BackgroundDisease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies.ResultsWe sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae.ConclusionsOur results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

BackgroundThe Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees.ResultsAfter confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect.ConclusionsOur data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1193-6) contains supplementary material, which is available to authorized users.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype–based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

Social organisms combat pathogens through individual innate immune responses or through social immunity—behaviors among individuals that limit pathogen transmission within groups. Although we have a relatively detailed understanding of the genetics and evolution of the innate immune system of animals, we know little about social immunity. Addressing this knowledge gap is crucial for understanding how life-history traits influence immunity, and identifying if trade-offs exist between innate and social immunity. Hygienic behavior in the Western honey bee, Apis mellifera , provides an excellent model for investigating the genetics and evolution of social immunity in animals. This heritable, colony-level behavior is performed by nurse bees when they detect and remove infected or dead brood from the colony. We sequenced 125 haploid genomes from two artificially selected highly hygienic populations and a baseline unselected population. Genomic contrasts allowed us to identify a minimum of 73 genes tentatively associated with hygienic behavior. Many genes were within previously discovered QTLs associated with hygienic behavior and were predictive of hygienic behavior within the unselected population. These genes were often involved in neuronal development and sensory perception in solitary insects. We found that genes associated with hygienic behavior have evidence of positive selection within honey bees ( Apis ), supporting the hypothesis that social immunity contributes to fitness. Our results indicate that genes influencing developmental neurobiology and behavior in solitary insects may have been co-opted to give rise to a novel and adaptive social immune phenotype in honey bees.

27We present a novel way to select for highly polygenic traits. For millennia, 28 humans have used observable phenotypes to selectively breed stronger or more 29 productive livestock and crops. Selection on genotype, using single-nucleotide 30 polymorphisms (SNPs) and quantitative trait loci (QTLs), is also now applied broadly in 31 livestock breeding programs; however, selection on protein or mRNA expression 32 markers have not been proved useful yet. Here we demonstrate the utility of protein 33 markers to select for disease-resistant behaviour in the European honey bees (Apis 34 mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating 35 cis and trans effects from many genomic loci, may overcome limitations of genomic 36 markers to allow for selection. After three generations of selection, the resulting stock 37 performed as well or better than bees selected using phenotype-based assessment of 38 this trait, when challenged with disease. This is the first demonstration of the efficacy of 39 protein markers for selective breeding in any agricultural species, plant or animal. 40 41 Significance statement 42The honey bee has been in the news a lot recently, largely because of world-43 wide die-offs due to the parasitic Varroa mite, which is becoming resistant to the 44 chemical controls the bee industry uses. In this study, we show that robust expression 45 biomarkers of a disease-resistance trait can be used, in an out-bred population, to select 46 for that trait. After three generations of selection, the resulting stock performed as well or 47 better than bees selected using the phenotypic best method for assessing this trait when 48 challenged with disease. This is the first demonstration of an expression marker for 49 selective breeding in any agricultural species, plant or animal. This also represents a 50 completely novel way to select for highly polygenic traits. 51Bees do, however, have some effective disease-resistance traits, which also 67 happen to be highly polygenic: one example is the social immunity function known as 68 hygienic behaviour. Bees exhibiting hygienic behaviour are more efficient at removing 69 dead, diseased, or dying brood from the hive 9, 10 , enabling them to resist or at least co-70 exist with pathogens such as American Fouldbrood (Paenibacillus larvae) or parasites 71 such as Varroa mites (V. destructor) that would otherwise kill the colony. A closely 72 related but distinct trait known as Varroa-sensitive hygiene enables bees to detect and 73 disrupt the life cycle of reproductive female Varroa mites 11 . Both hygienic behaviour and 74Varroa-sensitive hygiene are heritable 12, 13 and can therefore be selectively bred for; 75 likewise, QTLs and SNPs have been linked to each behaviour 14, 15