Social and Individual Immune Responses of Western Honey Bees

Brian McKee
BI-494 Immunology, Northern Michigan University, Marquette, Michigan, United States of America

Introduction.
Western honey bees (Apis mellifera) congregate in colonies that form model super-organisms for social immunity (Evans, 2004; Parker et al., 2012). Honey bee hives are known to be warm, moist, and densely crowded which can facilitate the breeding of pathogenic organisms. Individual honey bees, and the collective colonies they establish, have immune responses ranging from colony-wide behavior to microscopic chemical action. These physiological subdivisions thus overlap between the macro- and micro-scale. At the colony level, masses of bees can thermoregulate the hive by shaking simultaneously to maintain homeostasis or induce fever (Starks et al., 2000). Further, temporal polyethism is an agedependent division of labor which affects bees immunologically in that immune system components change between brood and adults (Alaux et al., 2010; Schmid et al., 2008; Wilson-Rich, et al., 2008;). Behavioral defenses may include antiseptic removal of terminally infected nestmates (Baracchi et al., 2012), and mutual grooming to remove Varroa spp. mites (Navajas et al., 2008; Spivak, 1996). Honey bees have been selectively bred, in two distinct cases, to display hygienic grooming and subsequent mite removal behavior (Harbo and Harris, 2009; Rinderer et al., 2010). Propolis is the resinous mixture that honey bees collect from botanical sources and mix with their saliva in order to employ as a sealant for unwanted open spaces in the hive. Small gaps are filled with this material while beeswax fills larger gaps . Sealing up the hive is a preemptive action which helps prevent pathogens from entering the hive. Beyond this, honey bees have the ability to collect antimicrobial compounds from their environment (Simone et al., 2009). These compounds are mainly obtained from leaf buds and wounds. A supplementary example of an individual bees physical defense is its chitinous exoskeleton. 1|Page

At the microscopic level, bee saliva contains antimicrobial enzymes that are immunologically beneficial. The first step of an individuals immune response is recognizing the difference between self and foreign. Foreign pathogenic compounds bind to pattern recognition receptors (PRRs) which lead to a variety of cascades. Bees have Toll-like receptors (TLRs) in addition to Immune deficiency (Imd), cJun N-terminal kinases (JNK), and Janus kinase/ Signal transducer and activator of transcription (JAK/STAT) pathways. These together discriminate foreign pathogen-associated molecular patterns and constitute the honey bee innate immune system. Evans et al., (2006) compared the above mechanisms to Drosophila melanogaster and Anopheles gambiae flies. This comparative experimental design does not, however, fully represent the consequent activation of these immune mechanisms in honey bees . Symbiotic immune cells are linked directly to chemical defenses which include antimicrobial peptides (AMPs) being released by immune gene up-regulation (Evans, 2004; Gregory et al., 2005). Another defense involves phagocytic hemocytes and secreted enzymes which circulate the hemolymph (Parker et al., 2012). Hemocytes signal phenoloxidase enzymes to produce indole groups which successively polymerize into melanin. This innate cascading process is called melanization which results in the encapsulation or further destruction of harmful foreign material. AMPs, complement-like fragments, cytokine-like, and chemokine-like constituents establish the innate response in this arthropod (Esche, et al., 2005). An example chemical cascade involves releasing signaling proteins which penetrate foreign cells, causing them to up-regulate immune response genes. These genes subsequently transcribe mRNA to exit the nucleus of the foreign cell and translate genetic orders into AMPs by ribosomal action. The cell thus dies via apoptosis or autophagy. Beyond the encapsulating destruction of foreign compounds, phenols, quinines, and reactive oxygen species may be employed (Gonzlez-Santoyo & Crdoba-Aguilar, 2012). Biocide is a term which includes all anthropomorphically implemented chemicals which act to kill an organism. These biocides include pesticides, acaricides (miticides), insecticides, herbicides, etc. 2|Page

Diverse lethal and sublethal effects resulting from these chemicals have shown both benefit and hindrance to bees in distinct cases (Dainat et al. 2012; Vidau et al., 2011).

Methodology.
All literature employed in this review was located via Google Scholar. The defining criteria was that cited journal articles must directly apply to Apis mellifera and their immunological responses to varying stimuli. Social & Behavioral immune action and Cellular & Chemical action are grouped together in order to simplify the myriad subdivisions of western honey bee immune-related responses. Overlaps between the two subdivisions did occur but are presented in logical fashion. Zotero Standalone application was employed to organize references (https://www.zotero.org/).

