By Geoff Tribe, Karin Sternberg and Jenny Cullinan

An interesting pattern discerned in the natural nests of wild honeybees in pristine Coastal Fynbos and Succulent Karoo has been the lavish use of propolis to form an enclosing wall at the entrance to the nest.
 The vast majority of nests in the coastal fynbos are located under boulders and half of those in the succulent Karoo within deserted aardvark burrows. These nests have been completely sealed off except for the few exit holes within the propolis sheath (Fig. 1a+b).

The nest within a cliff in the coastal fynbos was partially propolised with part of the combs still exposed. The value of such propolis barriers could be gauged from the behaviour of the bees which started rebuilding these protective shields soon after a wild fire had passed through and destroyed them (Fig.2). The choice of nesting sites in the two biomes differs considerably as a result of the climate and topography and yet this phenomenon occurs in over 95% of the nests under rocks or in burrows. The propolis forms an immediate mechanical barrier but due to its chemical properties, imparts social immunity to honeybees through both contact and volatile emissions. Propolis is used by honeybees for a variety of purposes and certain races of the Western Honeybee (Apis mellifera) are known to use propolis more abundantly than others, particularly colonies of wild bees. Why should this be so, and what are the main cues for using propolis? A look at African races of honeybees that do or don’t use propolis when related to their environment, and where and when propolis is used may give some insight into this behaviour.

Use of propolis

A colony of honeybees collects 150-700g of propolis per hive/annum (Ghisalberti 1979, Prost-Jean 1985). Various reasons have been suggested for the ‘excessive’ use of propolis by certain races of honeybees and the Greeks very early named the substance for what they regarded as its major function – pro = before or in defence of, polis = the city.

Several environmental cues have been suggested that initiate the collection and use of propolis. The one most often touted is the enclosure of the nest with the onset of winter which is initiated by shortening day length and dropping temperatures. However, a direct correlation has never been established between the two although there is an increasing use of propolis with the onset of winter, possibly due to a reduction in nectar flow and the availability of excess foragers. This has also been observed in the African bee. If the distribution of propolis within a hive were to be quantified, the amount used in building a wall to enclose the colony, thus enhancing increased control of temperature and humidity within the hive, would account for at least half of all the propolis used in the hive. But this is not always so in the case of a wild colony in a cavity in which much propolis is used to line the cavity walls, the possible reason for this will be explained below.

Dead mice, decaying death’s head moths and other intruders are often coated with propolis which could be regarded as disease control because they are too large to remove. The large hive beetle (Hoplostomus fuligineus) of which up to 750 have been recorded in a hive, are impervious to stings (Fig.3) and cannot be removed by the bees. They burrow through comb eating bee larvae and are most destructive. The only recourse the bees have is to coat them with propolis and this has the effect of preventing them from exiting through the narrow hive entrance where they then die. The beetles lay their eggs in dung and those which do escape can often not fly as their elytra are fused together with propolis (Tribe 2009).

Constituents of propolis

Propolis is the generic name for the resinous substance collected by honeybees from various plant sources whose composition varies depending on its origin. The chemical composition of resins is complex and variable within and among plant families, traits that make resin production a good defence against rapidly evolving pests and pathogens (Wilson et al. 2013). Exudate from trees, plant wounds, and waxes from buds, such as from Protea repens and Leucadendron, are major sources of propolis, the waxes protecting the delicate new buds against harmful ultraviolet radiation.     These plant exudates are present on all continents and honeybees introduced to continents where they were never present (such as Australia, New Zealand and the Americas) find no difficulty in obtaining propolis. The genus Populus is widely regarded as a preferential source of resin for honey bees in temperate regions (Wilson et al. 2013). Apis mellifera scutellata has in addition been recorded collecting pieces of leaves of Baccharis dracunculifolia (Compositae) with which to press the propolis into the corbiculae of the hind legs (Yoneda et al. 2001). Tree resins and developing leaves and buds have a high concentration of a wide variety of polyphenols which may differ radically from each other from different parts of the world.

Propolis may vary tremendously depending upon what is available in the immediate vicinity of the honeybee colony but consists of waxes, resins, balsams, aromatic and ethereal oils, pollen and other organic material in a ratio of say, 30% waxes, 55% resins and balsam, 10% ethereal oils and 5% pollen (Bee World 1973). Propolis may vary in colour from mustard-yellow to dark brown and the colour from the same source may vary depending on the season and the state of development of the buds. What bees are collecting are the chemical defences of plants used against fungi, bacteria and various predators. Resins of trees for example function to expel or encapsulate bark beetles while at the same time protecting the wound from fungal pathogens. The medicinal uses of garlic for humans are well known where, following the wounding of the corm, the volatiles mix with oxygen to form a potent compound which acts as an enhanced deterrent. Propolis is hard and brittle when cold, but becomes sticky when warm. At temperatures of 25-45°C propolis is soft and pliable and most varieties will melt between 60-70°C and some only at 100°C. Beeswax melts at about 63°C. Honeybees maintain a hive temperature of 34°C within the brood area. The yellow colour imparted to beeswax is known to be due to the presence of some constituents of propolis (Ghisalberti 1979).

