers despite developing from the same genome in the same colony environment. Thus, the different social roles of the two female castes are critical for the profound phenotypic plasticity. In several special cases, such as the reproductive workers of Apis mellifera capensis, within-caste plasticity enables further studies of the fecundity-longevity syndrome in honey bees. At present, molecular evidence suggests that a reorganization of physiological control pathways may facilitate longevity of reproductive individuals. However, the social role and social environment of the different colony members are also very important and one of the key future questions is how much social facilitation versus internal regulation is responsible for the positive association between fecundity and longevity in honey bees., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Host-pathogen coevolution leads to reciprocal adaptations, allowing pathogens to increase host exploitation or hosts to minimise costs of infection. As pathogen resistance is often associated with considerable costs, tolerance may be an evolutionary alternative. Here, we examined the effect of two closely related and highly host dependent intracellular gut pathogens, Nosema apis and Nosema ceranae, on the energetic state in Nosema tolerant and sensitive honeybees facing the infection. We quantified the three major haemolymph carbohydrates fructose, glucose, and trehalose using high-performance liquid chromatography (HPLC) as a measure for host energetic state. Trehalose levels in the haemolymph were negatively associated with N. apis infection intensity and with N. ceranae infection regardless of the infection intensity in sensitive honeybees. Nevertheless, there was no such association in Nosema spp. infected tolerant honeybees. These findings suggest that energy availability in tolerant honeybees was not compromised by the infection. This result obtained at the individual level may also have implications at the colony level where workers in spite of a Nosema infection can still perform as well as healthy bees, maintaining colony efficiency and productivity.

Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony ("drifting"). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and Nosema spp., and Varroa mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or Nosema spp. were associated with the frequency of drifting. Only colony infestation with Varroa was associated with significantly enhanced drifting. More specifically, colonies with high Varroa infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since Varroa-infested colonies show an enhanced attraction of drifting workers, and not only those infected with Varroa and its associated pathogens, infestation by Varroa may also facilitate the uptake of other pests and parasites.

Apoptosis is not only pivotal for development, but also for pathogen defence in multicellular organisms. Although numerous intracellular pathogens are known to interfere with the host's apoptotic machinery to overcome this defence, its importance for host-parasite coevolution has been neglected. We conducted three inoculation experiments to investigate in the apoptotic respond during infection with the intracellular gut pathogen Nosema ceranae, which is considered as potential global threat to the honeybee (Apis mellifera) and other bee pollinators, in sensitive and tolerant honeybees. To explore apoptotic processes in the gut epithelium, we visualised apoptotic cells using TUNEL assays and measured the relative expression levels of subset of candidate genes involved in the apoptotic machinery using qPCR. Our results suggest that N. ceranae reduces apoptosis in sensitive honeybees by enhancing inhibitor of apoptosis protein-(iap)-2 gene transcription. Interestingly, this seems not be the case in Nosema tolerant honeybees. We propose that these tolerant honeybees are able to escape the manipulation of apoptosis by N. ceranae, which may have evolved a mechanism to regulate an anti-apoptotic gene as key adaptation for improved host invasion.

The ectoparasitic mite Varroa destructor is a major global threat to the Western honeybee Apis mellifera. This mite was originally a parasite of A. cerana in Asia but managed to spill over into colonies of A. mellifera which had been introduced to this continent for honey production. To date, only two almost clonal types of V. destructor from Korea and Japan have been detected in A. mellifera colonies. However, since both A. mellifera and A. cerana colonies are kept in close proximity throughout Asia, not only new spill overs but also spill backs of highly virulent types may be possible, with unpredictable consequences for both honeybee species. We studied the dispersal and hybridisation potential of Varroa from sympatric colonies of the two hosts in Northern Vietnam and the Philippines using mitochondrial and microsatellite DNA markers. We found a very distinct mtDNA haplotype equally invading both A. mellifera and A. cerana in the Philippines. In contrast, we observed a complete reproductive isolation of various Vietnamese Varroa populations in A. mellifera and A. cerana colonies even if kept in the same apiaries. In light of this variance in host specificity, the adaptation of the mite to its hosts seems to have generated much more genetic diversity than previously recognised and the Varroa species complex may include substantial cryptic speciation.

The evolution of eusociality is one of the major transitions in evolution, but the underlying genomic changes are unknown. We compared the genomes of 10 bee species that vary in social complexity, representing multiple independent transitions in social evolution, and report three major findings. First, many important genes show evidence of neutral evolution as a consequence of relaxed selection with increasing social complexity. Second, there is no single road map to eusociality; independent evolutionary transitions in sociality have independent genetic underpinnings. Third, though clearly independent in detail, these transitions do have similar general features, including an increase in constrained protein evolution accompanied by increases in the potential for gene regulation and decreases in diversity and abundance of transposable elements. Eusociality may arise through different mechanisms each time, but would likely always involve an increase in the complexity of gene networks., (Copyright © 2015, American Association for the Advancement of Science.)

