Recent investigations associate Varroa destructor (Mesostigmata: Varroidae) parasitism and its associated pathogens and agricultural pesticides with negative effects on colony health, resulting in sporadic global declines in domestic honeybee (Apis mellifera) populations. These events have motivated efforts to develop research tools that can offer insight into the causes of declining bee health as well as identify biomarkers to guide breeding programs. Here we report the development of a bee-specific peptide array for characterizing global cellular kinase activity in whole bee extracts. The arrays reveal distinct, developmentally-specific signaling profiles between bees with differential susceptibility to infestation by Varroa mites. Gene ontology analysis of the differentially phosphorylated peptides indicates that the differential susceptibility to Varroa mite infestation does not reflect compromised immunity; rather, there is evidence for mite-mediated immune suppression within the susceptible phenotype that may reduce the ability of these bees to counter secondary viral infections. This hypothesis is supported by the demonstration of more diverse viral infections in mite-infested, susceptible adult bees. The bee-specific peptide arrays are an effective tool for understanding the molecular basis of this complex phenotype as well as for the discovery and utilization of phosphorylation biomarkers for breeding programs.

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees.

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the mite Varroa destructor is a serious threat to honeybee populations. Selective breeding for Varroa mite tolerance could be accelerated by biomarkers within individual bees that could be applied to evaluate a colony phenotype. Previously, we demonstrated differences in kinase-mediated signaling between bees from colonies of extreme phenotypes of mite susceptibility. We expand these findings by defining a panel of 19 phosphorylation events that differ significantly between individual pupae from multiple colonies with distinct Varroa mite tolerant phenotypes. the predictive capacity of these biomarkers was evaluated by analyzing uninfested pupae from eight colonies representing a spectrum of mite tolerance. The pool of biomarkers effectively discriminated individual pupae on the basis of colony susceptibility to mite infestation. Kinome analysis of uninfested pupae from mite tolerant colonies highlighted an increased innate immune response capacity. The implication that differences in innate immunity contribute to mite susceptibility is supported by the observation that induction of innate immune signaling responses to infestation is compromised in pupae of the susceptible colonies. collectively, biomarkers within individual pupae that are predictive of the susceptibility of colonies to mite infestation could provide a molecular tool for selective breeding of tolerant colonies.Prioritizing bees at the dark-eyed pupae stage of development minimizes potential signaling variability arising from different castes or environmental influences in adult bees. All samples were analyzed within the same kinome assay to minimize technical effects due to inter-assay variability. Individual frozen pupae were placed in a sealed plastic bag in the presence of 300 μl of lysis buffer 25 . The pupae were pulverized with a rubber mallet and the resulting suspension was centrifuged at 10,000 × g for 10 min. Supernatants were used for kinome analysis 25 . Data analysis. The dataset for each array contains signal intensities associated with the nine technical replicates for each of the 299 peptides spotted on each array. Kinome data were processed through PIIKA 2, a pipeline for processing kinome array data 45 .Identification of peptide biomarkers of varroa mite susceptibility. A one-sided paired student's t-test of normal distribution was used to compare normalized signal intensity values for each of the 299 peptides of individual pupae (n = 5) representing the two high and low colony phenotypes of Varroa mite susceptibility. Specifically, mite tolerance was represented by pupae from the S88 (n = 3) and S23A (n = 2) colonies while mite susceptibility was represented by pupae from the G4 (n = 3) and S88-4 (n = 2) colonies in the absence of mite infestation. Peptides with significant (P-value < 0.01) differences in levels of phosphorylation between pupae of the two phenotypes were classified as potential biomarkers.