BackgroundThere is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection.ResultsUsing quantitative proteomics, we compared the hemolymph (insect blood) of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO), lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection.ConclusionWe propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct defensive measures, such as the massive enhancement of PO activity.

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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.

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A laboratory bioassay was developed to evaluate miticides to control Varroa jacobsoni (Oudemans), an important parasite of the honey bee, Apis mellifera L. Bees and mites were exposed to applications of essential oil constituents in petri dishes (60 by 20 mm). The registered mite control agents tau-fluvalinate (Apistan) and formic acid also were evaluated as positive controls. Treatments that caused high mite mortality (> 70%) at doses that produced low bee mortality (< 30%) were considered mite selective. The six most selective of the 22 treatments tested (clove oil, benzyl acetate, thymol, carvacrol, methyl salicylate, and Magic3) were further evaluated to estimate LD50 values and selectivity ratios (A. mellifera LD50/V. jacobsoni LD50) at 24, 43, and 67 h after exposure. Tau-fluvalinate was the most selective treatment, but thymol, clove oil, Magic3, and methyl salicylate demonstrated selectivity equal to or greater than formic acid. The effect of mode of application (complete exposure versus vapor only) on bee and mite mortality was assessed for thymol, clove oil, and Magic3 by using a 2-chambered dish design. Estimated V. jacobsoni LD50 values were significantly lower for complete exposure applications of thymol and Magic3, suggesting that both vapor and topical exposure influenced mite mortality, whereas estimated values for clove oil suggested that topical exposure had little or no influence on mite mortality. These results indicate that essential oil constituents alone may not be selective enough to control Varroa under all conditions, but could be a useful component of an integrated pest management approach to parasitic mite management in honey bee colonies.