Literature review.
Social and Behavioral Honey bee colonies are deemed super-organisms because the entire

colony exhibits unique phenomena produced and governed by the collective of individual bees; these phenomena cannot be displayed by individual bees alone. One of the most notable macro-scale immune responses includes inducing a hive-wide fever by shaking simultaneously. Raising the temperature of the nest prevents the growth of some heat-sensitive pathogens such as Ascosphaera apis (Starks et al., 2000). A. apis is a fungus that penetrates into brood comb and causes chalkbrood disease. It was demonstrated that brood comb temperature is positively correlated with ambient temperature increase resulting from self-induced fever in treatment colonies introduced to A. apis. This response is also imperative in fending off large arthropods which may enter the hive. Bees have developed a behavior of conglomerating on an intruding insect and shaking until the intense heat subdues it (Ono et al., 1995). This heat-raising behavior has three main effects: defense against pathogens, maintaining homeostasis (especially over winter), and catalyzing brood development.

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Despite the association between social organisms and the increased risk of disease transmission, honey bees have displayed behavioral responses of removing bees infected with deformed wing virus (DWV). The hive-wide antiseptic response to this virus reinforces the idea that bee colonies can be viewed as super-organisms. Baracchi et al. (2012) suggests that infected bees may be detected through the recognition of cuticular hydrocarbon profiles of infected bees. They also noted that DWV treatment colonies were more susceptible to increasing risks of epidemics from introduced infected bees, despite

showing increased hygienic behavior, when compared to healthy control colonies, (Figure 1).

Figure 1 | The number of DMW and healthy bees dragged out of the hives 10 minutes post-introduction. 2 test results are reported for the overall samples taken together in healthy and infested colonies (first and second main columns). 2 results for healthy and infested colonies were then grouped together in which P = 0.001 (third main column). DOI: 10.1016/j.jinsphys.2012.09.010 Due in part by temporal polyethism, honey bee immune systems alter with age. The honey bee lifecycle involves proceeding from immature brood, to nurse bees, to mature forager bees. Forager bees have strongly reduced hemocyte numbers compared to younger nurse bees and brood. Bull et al. (2012) explain that young adult bees mask their increased susceptibility to infection when compared to older bees. Nurse bees define their niche by tending to brood and performing hygienic behavior within the hive . Nurse bees cap new pupae in the brood comb with wax until eclosion. Immune response tactics typically

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shift from hemocytic to phenoloxidase-based immunity with age (Schmid et al., 2008). This might be because mature forager bees do not have much of a reason to expend energy on such a long-term metabolically-expensive response. Forager bees typically encounter increased pathogen exposure because they frequently leave the hive. In the absence of foragers, potentially implicating colony collapse, younger bees can be forced to take the place of older bees. Foraging bees may act as vaccinated individuals that provide a type of herd immunity. With their absence, the disease resistance capacity of the colony is likely hindered. It has additionally been found that when comparing both newly-emerged and mature forager bees, experimentally infected with Metarhizium anisopliae, young bees were more susceptible to infection. Older bees did not appear to up-regulate their immune response to the same degree as young bees. This may suggest that older bees are innately more resilient to initial infection. Conversely, conflicting evidence shows that nurse bees are not as immunologically competent as foraging bees (Wilson-Rich et al., 2008). Varroa mites (Varroa spp.) are honey bee parasites that cause varroosis. They are known to be associated with other honey bee pathogens and are confirmed vectors of single stranded RNA viruses . Examples include DWV and Kashmir bee virus (KBV) (Bowen-Walker et al., 1999; Chen et al., 2004). Mites produce wounds through the exoskeleton of bees while feeding. These wound sites can harbor infections of Melissococcus pluton, a precursor of the brood disease European foulbrood (Kanbar and Engels 2003). Navajas et al. (2008) measured differences in gene expression in two colonies of Varroasusceptible and two colonies of Varroa-tolerant bees. They identified 148 genes with significantly different patterns of expression: 32 varied with the presence of Varroa jacobsoni, 116 varied with bee genotype, and 2 varied with both. Expression of genes related to embryonic development, cell metabolism, and immunity were thus changed as a result of Varroa parasitism. Varroa-tolerant bees were chiefly characterized by differences in the expression of genes regulating neuronal development, neuronal 5|Page