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats., Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits., Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.

Microbial pathogens are thought to have a profound impact on insect populations. Honey bees are suffering from elevated colony losses in the northern hemisphere possibly because of a variety of emergent microbial pathogens, with which pesticides may interact to exacerbate their impacts. To reveal such potential interactions, we administered at sublethal and field realistic doses one neonicotinoid pesticide (thiacloprid) and two common microbial pathogens, the invasive microsporidian Nosema ceranae and black queen cell virus (BQCV), individually to larval and adult honey bees in the laboratory. Through fully crossed experiments in which treatments were administered singly or in combination, we found an additive interaction between BQCV and thiacloprid on host larval survival likely because the pesticide significantly elevated viral loads. In adult bees, two synergistic interactions increased individual mortality: between N. ceranae and BQCV, and between N. ceranae and thiacloprid. The combination of two pathogens had a more profound effect on elevating adult mortality than N. ceranae plus thiacloprid. Common microbial pathogens appear to be major threats to honey bees, while sublethal doses of pesticide may enhance their deleterious effects on honey bee larvae and adults. It remains an open question as to whether these interactions can affect colony survival., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The mite Varroa destructor is one of the most dangerous parasites of the Western honeybee (Apis mellifera) causing enormous colony losses worldwide. Various chemical treatments for the control of the Varroa mite are currently in use, which, however, lead to residues in bee products and often to resistance in mites. This facilitated the exploration of alternative treatment methods and breeding for mite resistant honeybees has been in focus for breeders in many parts of the world with variable results. Another approach has been applied to a honeybee population on Gotland (Sweden) that was exposed to natural selection and survived Varroa-infestation for more than 10years without treatment. Eventually this population became resistant to the parasite by suppressing the reproduction of the mite. A previous QTL mapping study had identified a region on chromosome 7 with major loci contributing to the mite resistance. Here, a microsatellite scan of the significant candidate QTL regions was used to investigate potential footprints of selection in the original population by comparing the study population on Gotland before (2000) and after selection (2007). Genetic drift had caused an extreme loss of genetic diversity in the 2007 population for all genetic markers tested. In addition to this overall reduction of heterozygosity, two loci on chromosome 7 showed an even stronger and significant reduction in diversity than expected from genetic drift alone. Within the selective sweep eleven genes are annotated, one of them being a putative candidate to interfere with reduced mite reproduction. A glucose-methanol-choline oxidoreductase (GMCOX18) might be involved in changing volatiles emitted by bee larvae that might be essential to trigger oogenesis in Varroa., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The allocation of large numbers of workers facilitates the swift intake of locally available resources which is essential for ant colony survival. To organise the traffic between nest and food source, the black-meadow ant Formica pratensis establishes permanent trunk trails, which are maintained by the ants. To unravel the ant organisation and potential traffic rules on these trails, we analysed velocity and lane segregation under various densities by experimentally changing feeding regimes. Even under the highest ant densities achieved, we never observed any traffic jams. On the contrary, velocity increased after supplementary feeding despite an enhanced density. Furthermore, inbound ants returning to the nest had a higher velocity than those leaving the colony. Whilst at low and medium density the ants used the centre of the trail, they used the full width of the trail at high density. Outbound ants also showed some degree of lane segregation which contributes to traffic organisation.

The giant honeybee Apis dorsata often forms dense colony aggregations which can include up to 200 often closely related nests in the same location, setting the stage for inbred matings. Yet, like in all other Apis species, A. dorsata queens mate in mid-air on lek like drone congregation areas (DCAs) where large numbers of males gather in flight. We here report how the drone composition of A. dorsata DCAs facilitates outbreeding, taking into the account both spatial (three DCAs) and temporal (subsequent sampling days) dynamics. We compared the drones' genotypes at ten microsatellite DNA markers with those of the queen genotypes of six drone-producing colonies located close to the DCAs (Tenom, Sabah, Malaysia). None of 430 sampled drones originated from any of these nearby colonies. Moreover, we estimated that 141 unidentified colonies were contributing to the three DCAs. Most of these colonies were participating multiple times in the different locations and/or during the consecutive days of sampling. The drones sampled in the DCAs could be attributed to six subpopulations. These were all admixed in all DCA samples, increasing the effective population size an order of magnitude and preventing matings between potentially related queens and drones.

