The first line of social defense is to pre-vent the “uptake” of parasites by individual nest members. As biotic threats to a nest are by definition external to the nest, the mem-bers most prone to parasite uptake are the foragers. In honey bees, one way to limit parasite uptake is to narrow the range of individuals engaged in this risky behavior, and in a normal colony foraging is indeed restricted to the oldest, and most expend-able individuals. If an old forager becomes infected, its short remaining lifetime limits its opportunity for spreading the parasite.
A second line of defense is to prevent or reduce parasite “intake” – the entry of parasites into the nest. At a basic level this is exercised in the choice of bees to occupy cavities. Entrances to these cavities tend to be small, ranging from 10-40 cm2 in area10, which restricts access points for nest invad-ers and limits the surveillance demands on guard bees. However, as we mentioned, many parasites gain entry to colonies not by direct assault but by catching a ride on a forager. This is the exclusive mode of entry for the Varroa mite which is otherwise in-capable of independent movement between colonies. Guard bees inspect returning for-agers and repel those infected with patho-gens including viruses, but I am unaware of any evidence that guard bees restrict entry of Varroa-laden nestmates. Nevertheless, the choice of nest site and guard policing behaviors constitute an important line of social defense.
If these first lines of defense fail, or if the parasite gains a foothold through vertical transmission, then the third line of defense focuses on preventing the parasite from get-ting established in the nest. This is where we see a battery of hygienic behaviors come into play, and for a beekeeping audience I must clarify that I’m talking about general
hygiene – not the specific form that has be-come a familiar management tool against Varroa; that one comes later.
For now, we’re including one of the most well-known examples of insect-applied an-timicrobials – the use of plant resins. Honey bees collect tree resins (Fig. 1), return them to the nest, mix them with beeswax, and apply them inside cells and onto nest cav-ity walls, at which point we call the sub-stance propolis (Fig. 2). The subsub-stance has antimicrobial properties which serve to reduce pathogen load in the nest environ-ment. However, it has also been recently shown that propolis reduces the expression of immune response genes in 7-day old bees. The significance of a reduction in im-mune response was due to an overall reduc-tion in bacterial loads in the experimentally propolis-treated colonies, but moreover, a highly charged immune system is not only indicative of a pathogen problem, but it is also exhausting on the bees to sustain the response.11 Propolis therefore “turns the temperature down” on a stressful situation – first by direct antimicrobial action and second by reducing the need for the bees to ramp up a costly immune reaction. Colonies whose bees are in a sustained state of im-mune response produce less brood.12
There are other behaviors in the colony that constitute hygienic resistance to para-site establishment in the nest. These include the well-known “undertaker” bees who re-move corpses of dead nestmates from the nest. And venom, it turns out, has more uses than its well-known function in defense;
there is evidence that bees apply it to their beeswax combs and onto their own integu-ments, apparently benefiting from venom’s antimicrobial properties.13 And lastly, there is evidence that the Cape Honey Bees of southern Africa, Apis mellifera capensis,
“socially encapsulate” invasive small hive beetles with propolis prisons in an action analogous to scarring and abscess formation in mammals.14
In the event a parasite becomes estab-lished in the nest, then the colony attempts a fourth line of defense – limiting the para-site’s spread between colony groups. The probability of a healthy colony member becoming infected is a product of its sus-ceptibility, its contact rate with an infected individual, and the infectivity of that indi-vidual (number of infectious propagules it carries). The most direct way to reduce in-fectious propagules is to pick them off and kill them – and the best example of this for bees is the well-known grooming behavior against Varroa mites. Bees expressing this heritable trait can detect and remove mites off their own bodies or bodies of nestmates and sometimes lethally bite them15. A similar strategy is employed with so-called hygienic lines of bees that are capable of detecting compromised cells of brood, opening them up, and removing the infected pupa and its associated pathogens or parasites.16
A higher-order expression of this fourth line of defense happens with the fact that members of a colony do not randomly dis-tribute themselves throughout a nest, but instead compartmentalize themselves into recognizable zones based on age and re-productive status. Young bees, the brood, and the queen are always central in the nest whereas older hive bees and foragers pre-dominate at the periphery. As social interac-tions are more common within, rather than across, these compartments, this has the ef-fect of localizing parasites and limiting their spread (Fig. 3). This has been called “orga-nizational immunity17,” and readers of this column will recognize it as an easy example of an emergent property – the kind of order that emerges spontaneously given enabling pre-existing conditions.