Medicinal uses

Propolis has antibacterial, antimicrobial and antifungal activity; the largest group of compounds isolated are flavonoid pigments which are ubiquitous in the plant kingdom. Myrrh and frankincense of ancient times were aromatic resins obtained from trees either through bark incision or extraction from beekeeper’s propolis derived from balsam (Iannuzzi 1983a). Propolis may have been used by ancient Egyptians for embalming purposes. The Biblical ‘balm of Gilead’ was a bee-collected resinous material i.e. propolis used to heal wounds.

During the Anglo-Boer War a preparation of propolis and Vaseline, ‘propolisin vasogen’, was used as a medication due to its antibacterial properties in aiding the healing of wounds and tissue regeneration (Ghisalberti 1979). Propolis is effective against 38 skin fungi and on second degree burns. Propolis also has a surface anaesthetic action with negligible penetrating power in which the active principle is suggested to come from its essential oil. An alcohol extract was reported to be 3.5 times as strong as cocaine and was used in dental practice in the USSR in 1953.

Control of American Foulbrood

Lindenfelser (1969) discovered that an alcohol extract of propolis inhibited the growth of American Foul Brood (Paenibacillus larvae) disease in honeybees. The extract was fed directly, or mixed in dilute honey, or sprayed as an aqueous or saline solution on to the combs. At 500μg/ml the disease was controlled only during treatment, but higher concentrations destroyed healthy larvae and caused deformities. Resin from different species of plants varied in their ability to inhibit P. larvae with North American poplars differentially inhibiting the growth of P. larvae (Wilson et al. 2013). Thus, a bee’s choice of resin could have profound consequences for their ability to reduce the overall microbe load within the nest cavity. Stingless bees increase resin foraging in response to ant attacks, while honey bees increase resin foraging when intentionally exposed to the larval fungal pathogen Ascosphaera apis, the cause of chalkbrood (Simone-Finstrom & Spivak 2012).

Collection of propolis

Honeybees scrape the waxes and resins from various plants and pack it into their corbiculae or pollen baskets and return to the hive where they wait in a remote part of the hive until bees needing propolis come and pull pieces off her loads (Bee World 1973). The individual bees that both use it and collect it are specialists of foraging age. Bees that normally forage propolis also use it in the hive and are middle-aged, the latter known as ‘cementing’ bees. Resin collection usually takes place in the warmer part of the day between 10h00 and 15h30 on sunny days when it is more pliable. Honey bees have a high fidelity to a single botanical source of resin during a single foraging trip and it appears that availability, proximity, and perhaps toxicity may play roles in the selection of resins by bees (Wilson et al. 2013). Propolis may contain as many as 300 different chemicals which make it difficult for an organism to develop resistance.

The small hive beetle

The small hive beetle, Aethina tumida (Fig.4.), is native to sub-Saharan Africa and is found to a lesser or greater degree in most colonies of honeybees. If the colony is strong and healthy, these beetles are kept in check and harassed by the bees and thrown out of the hive if the bees are able to get a grip on them. But the beetle is highly adapted to its life within the hive where it is able to fit into a cell and feeds on honey and the eggs of bees. The hemispherical beetles are hairless, half the size of a worker bee, and they tuck their legs under their bodies and tightly adhere to the substrate so that a bee may not dislodge it (Tribe 2009). As the bee attempts to bring its sting into operation, it must release the beetle it has cornered which then takes the opportunity to scoot away. If a colony is diseased or begins to fail, then the beetles immediately become active, mate, and lay copious numbers of eggs. The resultant larvae devour the entire contents of the hive, causing a characteristic stink and then bail out and pupate within the soil in front of the hive. They function as the scavengers of the honeybee world where, for example, diseased colonies are neutralized and destroyed. Propolis however, is rarely eaten by the small or large hive beetles or by wax moths.

The presence of the small hive beetle appears also to result in excessive use of propolis within the hive (Tribe 2000). Researchers at Rhodes University have shown that honeybees keep the beetles at bay by encasing them in propolis ‘igloos’ or prisons if they cannot extract them from narrow crevices. But the beetles are able to survive in the hive by mimicking the begging of food by another bee, in which certain bees are deceived into feeding them (Neumann et al. 2001). In wild nests, especially in decaying holes in trees, the inside of the cavity may be totally lined with propolis which denies hiding places for these beetles.