Honeybee colonies offer an excellent environment for microbial pathogen development. The highest virulent, colony killing, bacterial agents are Paenibacillus larvae causing American foulbrood (AFB), and European foulbrood (EFB) associated bacteria. Besides the innate immune defense, honeybees evolved behavioral defenses to combat infections. Foraging of antimicrobial plant compounds plays a key role for this "social immunity" behavior. Secondary plant metabolites in floral nectar are known for their antimicrobial effects. Yet, these compounds are highly plant specific, and the effects on bee health will depend on the floral origin of the honey produced. As worker bees not only feed themselves, but also the larvae and other colony members, honey is a prime candidate acting as self-medication agent in honeybee colonies to prevent or decrease infections. Here, we test eight AFB and EFB bacterial strains and the growth inhibitory activity of three honey types. Using a high-throughput cell growth assay, we show that all honeys have high growth inhibitory activity and the two monofloral honeys appeared to be strain specific. The specificity of the monofloral honeys and the strong antimicrobial potential of the polyfloral honey suggest that the diversity of honeys in the honey stores of a colony may be highly adaptive for its "social immunity" against the highly diverse suite of pathogens encountered in nature. This ecological diversity may therefore operate similar to the well-known effects of host genetic variance in the arms race between host and parasite.

The unique nomadic life-history pattern of army ants (army ant adaptive syndrome), including obligate colony fission and strongly male-biased sex-ratios, makes army ants prone to heavily reduced effective population sizes (Ne). Excessive multiple mating by queens (polyandry) has been suggested to compensate these negative effects by increasing genetic variance in colonies and populations. However, the combined effects and evolutionary consequences of polyandry and army ant life history on genetic colony and population structure have only been studied in a few selected species. Here we provide new genetic data on paternity frequencies, colony structure and paternity skew for the five Neotropical army ants Eciton mexicanum, E. vagans, Labidus coecus, L. praedator and Nomamyrmex esenbeckii; and compare those data among a total of nine army ant species (including literature data). The number of effective matings per queen ranged from about 6 up to 25 in our tested species, and we show that such extreme polyandry is in two ways highly adaptive. First, given the detected low intracolonial relatedness and population differentiation extreme polyandry may counteract inbreeding and low Ne. Second, as indicated by a negative correlation of paternity frequency and paternity skew, queens maximize intracolonial genotypic variance by increasingly equalizing paternity shares with higher numbers of sires. Thus, extreme polyandry is not only an integral part of the army ant syndrome, but generally adaptive in social insects by improving genetic variance, even at the high end spectrum of mating frequencies.

To date five different theories compete in explaining the biological mechanisms of senescence or ageing in invertebrates. Physiological, genetical, and environmental mechanisms form the image of ageing in individuals and groups. Social insects, especially the honeybee Apis mellifera, present exceptional model systems to study developmentally related ageing. The extremely high phenotypic plasticity for life expectancy resulting from the female caste system provides a most useful system to study open questions with respect to ageing. Here, we used long-lived winter worker honeybees and measured transcriptional changes of 14 antioxidative enzyme, immunity, and ageing-related (insulin/insulin-like growth factor signaling pathway) genes at two time points during hibernation. Additionally, worker bees were challenged with a bacterial infection to test ageing- and infection-associated immunity changes. Gene expression levels for each group of target genes revealed that ageing had a much higher impact than the bacterial challenge, notably for immunity-related genes. Antimicrobial peptide and antioxidative enzyme genes were significantly upregulated in aged worker honeybees independent of bacterial infections. The known ageing markers vitellogenin and IlP-1 were opposed regulated with decreasing vitellogenin levels during ageing. The increased antioxidative enzyme and antimicrobial peptide gene expression may contribute to a retardation of senescence in long-lived hibernating worker honeybees., (© The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the honeybee, Apis mellifera, the queen larvae are fed with a diet exclusively composed of royal jelly (RJ), a secretion of the hypopharyngeal gland of young worker bees that nurse the brood. Up to 15% of RJ is composed of proteins, the nine most abundant of which have been termed major royal jelly proteins (MRJPs). Although it is widely accepted that RJ somehow determines the fate of a female larva and in spite of considerable research efforts, there are surprisingly few studies that address the biochemical characterisation and functions of these MRJPs. Here we review the research on MRJPs not only in honeybees but in hymenopteran insects in general and provide metadata analyses on genome organisation of mrjp genes, corroborating previous reports that MRJPs have important functions for insect development and not just a nutritional value for developing honeybee larvae., (© 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society.)