Another higher-order example of limiting a parasite’s spread invokes genetic diversity, and here we harken back to polyandry, the subject of my installment in May 2016 – the queen’s habit of mating with many males which causes her workers to be genetically diverse. Genetic homogeneity, sameness, would be a dangerous situation in a dense aggregation of individuals like a social in-sect colony. One virulent pathogen could sweep through the nest with devastating results. But genetic diversity not only in-creases the likelihood that individuals will possess innate resistance mechanisms to a variety of pathogens, it also increases behav-ioral repertoires that add up to social immu-nity. A good example is hygienic behavior.
It is not just one behavior, but rather a suite of behaviors – the ability to detect abnormal brood, the ability to uncap it, the ability to remove the contents, and a low tolerance threshold for abnormal brood that stimulates the possessor to engage in the process. There are at least six genetic regions responsible for these behaviors18, and a multiply-mated queen has a better chance of delivering all necessary genes to her colony. It is no Figure 2. Bees mix plant resins with beeswax to produce propolis. They coat
it on the interiors of nest cavities and brood cells. Although its initial function may have been to give structural strength to combs, propolis also confers an-timicrobial benefits to the colony and reduces energetic costs associated with innate immune responses. This image shows the interior of a Langstroth hive in South Africa in which the bees have severely restricted the entrance size with a sheet of propolis.
surprise that high rates of queen polyandry have been associated with lower disease in-cidence in colonies.19
As a fifth and final line of defense we can hypothesize on colony strategies that limit vertical transmission of parasites to a colony’s swarm offspring. One line of evidence for this is the fact that Nosema-infected workers remove themselves from tending the queen20; as it is the old queen that moves with a swarm, this can be inter-preted as a strategy for reducing Nosema risk to the swarm offspring. The existence of natural selection against horizontal transmis-sion seems less likely, at least from the point of view of an infected colony. There is no obvious evolutionary advantage to protect-ing a neighborprotect-ing colony from your infec-tion unless that colony is closely related or its proximity increases the chance for your re-infection. If natural selection responds to horizontal transmission at all, it is probably active as a defensive measure in step one above – reducing uptake of parasites. This may be one explanation why colonies in na-ture separate themselves from one another at rather large distances, ranging from 304 - 4848 meters.21
By now it is apparent that social immu-nity is yet another one of those complex and interacting pods of biologic phenomena that collectively make up the honey bee super-organism. Like caste differentiation, comb
construction, mating behavior, group deci-sion-making, and so many others, the social immunity pod is an outcome of interacting behaviors, pre-existing conditions, and emer-gent properties. This month we covered some of its mechanisms, but we haven’t yet talked about how it is regulated or how it evolved.
References
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relationships in the genus Apis inferred from mitochondrial DNA sequence data.
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4 Cremer, S. and M. Sixt. 2009. Analo-gies in the evolution of individual and social immunity. Philosophical Transac-tions of the Royal Society B 364: 129-142 DOI:10.1098/rstb.2008.0166 5 Hoffmann, J.A. 2003. The immune
re-sponse of Drosophila. Nature 426: 33-38 6 The Honeybee Genome Sequencing
Consortium. 2006. Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443(7114): 931-949 DOI: 10.1038/nature05260 7 Evans, J.D. et al. 2006. Immune
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8 Cremer, S. et al. 2007. Social immu-nity. Current Biology 17: R693-R702 DOI: 10.1016/j.cub.2007.06.008 9 Bull, J.J., et al. 1991. Selection of
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10 Seeley, T.D. and R.A. Morse. 1976.
The nest of the honey bee (Apis mellifera L.). Insectes Sociaux 23: 495-512 11 Simone-Finstrom, M. and M. Spivak.
2010. Propolis and bee health: the natu-ral history and significance of resin use by honey bees. Apidologie 41: 295-311 12 Evans J.D. and J.S. Pettis. 2005. Col-ony-level impacts of immune respon-siveness in honey bees, Apis mellifera.
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13 Baracchi, D. and S. Turillazzi. 2010.