sensitivity, and olfaction. Olfaction may be related to increased hygienic and grooming behavior and is displayed by Varroa-tolerant mites. In other cases, Varroa mites do not seem to affect particular honey bee colony population abundances (Gregory et al., 2005). These results, highlight the potential importance of behavioral mechanisms in response to Varroa jacobsoni and suggest that a study focused on bee genealogy and neurology is of importance in the future. Specific behavioral and physiological changes found in parasitized bees were indicated which provide an initial step toward better understanding behavioral and genomic responses involved in these complex host-parasite immune relationships. Cellular and Chemical Continuing deeper into the microscopic scale, honey bee pupae

collected from moderately Varroa-parasitized colonies showed a significant down-regulation of immunerelated transcripts (Gregory et al., 2005). Interestingly, this suppression of immune-gene transcripts disappeared when bee pupae encountered higher mite loads. To explore whether the presence of V. destructor affects the humoral immune response, transcript levels for abaecin and defensin were examined. Both of these AMPs are innately present in honey bees. Several non-exclusive mechanisms might explain the relationship between Varroa parasitism and immune responses. Honey bees appear to mount cellular immune responses at wound sites caused by V. destructor (Kanbar and Engels 2003). Pupae with heavy mite loads were infested for longer, potentially resulting in a stronger immune response. Abaecin and defensin have also been tested in regard to the bacterial pathogen Paenibacillus larvae (Evans, 2004). It was shown that honey bee larvae are highly susceptible to this bacteria only during the first instar of larval development. Given that mites can transmit disease, it might be adaptive for bees to respond by up-regulating their immune responses. Likewise, mites could benefit by inhibiting the immune responses of bees which negatively impact the feeding of the mites. Immune genes and AMPs are known to be up-regulated in 6|Page

dying colonies as well in bees with bacterial oral infections (Dainat et al., 2012; Evans and Lopoez, 2004). Finally, bees that are parasitized by mites could be less effective in mounting an immune response due to physiological costs of both parasitism and the immune response itself. Activity against gram-positive and gram-negative bacteria have both been displayed by honey bees (Casteels et al., 1994). Beyond this, bees demonstrate the ability to collect antimicrobial compounds from their environment (Simone et al., 2009). It has further been proposed that nonpathogenic bacteria can be used as a probiotic to enhance honey bee immunity, helping bee larvae and other life stages survive attacks from encountered pathogens (Evens and Lopez, 2004). This identified bee-bacteria symbiosis elucidates yet another layer of honey bee immunology. Bee larvae are also able to cope with E. coli infection, and winter bees may be especially well-prepared for bacterial infections (Gtschenberger et al., 2013). The expression of the genes for metabolizing enzymes, needed to process nectar into honey, are created in the hypopharyngeal gland alone. Tying back to the previous subsection, the secretion of these enzymes is associated with the age-dependent role change of the worker (Ohashi et al., 1999). Glucose makes up a significant portion (~31.28%) of honey (White, 1980), and honey is an important overwinter food source. Ohashi et al. (1999) were the first to describe the purification and cDNA cloning of a glucose dehydrogenase with animal origin. Honey bee saliva contains both amylase, and glucose oxidase which oxygenizes glucose into hydrogen peroxide, a reactive oxygen species . Constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether specifically induce honey bee detoxification genes. These inducers are primarily found in pollen and propolis. Massively parallel RNA sequence analysis showed that p-coumaric acid up-regulates all classes of detoxification genes as well as select AMP genes. This up-regulation is significant in that that adding p-coumaric acid to a diet of sucrose increases the metabolism of coumaphos, a widely used in-hive miticide, by 60% (Mao 7|Page

et al., 2013). Due to the fact that p-coumaric acid is a component of pollen grain cell walls, it is abundant in the natural diet of honey bees and may function as a nutraceutical that regulates immune and detoxification processes. Sucrose constitutes a relatively small (~1.31%), yet significant portion of honey (White, 1980). This evidence highlights the importance of leaving a sufficient portion of honey in managed hives during cold months so that hydrogen peroxide can be oxygenized and actively implemented defensively by bees. The widespread use of honey substitutes as overwintering food sources, may compromise the ability of honey bees to cope with certain pathogens and biocides. Fat bodies are known to decrease with age and are major factors in the production of AMPs (Wilson-Rich et al., 2008). Forager bees are the final stage of the honey bee lifecycle and do not maintain their fat bodies. This is because forager bees are out of the hive restlessly collecting pollen. As a supplement, bees deprived of protein are likewise unable to develop their fat bodies fully (Alaux et al., 2010). This study also noted that hemocyte concentration and fat body contents decreased with age while