Both climatic and geographical factors play an important role for the biogeographical distribution of species. The Carpathian mountain ridge has been suggested as a natural geographical divide between the two honeybee subspecies Apis mellifera carnica and A. m. macedonica. We sampled one worker from one colony each at 138 traditional apiaries located across the Carpathians spanning from the Hungarian plains to the Danube delta. All samples were sequenced at the mitochondrial tRNA(Leu)-cox2 intergenic region and genotyped at twelve microsatellite loci. The Carpathians had only limited impact on the biogeography because both subspecies were abundant on either side of the mountain ridge. In contrast, subspecies differentiation strongly correlated with the various temperature zones in Romania. A. m. carnica is more abundant in regions with the mean average temperature below 9 °C, whereas A. m. macedonica honeybees are more frequent in regions with mean temperatures above 9 °C. This range selection may have impact on the future biogeography in the light of anticipated global climatic changes., (© 2014 John Wiley & Sons Ltd.)

Nosema is a microsporidian parasite of the honeybee, which infects the epithelial cells of the gut. In Denmark, honeybee colonies have been selectively bred for the absence of Nosema over decades, resulting in a breeding line that is tolerant toward Nosema infections. As the tolerance toward the Nosema infection is a result of artificial selection, we screened chromosome 14 for a selective sweep with microsatellite markers, where a major quantitative trait locus (QTL) had been identified to be involved in the reduction in Nosema spores in the honeybees. By comparing the genetic variability of 10 colonies of the selected honeybee strain with a population sample from 22 unselected colonies, a selective sweep was revealed within the previously identified QTL region. The genetic variability of the swept loci was not only reduced in relation to the flanking markers on chromosome 14 within the selected strain but also significantly reduced compared with the same region in the unselected honeybees. This confirmed the results of the previous QTL mapping for reduced Nosema infections. The success of the selective breeding may have driven the selective sweep found in our study., (© 2013 Stichting International Foundation for Animal Genetics.)

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

Background: In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee's genome., Results: We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals., Conclusions: The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

Honeybees have been studied for centuries, starting with Aristotle, who wrote the first book about bee breeding. More than 2000 years later, the honeybee entered the genomic era as the first social insect whose genome was sequenced, leading to significant insight into the molecular mechanisms underlying social behavior. In addition, gene expression studies and knockdown using RNAi have extended the understanding of social interactions. Much of the work has focused on caste determination - the mechanism that results in reproductive division of labor, division of labor within the worker caste, and worker reproduction - an essential process underlying eusociality. Here we review the molecular factors involved in caste determination and the differential regulation of caste-specific genes. Recent findings suggest that division of labor is influenced by a small number of loci showing high levels of pleiotropy, suggesting that changes in a small number of genes lead to large changes in the phenotype., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The article by Harpur et al. (2012) 'Management increases genetic diversity of honey bees via admixture' concludes that '…honey bees do not suffer from reduced genetic diversity caused by management and, consequently, that reduced genetic diversity is probably not contributing to declines of managed Apis mellifera populations'. In the light of current honeybee and beekeeping declines and their consequences for honeybee conservation and the pollination services they provide, we would like to express our concern about the conclusions drawn from the results of Harpur et al. (2012). While many honeybee management practices do not imply admixture, we are convinced that the large-scale genetic homogenization of admixed populations could drive the loss of valuable local adaptations. We also point out that the authors did not account for the extensive gene flow that occurs between managed and wild/feral honeybee populations and raise concerns about the data set used. Finally, we caution against underestimating the importance of genetic diversity for honeybee colonies and highlight the importance of promoting the use of endemic honeybee subspecies in apiculture., (© 2013 John Wiley & Sons Ltd.)

We present RESTseq, an improved approach for a cost efficient, highly flexible and repeatable enrichment of DNA fragments from digested genomic DNA using Next Generation Sequencing platforms including small scale Personal Genome sequencers. Easy adjustments make it suitable for a wide range of studies requiring SNP detection or SNP genotyping from fine-scale linkage mapping to population genomics and population genetics also in non-model organisms. We demonstrate the validity of our approach by comparing two honeybee and several stingless bee samples.

Microsatellites, or simple sequence repeats (SSRs), are common and widespread DNA elements in genomes of many organisms. However, their dynamics in genome evolution is unclear, whereby they are thought to evolve neutrally. More available genome sequences along with dated phylogenies allowed for studying the evolution of these repetitive DNA elements along evolutionary time scales. This could be used to compare rates of genome evolution. We show that SSRs in insects can be retained for several hundred million years. Different types of microsatellites seem to be retained longer than others. By comparing Dipteran with Hymenopteran species, we found very similar patterns of SSR loss during their evolution, but both taxa differ profoundly in the rate. Relative to divergence time, Diptera lost SSRs twice as fast as Hymenoptera. The loss of SSRs on the Drosophila melanogaster X-chromosome was higher than on the other chromosomes. However, accounting for generation time, the Diptera show an 8.5-fold slower rate of SSR loss than the Hymenoptera, which, in contrast to previous studies, suggests a faster genome evolution in the latter. This shows that generation time differences can have a profound effect. A faster genome evolution in these insects could be facilitated by several factors very different to Diptera, which is discussed in light of our results on the haplodiploid D. melanogaster X-chromosome. Furthermore, large numbers of SSRs can be found to be in synteny and thus could be exploited as a tool to investigate genome structure and evolution.