Differences in venom and cuticular pep-tides in individuals of Apis mellifera (Hymenoptera: Apidae) determined by MALDI-TOF MS. Journal of Insect Physiology 56: 366-375
14 Neumann, P. et al. 2001. Social encap-sulation of beetle parasites by Cape hon-eybee colonies (Apis mellifera capensis Esch.). Naturwissenschaften 88: 214-216 15 Guzman-Novoa, E. et al. 2012. Ge-notypic variability and relationships between mite infestation levels, mite damage, grooming intensity, and re-moval of Varroa destructor mites in se-lected strains of worker honey bees (Apis mellifera L.). Journal of Invertebrate Pa-thology 110: 314-320
16 Spivak, M. and M. Gilliam. 1998. Hy-gienic behaviour of honey bees and its application for control of brood diseases and Varroa. Part II. Studies on hygienic behaviour since the Rothenbuhler era.
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quantita-tive trait loci influence task thresholds for hygienic behaviour in honeybees (Apis mellifera). Molecular Ecology 19: 1452–1461. DOI:10.1111/j.1365-294X.2010.04569.x
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10.1007/s13592-016-0443-9 Figure 3. Bees in a colony compartmentalize themselves into zones based on
age and reproductive status. Young bees, the brood, and the queen (with gold halo) are centered in the nest (light zone) whereas older maintenance workers and foragers predominate at the periphery (red zone). Social interactions (shown by connecting lines) are more common within, rather than across, these compartments; this has the effect of localizing parasites and limiting their spread, an outcome called “organizational immunity.” The zones are not equal in value. The brood, queen, and young bees are more important than the older bees on the edge. It is from the older edge cohorts that foragers are drawn that leave the colony (lower left), and as the most expendable cohort in the colony, it is foragers that are most likely to experience parasite uptake. By extension, foragers’ tendency to linger in the periphery tends to restrict infected individu-als ( ) to the periphery. Image re-drawn from Cremer et al.8
P
ropolis is commonly considered by beekeepers the bane of their exis-tence. It gums up the hive making it difficult to perform even the most simple tasks. As a result, beekeepers have selected against this trait and today few commercial queen breeders purposefully produce queens whose progeny are propolizers. When I capture swarms from areas where bees are actively managed, the lack of propolis production is a common trait. In contrast swarms that I capture from areas with little active beekeeping activity, and I presume to be for the most part feral, typically gum up my equipment within weeks. As a molecu-lar biologist, this suggested to me that there remains a fairly strong natural selection pro-cess for propolis production and that human selective forces interferes with the natural selection process. My research experience told me that organisms rarely endeavor in costly energetic behavior unless it providesa benefit in survival. In 2010 a comprehen-sive review of propolis and honey bee health was published. This review was a mere tease, providing researchers many more questions for future research.1 Since this review was published, researchers world-wide have started to explore propolis and its action within the hive. And the research is beginning to suggest that it may not be co-incidence that honey bee health has declined as we humans have selected against propolis production.
WHAT IS PROPOLIS Propolis is a complex mixture of com-pounds produced by honey bees and certain other insects, the exact mixture varies from country to country, region to region, and is dependent on the local fauna. The general mixture, however, remains fairly consis-tent. Honey bee propolis generally consists of 50% botanical compounds, 30% waxes,
10% essential oils, 5% pollen, and 5% other organic compounds and displays common biological activities.2 Those activities, rela-tive to honey bees, are; antibacterial, anti-oxidant, antiparasitic, antifungal, antiviral, anti-inflammatory, and immunomodulatory.
In temperate climates in the United States the primary source for the plant resins col-lected by honey bees are poplar, birch, horse chestnut, alder, beech, and some conifers. A Typical propolis sample from temperate re-gions contains over 300 unique compounds.
The primary compound types in this propo-lis are; phenolic acids and esters, flavonoids, terpenes, lignans, aromatic aldehydes and alcohols, fatty acids, stilbenes, and steroids.
Propolis from other climates or regions con-tain a totally different mix of compounds;
yet, demonstrate similar biological activity.
Interestingly, just as with nectar, honey bees appear to be just as selective in col-lecting plant resins for use in propolis. A
(l) Propolis deposited by bees on the top of new frames of a hive that has been selected for propolis production. (r) Propo-lis deposited on the top of new frames as well as being deposited to seal the seams between frames and the hive body.