Chemical Communication in the Honey Bee Colony
Honey bee have two forms of communication: mechanical and chemical. The former includes “piping”, buzzing and waggle dancing. The latter relates to pheromones released by the brood and adult bees, and will be examined here. By definition, pheromones are signalling chemicals released by an animal (e.g. queen) to control the behaviour of other animals in the vicinity (e.g. workers and drones).
This is an overview article of the known honey bee pheromones, written for beekeepers who want to know what scents are produced in the hive (or on your skin after a sting). A note: you’ll see references to “primer” and “releaser” pheromones. Primer pheromones are compounds that impact the bee’s physiology (e.g. suppression of worker egg laying from brood pheromones), whereas “releaser” pheromones influence their behaviour (e.g. increased pollen forage, also from brood pheromones). Remember, P for physiology (primer) and R for response (releaser).
Brood Pheromones
“Brood pheromone” is a blend of 10 fatty-acid esters, with the compounds being individually capable of triggering a degree of brood pheromone effects on adult bees (Le Conte et al. 2001). The components are listed for your reference: methyl palmitate, methyl oleate, methyl stearate, methyl linoleate, methyl linolenate, ethyl palmitate, ethyl oleate, ethyl stearate, ethyl linoleate, and ethyl linolenate. Fortunately, beekeepers are not chemists.
One signal of brood pheromone is “hunger”. Upon detection, nurse bees are prompted to feed the larvae (bee bread or nectar), and foragers begin gathering pollen (Pankiw et al., 1998 & Pankiw, 2004). The higher the pheromone concentrations, the larger the pollen loads, and the more nurse bees recruited for feeding activities.
A combination of brood and queen pheromones contributes to the inhibition of worker bee ovary development. This prevents the emergence of egg-laying workers and maintains order in the hive; the colony’s resources are dedicated to rearing bees from queen-laid eggs.
Alaux and colleagues found that exposure to brood pheromone delayed the transition of young bees from nursing to foraging roles1 (Alaux et al., 2009). Analysis showed a decrease in forager-related genes, and increases in nursing-related genes, up until those bees became foragers at 15 days of age. 15 days is quite young for a forager (~21 days is the average); it could be due an age-dependent effect of brood pheromones. Young bees do more nursing until a certain age, at which point the brood pheromone effects reverse and they perform more foraging.
Brood Pheromone Decreases Honey Bee Lifespan
One reason why winter bees live longer is due to brood absence. Smedal and colleague showed that in generally broodless condition (as in a winter cluster), worker lifespan averaged 20 weeks, unlike the 3-6 weeks of spring and summer bees (Smedal et al., 2009). Workers exposed to synthetic brood pheromones consumed more pollen, and had more active hypopharyngeal glands, and lowered vitellogenin stores (a egg-yolk precursor protein used in the production of royal jelly).
By comparison, colonies overwintered in the presence of brood pheromone had decreased longterm survival. The authors suggest that it is the pheromone, not the workload placed on nurse bees (and subsequent wear and tear), that decreases the overall adult lifespan. In other words, lifespan is not compared against “the metabolic costs of caregiving” (Amdam et al., 2009).
Vitellogenin Accumulation in Worker Bees
A drawback of Smedal & colleagues’ research is measuring the fat body’s vitellogenin stores as a survival marker, whereas other factors like stored lipids and carbohydrates may further influence bee survival (Smedal et al., 2009). When brood pheromone was removed, workers retained more vitellogenin, possibly for its antioxidant and immune defence properties, rather than to prepare for brood rearing (Seehuus et al., 2006). Vitellogenin may be one factor involved in extended worker bee lifespan, since the nutritional content of insect fat bodies is tied to their overall lifespan (Hahn & Denlinger, 2007).
Queen Bee Pheromones
Queen Mandibular Gland Pheromone
The major colony pheromone is the queen mandibular pheromone. The principally active component is (E)-9-oxodec-2-enoic-acid (9-ODA), with other compounds consisting of hydroxydec-2-enoic acid (9-HDA), methyl p-hydroxybenzoate (HOB) and 4-hydroxy-3-methoxy-phenylethanol (homovanillyl alcohol [HVA]). These five chemicals blend to form the basis of the mandibular pheromone secretion and signals queen presence (Slessor et al., 1988).