Drone aggregations are a widespread phenomenon in many stingless bee species (Meliponini), but the ultimate and proximate causes for their formation are still not well understood. One adaptive explanation for this phenomenon is the avoidance of inbreeding, which is especially detrimental for stingless bees due to the combined effects of the complementary sex-determining system and the small effective population size caused by eusociality and monandry. We analyzed the temporal genetic dynamics of a drone aggregation of the stingless bee Scaptotrigona mexicana with microsatellite markers over a time window of four weeks. We estimated the drones of the aggregation to originate from a total of 55 colonies using sibship re-construction. There was no detectable temporal genetic differentiation or sub-structuring in the aggregation. Most important, we could exclude all colonies in close proximity of the aggregation as origin of the drones in the aggregation, implicating that they originate from more distant colonies. We conclude that the diverse genetic composition and the distant origin of the drones of the S. mexicana drone congregation provides an effective mechanism to avoid mating among close relatives.

Propolis is derived from plant resins, collected by honeybees (Apis mellifera) and renown for its antibacterial properties. Here we test the antibacterial effects of ethanolic extracts of propolis from different origins on Paenibacillus larvae, the bacterial pathogen that causes American Foulbrood, a larval disease that can kill the honeybee colony. All tested propolis samples inhibited significantly the growth of P. larvae tested in vitro. The extracts showed major differences in the content of total flavonoids (ranging from 2.4% to 16.4%) and the total polyphenols (ranging between 23.3% and 63.2%). We found that it is not only the content of compounds in propolis, which influences the strength of antimicrobial effects but there is also a significant interaction effect among flavonoids of the propolis extracts. We propose that interaction effects among the various chemical compounds in propolis should be taken into account when considering the antibacterial effects against honeybee pathogens., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bumblebees are of profound ecological importance because of the pollination services they provide in natural and agricultural ecosystems. Any decline of these pollinators is therefore of great concern for ecosystem functioning. Increased parasite pressures have been discussed as a major factor for the loss of pollinators. One of the main parasites of bumblebees is Nosema bombi, an intracellular microsporidian parasite with considerable impact on the vitality of the host. Here we study the effect of host colony density and host genetic variability on N. bombi infections in natural populations of the bumblebee Bombus terrestris. We sampled males and workers from six B. terrestris populations located in an agricultural landscape in Middle Sweden to determine the prevalence and degree of N. bombi infections. All individuals were genotyped with five microsatellite markers to infer the colony densities in the sampled populations and the genetic variability of the host population. We confirmed that genetic variability and sex significantly correlate with the degree of infection with N. bombi. Males and workers with lower genetic variability had significantly higher infection levels than average. Also colony density had a significant impact on the degree of infection, with high density populations having higher infected individuals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Honey bee colonies (Apis mellifera) have been selected for low level of Nosema in Denmark over decades and Nosema is now rarely found in bee colonies from these breeding lines. We compared the immune response of a selected and an unselected honey bee lineage, taking advantage of the haploid males to study its potential impact on the tolerance toward Nosema ceranae, a novel introduced microsporidian pathogen. After artificial infections of the N. ceranae spores, the lineage selected for Nosema tolerance showed a higher N. ceranae spore load, a lower mortality and an up-regulated immune response. The differences in the response of the innate immune system between the selected and unselected lineage were strongest at day six post infection. In particular genes of the Toll pathway were up-regulated in the selected strain, probably is the main immune pathway involved in N. ceranae infection response. After decades of selective breeding for Nosema tolerance in the Danish strain, it appears these bees are tolerant to N. ceranae infections., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The microsporidium Nosema ceranae is a newly prevalent parasite of the European honey bee (Apis mellifera). Although this parasite is presently spreading across the world into its novel host, the mechanisms by it which affects the bees and how bees respond are not well understood. We therefore performed an extensive characterization of the parasite effects at the molecular level by using genetic and biochemical tools. The transcriptome modifications at the midgut level were characterized seven days post-infection with tiling microarrays. Then we tested the bee midgut response to infection by measuring activity of antioxidant and detoxification enzymes (superoxide dismutases, glutathione peroxidases, glutathione reductase, and glutathione-S-transferase). At the gene-expression level, the bee midgut responded to N. ceranae infection by an increase in oxidative stress concurrent with the generation of antioxidant enzymes, defense and protective response specifically observed in the gut of mammals and insects. However, at the enzymatic level, the protective response was not confirmed, with only glutathione-S-transferase exhibiting a higher activity in infected bees. The oxidative stress was associated with a higher transcription of sugar transporter in the gut. Finally, a dramatic effect of the microsporidia infection was the inhibition of genes involved in the homeostasis and renewal of intestinal tissues (Wnt signaling pathway), a phenomenon that was confirmed at the histological level. This tissue degeneration and prevention of gut epithelium renewal may explain early bee death. In conclusion, our integrated approach not only gives new insights into the pathological effects of N. ceranae and the bee gut response, but also demonstrate that the honey bee gut is an interesting model system for studying host defense responses.

