We have described here the discovery of several proteins whose expression levels may impact honey bee resistance to infestation by the Varroa
mite. Natural diversity in these behaviors was a prerequisite to this study and we observed that the levels of each behavior in any given colony were not random. As expected, there was a strong negative correlation between mite infestation levels and HB. At the expression level, several proteins were highly significant predictors of HB and mite infestation dynamics. Highlighted within these proteins were the putative ApoO homolog and a putative Tg. Apolipoproteins are called apolipophorins in insects, and they have diverse roles in lipid solubilization and the transport of small hydrophobic ligands [27
]. In innate immunity the apolipophorin ApoLp-III stimulates antimicrobial activity in the hemolymph, acting as a pattern recognition system for LPS and lipoteichoic acid (LTA) [29
]. Lastly, the strong correlation of Tg with both NDs and an increase in the ratio of phoretic mites to brood mites suggests that Tg activity could provide a measure of resistance to Varroa
is an ecto-parasite feeding communally and repeatedly on hemolymph of the honey bee through a bite wound in the cuticle [30
]. In insects' innate immunity the cuticle provides the first line of defense; once breeched, innate defense systems of the haemocoel cavity are orchestrated by hemocytes, the fat body and hemocoel [33
]. Normal wounds heal as hemocytes and plasmatocytes exocytose the clotting factors hemolectin and Eig71Ee [34
]. These molecules and other plasma-based factors such as fondue are cross-linked by Tgs in a Ca2+
dependent mechanism to form a primary clot. However, V. destructor
transmits bio-active compounds that prevent healing and allow continued feeding to occur at the same wound [35
]. In the tick arthropod-mammalian ecto-parasitic systems, 18 known bio-active suppressants target innate antiseptic defenses, including several immune cells types, inflammatory and coagulatory cascades [36
]. In honeybees, the effect V. destructor
elicits on the immune system is uncertain. Yang and Cox-Foster [37
] demonstrated that Varroa
parasitism increases the susceptibility of adult bees to bacterial infection, but no major immunosuppressive effects were revealed by transcriptomic studies on specific immune genes or in global analyses [38
]. More recently a study has reveled that salivary secretions from the Varroa
mite are able to damage hemocyte aggregation in the tomato moth, (Lacanobia oleracea
] but no known factors of either pathogen or host are identified. We report here that elevated expression of a putative key clotting factor (Tg) is found in the larva of Varroa
resistant bee colonies. These data indicate that honey bees have adapted to Varroa
, increasing the clotting capacity of hemolymph in order to limit mite reproduction.
While the experiments described here were clearly of sufficient power to permit the discovery of some correlations between protein expression and behavioral traits, the variability within such out-bred populations is very high. This is likely a significant limitation in fully defining the molecular mechanism of something as complex as a behavior. Practical limitations in the number of colonies that could be sampled and the depth to which the proteome could be measured across multiple samples were inherent problems here, as with any proteomics study. Even so, an exploratory approach was seen as an important step in generating new hypotheses in a currently poorly understood area of biology.
It is thought that the speed with which hygienic bees respond is driven by a lower limit of olfactory detection of the diseased brood odor [40
], which is in turn influenced by the neuromodulator octopamine [41
]. In the antennal lobe, octopamine concentration varies between behavioral state, being low in nurse bees and high in foragers. Juvenile hormone and brood pheromone both modulate behavioral responses to octopamine [42
] and both are involved in several aspects of behavioral maturation, with the best-understood system being the transition from nurse to forager. This maturation invokes physiological changes that are underpinned by increased neural processing which is required to interpret complex visual information for flight behavior. Anatomically, expansion of the mushroom body neurophil space in the brain and decrease in the volume of the olfactory glomeruli of the antennal lobes occurs during this transition [43
]. Olfactory sensory neurons from the antennae project onto the glomeruli of the antennal lobe via the antennal nerve, and olfactory information is processed and projected to higher-order brain centers such as the mushroom bodies or lateral protocerebrum.
The data presented here indicates that cells (most likely neurons with antennal axons) of bees performing rapid hygiene express different levels of proteins involved in adhesion and vesicle processing (Figure ), supporting the role of octopamine and maturation as an important control of this behavior. The cell adhesion proteins identified were all integrin proteins, some of which have been reported to regulate synaptic plasticity [44
]. Specifically, ankerin 2 stabilizes synaptic connections to the spectrin-actin cytoskeleton and laminin A, Zasp and Fas1 are involved in the assembly of functional integrin adhesion sites essential for growth cone extension in axon guidance during neurogensis [45
]. The increased expression of vesicle sorting proteins in hygienic bees indicates that while plasticity may be reduced, antennae of hygienic bees provide a strong input into higher brain function. These data could be explained by the environment of a hygienic nest bee, in which strong brood and queen-based olfactory cues are the major sensory inputs for bee development, behavior and social cohesion [43
] (Figure ). Dimorphism in neural plasticity has been well characterized in the antennae of drones, where the antennal sensory nerves are thicker but project into a smaller number of glomeruli than in workers [46
]. This configuration provides drones with the lower limit of detection for queen pheromone, enabling efficient queen finding during mating flights.
Summary model for proteins identified in antennae and larvae from correlation screen. For details see discussion.
VSH limits mite reproductive success in the brood by specifically detecting the presence of a post-ovipositional mite. As part of the bees' response, a sensitive adult uncaps and re-caps the cell, effectively inhibiting mite reproduction [48
]. The signal being sensed in this process remains unknown, although it peaks between three and five days after the cell is initially capped, leading to speculation that VSH adult bees respond to temporal fluxes in pathology mediated by oviposition, wounding related stress responses, infections, and olfactory cues [48
]. Correlation between VSH scores and two proteins encoding divergent members of the To/JHBP super-family suggest they may be functionally linked to the behavior; To/JHBPs contain a conserved ligand binding domain with differing affinities to small lipophilic molecules such as JH and the N-terminal signal peptide indicates that they are probably secreted into the hemolymph where they act as soluble receptors for their ligands [49
]. In the honey bee genome there are eight To/JHBP genes, located at two distinct loci, and we see one protein from each loci, one positively correlated with VSH and one negatively correlated (Figure ). Biologically, this separation in the genome suggests divergent functions and this is further supported by their differential regulation in our study. One of these proteins is completely uncharacterized but in Phormia regina
the ortholog of the other To/JHBP is thought to be involved in chemosensation in antennal olfaction and taste [49
] leading to the attractive hypothesis that it is playing a similar role in sensing brood.
That sensory and neuronal processes have a link to disease tolerant behavior may be expected but, intriguingly, a class of proteins involved in larval cuticle formation/structure also emerged as likely candidates. An arthropod's cuticle forms the primary physical barrier to the environment so while the cuticle plays an obvious role in pathogen defense, how it may contribute to social immunity mechanisms is less clear. Cuticular lipids differ between bees depending upon caste and attacks by V. destructor
can alter the composition in adults and larvae [51
]. The role of the cuticle in social immunity is supported by the data presented here, which indicates that several proteins involved in forming and maintaining the cuticle are significantly correlated with disease tolerance behaviors of nurse bees (Figure ).