Several wild swarms built under branches of trees have been recorded being totally encased within a propolis sheath (Tribe & Fletcher 1977). First a propolis layer is laid down under the branch or intertwining branches to which the combs are then attached. Here the main advantage appears to be the enhanced control of temperature and humidity, although the control of pests is also facilitated by this. These nests are usually so successful that the weight of their combs, which are weakened by high summer temperatures, eventually causes the entire colony to crash to the ground (Tribe 1979). Other pests with which the bees have to contend are the large hive beetles, wax moths, bee pirates and death’s head moths where narrow entrances in propolis sheaths are easily defended.

Propolis and races of Apis mellifera

Different races of Apis mellifera are recorded to use propolis to a greater or lesser degree (Ruttner 1986). The ‘Punic’ or ‘Tellian’ bee, Apis mellifera intermissa, is a uniform black race inhabiting the region of North Africa from the Atlas Mountain to the Mediterranean Sea and Atlantic Ocean (Tunisia, Morocco, and Algeria). This race is recorded to use excessive amounts of propolis. The coastal Mediterranean vegetation gives way to inland areas in which intense climatic extremes are experienced – a reason why European races of bees imported on a large scale (mostly from Italy and France) have failed to become established. Not only is there a huge daily variation in temperature which would warrant the lavish use of propolis as a means to control temperature and humidity within the nest, but the Tellian bees are known to be susceptible to brood diseases; a further reason for an abundant use of propolis.

Excessive use of propolis is also recorded for Apis mellifera iberica which is closely related to the Punic bee and A.m. mellifera which survives winter temperatures as low as -45°C and is adapted to a continental climate with its severe extremes of temperature.

Holistic defence mechanisms

Randy Oliver (2010) gives a good account of the honeybee immune system. As a complex super-organism, the honeybee colony is imbued with defences at various levels that are physical, chemical and behavioural at colony level but they are also endowed with an effective immune system at the level of the individual bee. The large honey stores of the Western honeybee which is necessary to see it through the dearth period, be it drought or winter, as well as their nutritious brood, represents a considerable food resource to predators. Firstly, the siting of nests in inaccessible clefts in rocks or high up in trees has immediate survival value. Mass attack following the marking with alarm pheromone of mammalian predators serves as a major deterrent at colony level. Smaller predators such as wax moth larvae, small hive beetles and Varroa mites are either stung or physically removed by biting with the mandibles.

Behavioural defence involves the use of undertaker bees which carry dead or dying bees beyond the boundaries of the colony; hygienic bees with their ‘washer-woman’ action remove fungi; while sick brood is detected before it becomes infective and is removed from the hive. Diseased bees voluntary leave the colony, never to return. At the individual level, eggs are laid in clean cells isolated from others and the larvae spin cocoons within the capped cells to further protect the pupae. Antimicrobial enzymes are added to the nectar to produce honey and pollen is inoculated with beneficial moulds and bacteria to preserve it within designated cells. Should parasites reach levels that are too high with which to cope, the swarm may simply abscond.

The stomach of the honeybee is possibly the most vulnerable to diseases such as AFB and nosema despite an arsenal of immune cells (haemocytes) and antimicrobial peptides to engulf and neutralise them. An additional weapon in the arsenal of the bees is self-medication using the defensive chemicals of plants.

Propolis vapours and contact as prophylactic medication?

One cost of social living is an increased rate of disease transmission among individuals, and honey bees are highly prone to a diverse set of pathogens and parasites (Wilson et al. 2013). Propolis deposited in the hive has important immunological benefits which exhibit phytoinhibitory and phytotoxic properties induced within the hive presumably from vapours because potato tubers kept in a hive did not sprout and after an extended period they suffered permanent inhibition (Ghisalberti 1979). An aqueous extract of propolis was also shown to inhibit germination. When comparing propolis treated colonies with controls, they were shown to have a significant reduction in the overall bacterial loads (Simone-Finstrom & Spivak 2010). Thus the presence of propolis in a honeybee colony may reduce the investment in the innate immune response by acting as an external immune defence mechanism i.e. the honeybee immune system is quieted in the presence of a layer of propolis enveloping the inside of a bee nest (McNeil 2010). This is the first direct evidence that the bees’ nest environment affects immune-gene expression (Simone-Finstrom & Spivak 2010). A degree of self-medication is evident where bees have been observed to embed strands of propolis in cleaned cells as a disease resistance mechanism and place propolis on the rims of cells. Cells are coated with a thin layer of propolis to sterilize them and bees entering or leaving the hive are additionally cleansed of microbes as they pass through various structures made of propolis.

Observations at natural nests have shown how bees utilize the innate antibacterial properties of propolis. In preparation for foraging trips and when the propolis is warm and sticky, bees have been seen licking at the propolis and ‘washing’ themselves with it. Bees walk across the propolis surfaces before leaving and after returning from foraging flights. It is thought that the propolis acts as a disinfecting zone. Because bees can “taste” through their feet this might be a form of protection through absorbing the antimicrobial properties of propolis prior to and after foraging (Sternberg, Cullinan, Tribe 2015).