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called "BeeDoctor", was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. "BeeDoctor" is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. "BeeDoctor" was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the "BeeDoctor", virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

Varroa destructor is a highly virulent ectoparasitic mite of the honey bee Apis mellifera and a major cause of colony losses for global apiculture. Typically, chemical treatment is essential to control the parasite population in the honey bee colony. Nevertheless a few honey bee populations survive mite infestation without any treatment. We used one such Varroa mite tolerant honey bee lineage from the island of Gotland, Sweden, to identify quantitative trait loci (QTL) controlling reduced mite reproduction. We crossed a queen from this tolerant population with drones from susceptible colonies to rear hybrid queens. Two hybrid queens were used to produce a mapping population of haploid drones. We discriminated drone pupae with and without mite reproduction, and screened the genome for potential QTL using a total of 216 heterozygous microsatellite markers in a bulk segregant analysis. Subsequently, we fine mapped three candidate target regions on chromosomes 4, 7, and 9. Although the individual effect of these three QTL was found to be relatively small, the set of all three had significant impact on suppression of V. destructor reproduction by epistasis. Although it is in principle possible to use these loci for marker-assisted selection, the strong epistatic effects between the three loci complicate selective breeding programs with the Gotland Varroa tolerant honey bee stock.

In eusocial insects the production of daughters is generally restricted to mated queens, and unmated workers are functionally sterile. The evolution of this worker sterility has been plausibly explained by kin selection theory [Hamilton W (1964) J Theor Biol 7:1-52], and many traits have evolved to prevent conflict over reproduction among the females in an insect colony. In honeybees (Apis mellifera), worker reproduction is regulated by the queen, brood pheromones, and worker policing. However, workers of the Cape honeybee, Apis mellifera capensis, can evade this control and establish themselves as social parasites by activating their ovaries, parthenogenetically producing diploid female offspring (thelytoky) and producing queen-like amounts of queen pheromones. All these traits have been shown to be strongly influenced by a single locus on chromosome 13 [Lattorff HMG, et al. (2007) Biol Lett 3:292-295]. We screened this region for candidate genes and found that alternative splicing of a gene homologous to the gemini transcription factor of Drosophila controls worker sterility. Knocking out the critical exon in a series of RNAi experiments resulted in rapid worker ovary activation-one of the traits characteristic of the social parasites. This genetic switch may be controlled by a short intronic splice enhancer motif of nine nucleotides attached to the alternative splice site. The lack of this motif in parasitic Cape honeybee clones suggests that the removal of nine nucleotides from the altruistic worker genome may be sufficient to turn a honeybee from an altruistic worker into a parasite.

RNA interference has been successfully used in adult honeybees, but there are only few reports about abdominal application of dsRNA/siRNA which have reached more distant tissues than the fat body. We studied systemic RNAi in honeybees by injecting fluorescent siRNA of the ubiquitously expressed honeybee homologue of the Glycerol-3-Phosphate Dehydrogenase (amGpdh) into the abdomens of adult bees and followed them by laser scanning microscopy and qPCR. The fat body was the sole tissue emitting fluorescence and showing a decreased gene expression, whereas the siRNA had apparently not reached the other tissues. Therefore, we conclude that certain genes in other tissues than the fat body cannot be easily reached by injecting siRNA into the body cavity. In particular, the lack of amGpdh knock down in ovaries after amGpdh dsRNA injection, supports that in some cases it may be particularly difficult to interfere with gene expression in ovaries by intra-abdominal injection. In these cases alternative inhibition techniques may be required to achieve an organismic non-lethal disruption of transcription., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Cooperation and conflict in social insects are closely linked to the genetic structure of the colony. Kin selection theory predicts conflict over the production of males between the workers and the queen and between the workers themselves, depending on intra-colonial relatedness but also on other factors like colony efficiency, sex ratios, cost of worker reproduction and worker dominance behaviour. In most bumblebee (Bombus) species the queen wins this conflict and often dominates male production. However, most studies in bumblebees have been conducted with only a few selected, mostly single mated species from temperate climate regions. Here we study the genetic colony composition of the facultative polyandrous neotropical bumblebee Bombus wilmattae, to assess the outcome of the queen-worker conflict over male production and to detect potential worker policing., Results: A total of 120 males from five colonies were genotyped with up to nine microsatellite markers to infer their parentage. Four of the five colonies were queen right at point of time of male sampling, while one had an uncertain queen status. The workers clearly dominated production of males with an average of 84.9% +/- 14.3% of males being worker sons. In the two doubly mated colonies 62.5% and 96.7% of the male offspring originated from workers and both patrilines participated in male production. Inferring the mother genotypes from the male offspring, between four to eight workers participated in the production of males., Conclusions: In this study we show that the workers clearly win the queen-worker conflict over male production in B. wilmattae, which sets them apart from the temperate bumblebee species studied so far. Workers clearly dominated male production in the singly as well the doubly mated colonies, with up to eight workers producing male offspring in a single colony. Moreover no monopolization of reproduction by single workers occurred.