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phenoloxidase and glucose oxidase activity increased with age (Figure 2). Figure 2 | Effects of a pollen diet on immunocompetence. Unfilled bars represent 5 day old bees while filled bars represent 10 day old bees. (a) Hemocyte concentration, (b) phenoloxidase activity, (c) fat body mass and (d) glucose oxidase activity. Eight bees per cage for each experimental group were analyzed for each immune parameter. The protein percentage of each pollen diet is indicated on the x-axis. Each letter indicates significant differences between diets (p < 0.05, NewmanKeuls post hoc tests). DOI: 10.1098/rsbl.2009.0986 In addition to pathological and nutritional deficiencies, biocides are another factor which may negatively affect honey bees immunologically. Responses to these chemicals vary widely, largely dependent on the employed biocide. Bee health may be compromised to the greatest degree by synergistic effects in which disease agents combine to produce exponential destructive results . A significant increase in bee mortality was noted in the case of Nosema ceranae fungal infection combined with sublethal amounts of the biocides fipronil and thiacloprid (Vidau et al., 2011). While the percent mortality of bees infected with N. ceranae alone reached a maximum of 47%, maximum percent mortalities for N. ceranae combined with thiacloprid and fipronil increased mortality rates to 71% and 82% respectively. Thiacloprid and fipronil are insecticides, while formic acid and oxalic acid can be implemented as miticides with minimal harm to bees (Dainat et al. 2012). Two populations infested with Varroa mites were studied in this experiment. The first population was made up of 13 infested colonies that did not survive the winter, of which 11 were left completely untreated with miticides. All hives which were left untreated collapsed completely. The second population consisted of 16 colonies, all of which received miticide treatment. Only 2 of 16 treated hives perished. Fourteen of 16 treated hives thus survived the winter. Remarkably, Cytochrome P450-mediated detoxification contributes to the tolerance of honey bees to another biocide, tau-fluvalinate (Mao et al., 2011). That is, CYP9Q metabolizes this miticide within bee guts, allowing bees to tolerate the chemical application of tau-fluvalinate while Varroa mites consequently perish. 9|Page

Controversy. I would like to explore the degree to which sharing salivary immune components with genetically and geographically distinct bees benefits the separate hive colonies.

Conclusion.
Western honey bees are social super-organisms that exhibit myriad immunologic responses. These responses to harmful foreign material occur from the cellular/chemical level up to the social/behavioral level. Indications exist that molecular mechanisms underlie social and innate immune behaviors. Examples of these adaptions involve neural remodeling, odorant recognition, and four immune receptor pathways. Immune system components and behaviors of bees were found to be influenced by age. It was discovered that bees exposed to low or moderate numbers of mites sharply reduce their immune-peptide transcripts, inversely compared to both heavily parasitized and unparasitized bees. Proper nutrition is also paramount to honey bee immune health in evading colony losses. Finally, anthropogenically applied chemicals can have both beneficial and hindering effects on honey bee health. In designing an experiment to test for immune system components which have be shared with separate colonies, it might be beneficial to selectively breed bees that range comparatively farther or display open interaction with bees of other colonies. Healthy control bee saliva would be inoculated into the foodstuff of infected colonies, which could theoretically be subsequently consumed. This may effectively transfer some of the immune system components from one colony to another. One inference is that the transferred bee saliva would contain sufficient amounts of stable enzymes, symbiotic bacteria, etc. Testing for effective transfer of these components would be measured in terms of decreased mortality rates or decreased pathogen abundance post-treatment; or more thoroughly, could involve polymerase 10 | P a g e

chain reaction to identify individually isolated components transferred to the treatment colony. Methods of teaching honey bees to interact with genetically distinct bees of other colonies should be explored as increased biodiversity is often linked to increased disease resistance and community stability.

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