We have demonstrated that immunostimulation of adult honey bee workers results in altered social interactions only in the case of bacteria-injected bees. However, all the treatment groups have significantly different cuticular hydrocarbon chemical profiles, and all treatments caused large scale changes in gene expression patterns in abdominal fat body and epithelial tissue, which includes oenocytes. It is important to note that these dramatic changes occurred in a relatively short timeframe, only six hours after immunostimulation. Importantly, this study demonstrates that expression of a large number of genes, not just those canonically associated with immune response pathways, are modulated by immunostimulation, as has been demonstrated by several other studies of genome-wide responses to immune challenges (for example,
[38–41]). However, comparisons with studies in Drosophila found limited overlap of gene expression patterns, and the handful of commonly regulated genes were key members of canonical immune response pathways.
It is somewhat perplexing that only bacteria-injected bees were subjected to significantly increased allo-grooming and aggression. All treatments caused changes in chemical profiles, and the changes did not appear to be substantially larger in bacteria-injected bees, though there was a trend for lower total quantity of branched alkanes in this group. However, previous studies have suggested that alkenes, rather than alkanes, play a significant role in nestmate recognition
. Based on the hierarchical clustering analysis, the gene expression patterns of bacteria-injected bees were clearly distinct from saline- and bead-injected bees. However, overall it did not appear that bacterial injection altered expression of an entirely unique set of genes (out of 302 transcripts, expression of 68 transcripts were significantly regulated only by bacterial injection, relative to controls, and expression of many of these genes changed non-significantly in the other treatments as well). Thus, perhaps bacteria injection did not cause change in expression of a large, unique subset genes, but rather caused more substantial changes in the magnitude of expression levels of some genes, as indicated by hierarchical clustering. Alternatively, it may be that expression of a few key genes and/or chemical profile components in bacteria-treated bees provoked behavioral changes in nestmates.
It remains to be determined if infection with honey bee specific parasites and pathogens causes similar changes in gene expression and behavioral responses, though there is some indication that infection with Nosema microsporidia does stimulate expression changes in a significantly overlapping set of genes (Holt, Aronstein and Grozinger, unpublished data). It also remains to be determined if these behavioral changes are adaptive: it is possible that bees have evolved to “match” the strength of the signal to the virulence of the pathogen, and thus infection with bacteria would elicit a greater change in the behavioral responses of nestmates than simple cuticular wounding. However, colony-level assays would need to be performed in order to determine if these changes in social interactions actually result in differences in the spread of pathogens through the colony and their impacts on infected individuals. For example, the increased grooming behavior we observed towards bacteria-injected individuals could facilitate the spread of pathogens through the colony (as observed in carpenter ants,
), slow the spread by causing isolation or removal of infected individuals
[56, 57] or reduce the impact on the infected individual
Our studies also demonstrate that immunostimulation elicits complex gene expression changes in the epithelial tissues in honey bee workers. Immunostimulation resulted in significant expression changes of 302 common transcripts in worker bees from the two colonies examined. Only 14 of these genes corresponded to previously annotated immune genes identified from the honey bee genome
. Several other biological processes were modified, including cell growth and proliferation, cytoskelatal structure, metabolism and components of the Notch signaling pathway. Cell growth, proliferation, and migration, particularly involving actin-mediated cytoskeletal changes, are required for repairing epithelial wounds
. The Notch signaling pathway has not yet been linked to immune response or wound repair, but wound repair uses many of the same developmental pathways that function during dorsal closure in Drosophila development, which does involve Notch signaling
. Alternatively, since the insect fat body is involved in regulating many key processes, including metabolism (which was commonly regulated in our study and by Varroa parasitization
), these changes may reflect general physiological changes after immunostimulation or stress
. Expression changes in three genes of the Notch signaling pathway (apterous, groucho, and pebbled) were confirmed using quantitative real-time PCR. Interestingly, expression was significantly higher in saline-injected bees relative to controls for all three genes, but only expression of apterous was affected in bacteria-injected bees, suggesting that changes in Notch signaling may be modulated temporally or by other signaling pathways.