Division of labour is a fundamental property of any social system. The specialization of different individuals in different tasks increases the overall work performance and efficiency. Specialization is thought to be the very foundation of the success of human societies but also in complex colonies of social insects. In human societies an advanced form of division of labour, especially since the industrialisation, is shift work, where individuals perform the same task but in subsequent cohorts in time. Although social insects can measure and are aware of time, shift work has not been documented in colonies of social insects so far. We observed foragers of two honeybee (Apis mellifera) colonies (approximately 140 workers each) and genotyped them with microsatellite DNA markers. We determined paternity and assigned them to the various subfamilies in the colony to test whether there is genetic variance for shift work in foraging honeybees. We could show that the patriline identity of the foragers had a significant effect on foraging either in the morning or evening. Individual foragers differed in their preference for the "early" or "late" shift, and shift work indeed existed in the colony.

Determining the extent and causes of barriers to gene flow is essential for understanding sympatric speciation, but the practical difficulties of quantifying reproductive isolation remain an obstacle to analysing this process. Social parasites are common in eusocial insects and tend to be close phylogenetic relatives of their hosts (= Emery's rule). Sympatric speciation caused by reproductive isolation between host and parasite is a possible evolutionary pathway. Socially parasitic workers of the Cape honeybee, Apis mellifera capensis, produce female clonal offspring parthenogenetically and invade colonies of the neighbouring subspecies A. m. scutellata. In the host colony, socially parasitic workers can become pseudoqueens, an intermediate caste with queenlike pheromone secretion. Here, we show that over an area of approximately 275.000 km², all parasitic workers bear the genetic signature of a clone founded by a single ancestral worker genotype. Any gene flow from the host to the parasite is impossible because honeybee workers cannot mate. Gene flow from the parasite to the host is possible, as parasitic larvae can develop into queens. However, we show that despite sympatric coexistence for more than a decade, gene flow between host and social parasite (F(st) = 0.32) and hybridizations (0.71%) are rare, resulting in reproductive isolation. Our data suggest a new barrier to gene flow in sympatry, which is not based on assortative matings but on thelytoky and reproductive division of labour in eusocial insects, thereby suggesting a new potential pathway to Emery's rule., (© 2010 The Authors. Journal of Evolutionary Biology © 2010 European Society For Evolutionary Biology.)

Background: The bumblebee Bombus terrestris is an ecologically and economically important pollinator and has become an important biological model system. To study fundamental evolutionary questions at the genomic level, a high resolution genetic linkage map is an essential tool for analyses ranging from quantitative trait loci (QTL) mapping to genome assembly and comparative genomics. We here present a saturated linkage map and match it with the Apis mellifera genome using homologous markers. This genome-wide comparison allows insights into structural conservations and rearrangements and thus the evolution on a chromosomal level., Results: The high density linkage map covers ~ 93% of the B. terrestris genome on 18 linkage groups (LGs) and has a length of 2'047 cM with an average marker distance of 4.02 cM. Based on a genome size of ~ 430 Mb, the recombination rate estimate is 4.76 cM/Mb. Sequence homologies of 242 homologous markers allowed to match 15 B. terrestris with A. mellifera LGs, five of them as composites. Comparing marker orders between both genomes we detect over 14% of the genome to be organized in synteny and 21% in rearranged blocks on the same homologous LG., Conclusions: This study demonstrates that, despite the very high recombination rates of both A. mellifera and B. terrestris and a long divergence time of about 100 million years, the genomes' genetic architecture is highly conserved. This reflects a slow genome evolution in these bees. We show that data on genome organization and conserved molecular markers can be used as a powerful tool for comparative genomics and evolutionary studies, opening up new avenues of research in the Apidae.