Propolis appears to be a multi-purpose substance which is used in a wide variety of situations according to its need, this largely being determined by its environmental circumstances especially where extreme fluctuations in temperature are experienced. An aspect of the value of propolis within a colony is its value as natural prophylactic medicine acting directly on the bee itself (McNeil 2010). On warm days the aromatic odour of the propolis which permeates the nest and the volatiles that fill the cavity could have a profound effect on reducing the overall microbe load within the nest. Is it perhaps possible that besides contact, the inhalation by the bees of these anti-biotic elements contributes to the general health of the bees within the colony? Thus the growing of plants with known antimicrobial resins around apiaries could possibly further promote bee health.

The honeybees of Africa have never been entirely domesticated, there being vastly more colonies in the wild than in hives. Thus the African bee still retains much of its natural health that made it so adaptable and vigorous in the wilds of Africa.

…to be continued.

The following video clip has been slowed down to 60% of the original speed and shows bees at one of the wild nests wiping themselves with propolis before flying off on their foraging trips. The entire wall over which the bees are walking is made of propolis:

 

The authors at work:

References

Bee World. 1973. Bee products: Propolis. Bee World 54(2): 71-73.

Ellis, J.D. 2002. Life behind bars: why honey bees feed small hive beetles. American Bee Journal 142(4): 267-269.

Ellis, J.D., Delaplane, K.S., Hepburn, H.R., and Elzen, P.J. 2002. Controlling small hive beetles (Aethina tumida Murray) in honey bee (Apis mellifera) colonies using a modified hive entrance. American bee Journal 142(4): 288-290.

Ellis, J.D. and Hepburn, H.R. 2003. A note on mapping propolis deposits in Cape honey bee (Apis mellifera capensis) colonies. African Entomology 11(1): 122-124.

Ghisalberti, E.L. 1979. Propolis: A review. Bee World 60(2): 59-84.

Iannuzzi, J. 1983a. Propolis: The most mysterious hive element. American Bee journal 123(8): 573-575.

Iannuzzi, J. 1983b. Propolis: The most mysterious hive element. American Bee Journal 123(9): 631-633.

Lindenfelser, L.A. 1969. In vivo activity of propolis against Bacillus in larvae. Invertebrate Pathology 12: 129-131.

McNeil, M.E.A. 2010. Marla Spivak: getting bees back on their own six feet. American Bee Journal, Part 1: September: 857-860, Part 2: October: 949-953.

Neumann P., Pirk C.W.W., Hepburn H.R., Solbrig A.J., Ratnieks F.L.W., Elzen P.J. and Baxter J.R. 2001. Social encapsulation of beetle parasites by Cape honeybee colonies (Apis mellifera capensis Esch.). Naturwissenschaften 88: 214-216.

Nicodemo, D., Couto, R., Malheiros, E., De Jong, D. 2012. Propolis production and its relation to wax production rate in Apis mellifera beehives. In: Científica , Jaboticabal, v.40, n.1, p.90 – 96, 2012.

Oliver,R. 2010. Sick Bees – Part 3. The Bee Immune System@ Scientific Beekeeping.

Ruttner, F. 1986. Geographical variability and classification. In: Bee Genetics and Breeding, Academic Press Inc. pp. 23-56.

Simone-Finstrom, M. and Spivak, M. 2010. Propolis and bee health: the natural history and significance of resin use by honey bees. Apidologie 41: 295-311.

Simone-Finstrom, M. and Spivak, M. 2012. Increased resin collection after parasite challenge: A case of self-medication in honey bees? PLoS One 7(3) e34601, doi:10.1371/journal.pone.0034601.

Tribe, G.D. 1979. The fate of the propolized nest. South African Bee Journal 51(6): 12-15.

Tribe, G.D. 2009. Creatures within the hive. Village Life 34: 38-43.

Tribe, G.D. 2000. A migrating swarm of small hive beetles (Aethina tumida Murray). South African Bee Journal 72(3): 121-122.

Tribe, G.D. and Fletcher, D.J.C. 1977. A propolized nest in the open. South African Bee Journal 49(4): 5-8.

Wilson, M.B., Spivak, M., Hegeman, A.D., Rendahl, A. and Cohen, J.D. 2013. Metabolomics reveals the origins of antimicrobial plant resins collected by honey bees. PLoS One 8(10):1-13.

Yoneda, M., Shibata, I. and Takahashi, S. 2001. Leaf collecting behaviour of Africanized honeybee. Poster: Apimondia, Durban, 28 Oct.-1 Nov., 2001.