We found significant expression changes of a number of key immune response genes (for a review of the function of these genes, see
[10, 23, 24]). The JAK/STAT pathway is regulated by the Domeless receptor; domeless expression was significantly regulated by immune stimulation in both genotypes of bees in our study. Activation of the IMD pathway requires cleavage of Relish by the caspase DREDD; both Dredd and Relish were significantly regulated in our study. We also observed significant regulation of PGRP-SC2 which suppresses activation the IMD pathway, with bacteria-injected bees showing the highest levels of PGRP-SC2 expression (data not shown). In fruit flies, PGRP-SC1 and PGR-SC2 may function in preventing over-activation of the IMD pathway
. The IMD pathway triggers the JNK pathway, which activates the transcriptional regulator AP-1 (which contains Kayak/D-fos), and AP-1 in turn negatively regulates Relish-dependent transcription. We found significant regulation of kayak in our study. Interestingly, we also found significant changes in expression of cabut, which is regulated by the JNK pathway but has not yet been linked to immune function
. The Toll pathway operates through transduction factors including spirit, which acts extracellularly and upstream of Spaetzle
 and the NF-κB protein Dorsal (which is negatively regulated by cactus). We found significant regulation of spirit and cactus in our study. Pale, which plays an important role in melanization and wound repair, and draper, which functions in phagocytosis, were also significantly regulated in our study. As a Gram-negative bacteria, it would be expected that E. coli would primarily stimulate activation of the IMD pathway. However, we observed changes in gene expression of members of the Toll pathway, including cactus, spirit and defensin-1, which exhibits Gram-positive antimicrobial activity and is not regulated by the IMD pathway
. Thus, there is likely considerable cross-talk between the pathways.
Our studies also identified several genes which may play a role in altering cuticular hydrocarbon patterns. Cuticular hydrocarbons are synthesized primarily in the oenocytes (reviewed in
), which are embedded in the fat body of adult honey bees
. Cuticular hydrocarbon biosynthesis involves activation of fatty acids by an acyl-CoA synthetase, chain elongations of fatty-acyl-CoAs to produce very long chain fatty acids, and subsequent conversion to a hydrocarbon, likely by a p450 enzyme
. Fatty acids are stored in lipid droplets in the adipoctye cells of the insect fat body
. Fatty acids can be released from droplets in the adipocytes and accumulate in the oenocytes; this occurs under starvation conditions in particular
. This process is mediated in part by lipid storage droplet-2 (Lsd-2): increased Lsd-2 expression in the fat bodies decreases lipid movement to the oenocytes. We found increased expression of Lsd-2 in immunostimulated bees (see Figure
6), suggesting reduced movement of lipids to oenocytes, and perhaps reduced levels of cuticular hydrocarbons. We did observe a decrease in the total relative quantity of all branched alkanes in bacteria-injected workers in both colonies but this difference was not significant.
Bubblegum (bgm) activates long chain fatty acids to form acyl-CoAs (reviewed in
), a key step in cuticular hydrocarbon biosynthesis. Bgm was originally described as a Drosophila mutant that resulted in elevated levels of very long chain fatty acids and neurodegeneration
. Bgm homologs have been identified in numerous species, including humans and mice, and have been demonstrated to activate long chain (C16) and very long chain (C24) fatty acids
. In our study, bgm expression was significantly decreased relative to controls (see Figure
Despite large differences in study designs and analysis methods, we found some overlap in gene expression with previous studies examining the effects of Varroa mite parasitization on honey bees
[38, 40]. Varroa-responsive genes were significantly associated with basic cellular processes, including cell organization, biogenesis and metabolism. We also found significant changes in functional categories associated with basic cellular processes, such as cell growth, proliferation and cytoskeletal structure. Varroa parasitization also caused changes in expression of pale. As discussed above, this may represent cellular mechanisms for wound-healing. Expression of potential hydrocarbon synthesis genes, namely bgm and Lsd-2, were also regulated by Varroa parasitization. Indeed, Varroa parasitized pupae and adults have modified cuticular hydrocarbon profiles
. These differences are likely responsible for stimulating hygienic behavior, in which diseased larvae are removed by adult worker bees, a key component of Varroa resistance
Comparison with two previous studies
[39, 41] examining the effects of immunostimulation on Drosophila global gene expression patterns revealed conserved changes in expression of key immune genes in Drosophila and honey bees (including relish, cactus, defensin-1, spirit, PGRP-SC2, and pale), but otherwise limited overlap in the significantly regulated genes. The lack of similarity could represent species-specific immune responses or simply technical differences – for example, Roxstrom-Lindquist
 orally infected young male flies with bacteria, fungi and microsporidia, and measured whole-body gene expression changes in only two replicates using Affymetrix microarrays.