We compared the mortality of honeybee (Apis mellifera) drone and worker larvae from a single queen under controlled in vitro conditions following infection with Paenibacillus larvae, a bacterium causing the brood disease American Foulbrood (AFB). We also determined absolute P. larvae cell numbers and lethal titres in deceased individuals of both sexes up to 8 days post infection using quantitative real-time PCR (qPCR). Our results show that in drones the onset of infection induced mortality is delayed by 1 day, the cumulative mortality is reduced by 10% and P. larvae cell numbers are higher than in worker larvae. Since differences in bacterial cell titres between sexes can be explained by differences in body size, larval size appears to be a key parameter for a lethal threshold in AFB tolerance. Both means and variances for lethal thresholds are similar for drone and worker larvae suggesting that drone resistance phenotypes resemble those of related workers., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Although pollinator declines are a global biodiversity threat, the demography of the western honeybee (Apis mellifera) has not been considered by conservationists because it is biased by the activity of beekeepers. To fill this gap in pollinator decline censuses and to provide a broad picture of the current status of honeybees across their natural range, we used microsatellite genetic markers to estimate colony densities and genetic diversity at different locations in Europe, Africa, and central Asia that had different patterns of land use. Genetic diversity and colony densities were highest in South Africa and lowest in Northern Europe and were correlated with mean annual temperature. Confounding factors not related to climate, however, are also likely to influence genetic diversity and colony densities in honeybee populations. Land use showed a significantly negative influence over genetic diversity and the density of honeybee colonies over all sampling locations. In Europe honeybees sampled in nature reserves had genetic diversity and colony densities similar to those sampled in agricultural landscapes, which suggests that the former are not wild but may have come from managed hives. Other results also support this idea: putative wild bees were rare in our European samples, and the mean estimated density of honeybee colonies on the continent closely resembled the reported mean number of managed hives. Current densities of European honeybee populations are in the same range as those found in the adverse climatic conditions of the Kalahari and Saharan deserts, which suggests that beekeeping activities do not compensate for the loss of wild colonies. Our findings highlight the importance of reconsidering the conservation status of honeybees in Europe and of regarding beekeeping not only as a profitable business for producing honey, but also as an essential component of biodiversity conservation.

Males of the honeybee (Apis mellifera) fly to specific drone congregation areas (DCAs), which virgin queens visit in order to mate. From the thousands of drones that are reared in a single colony, only very few succeed in copulating with a queen, and therefore, a strong selection is expected to act on adult drones during their mating flights. In consequence, the gathering of drones at DCAs may serve as an indirect mate selection mechanism, assuring that queens only mate with those individuals having a better flight ability and a higher responsiveness to the queen's visual and chemical cues. Here, we tested this idea relying on wing fluctuating asymmetry (FA) as a measure of phenotypic quality. By recapturing marked drones at a natural DCA and comparing their size and FA with a control sample of drones collected at their maternal hives, we were able to detect any selection on wing size and wing FA occurring during the mating flights. Although we found no solid evidence for selection on wing size, wing FA was found to be significantly lower in the drones collected at the DCA than in those collected at the hives. Our results demonstrate the action of selection during drone mating flights for the first time, showing that developmental stability can influence the mating ability of honeybee drones. We therefore conclude that selection during honeybee drone mating flights may confer some fitness advantages to the queens.

We present details and characteristics of 123 novel polymorphic microsatellite DNA loci for Bombus terrestris. Thirty-four of these loci have been tested in nine other Bombus species and 25 of them showed polymorphisms in at least one species. These microsatellite DNA loci together with the already established 60 loci will be useful for characterizing wild and managed populations of B. terrestris and other Bombus species as well as for detailed genetic studies in including mapping studies and genome annotations., (© 2009 Blackwell Publishing Ltd.)

Social cooperative spiders from diverse taxonomic families share life-history and demographic traits, including highly inbred colony structure. The combination of traits suggests constrained pathways for social evolution in spiders. The genus Stegodyphus has three independently evolved social species, which can be used as replicate samples to analyse population constraints in evolutionary time. We tested colony structure and population history of the social S. mimosarum from South and East Africa using mitochondrial DNA variation, and we compared the results to published data for the independently evolved social congener S. dumicola. S. mimosarum had many and diverse haplotypes (5-7% sequence divergence for ND1) but colonies were monomorphic and genealogically similar haplotypes occurred in abutting regions. These findings are nearly identical to results for S. dumicola and imply similar colony-level processes over evolutionary time in independently evolved social species. These population dynamics are discussed with respect to the apparent lack of cladogenesis in social spiders.