Mated queens have higher levels of queen mandibular pheromone, whereas virgin queens show a higher concentration of worker mandibular pheromones, 10-HDA & 10-HDAA. However, Strauss & colleagues found that 9-ODA concentrations are constant between virgin, drone-laying, and mated queens (Strauss et al., 2008).
The authors suggested that 9-ODA in virgin queens was used in drone attraction, whereas in mated queens it attracted a retinue. The fertility of queens was associated with increased ratios of 9-HDA, 10-HDAA, 10-HAD, and HVA. By contrast, the more fertile a queen the lower her 9-ODA levels.
Effects of Queen Mandibular Pheromone in Colonies
QMP maintains colony cohesion and signals Her Majesty’s presence. During mating flights, the virgin queen relies on QMP (specifically, 9-ODA) to transmit her location for the drones to mate. Depending on colony size, it can become saturated with QMP from a few hours to a day. When a worker bee bearing QMP interacts with another worker (via feeding or grooming), they will pass the QMP to them, and that worker will go on to pass it to others until the entire hive is familiar with her scent.
Grozinger & colleagues showed that QMP influences gene expression in the worker bee brain (Grozinger et al. 2003). Consistent with its effect on behaviour (delays transition from nursing to foraging), QMP further increases gene expression in the alleles already upregulated in nurses, and decrease the gene expression of those genes normally upregulated in foragers. Perhaps these behavioural changes make workers more attentive to the queen, dedicating more time to brood and queen care.
Further, the presence of brood and queen pheromones affect the reproductive capability of workers. We know from the aforementioned work of Strauss et al., that 9-ODA is not responsible for suppressing worker ovaries, as its concentrations decrease in more fertile queens, and increase in less fertile/virgin queens (Strauss et al., 2008). If 9-ODA was the suppressor, we would expect high levels in normal colonies, but the opposite is observed; queenright hives with laying workers usually have a weak or virgin queen and variable 9-ODA levels.
Queen Retinue Pheromone
One of the first articles I wrote was on raising queen bees. I mentioned that Queen Retinue Pheromone was its own compound, and failed to consider that it acts in synergy with the mandibular pheromone. Live and learn.
Keeling and colleagues found that four compounds synergised with QMP to form the retinue: coniferyl alcohol (produced by the mandibular glands), methyl oleate, hexadecane-1-ol, and linoleic acid (produced elsewhere in the body; Keeling et al., 2003). When workers are exposed to these compounds in isolation, no retinue response was triggered, whereas exposure in combination with QMP increased retinue activity.
Maisonnasee and colleagues showed that in queens can attract bees and form a retinue without QMP (Maisonnasse et al. 2010). Considering that the aforementioned latter three compounds are produced elsewhere from the queen’s body (Keeling et al., 2003), it is possible that a queen bee utilises other chemical substances to attract workers.
Like QMP, the retinue pheromone is secreted by the mandibular gland and is responsible for attracting and retaining the clusters of worker bees that attend to her needs. A decline in retinue pheromone has been suggested as another factor that determines swarming behaviour (reviewed in Grozinger et al., 2015).
Queen Dufour’s Gland Pheromone
The Dufour’s gland stimulates retinue behaviour, and establishes Her Majesty’s reproductive dominance by helping suppress worker ovaries along with QMP. The Dufour’s gland is located in the abdomen, clustered around the regions containing the sting sheath, venom and Koschevnikov glands. Previous research suggested that in queens, the gland provided eggs with a “queen-laid” scent, whereas it had defensive purposes in workers (lubricating the stinger; Martin et al. 2005). Further experiments by Martin and colleagues showed that when eggs are marked with the queen’s Dufour’s gland pheromone, workers removed them more often than the control (Martin et al., 2002).
Another hypothesis suggested that the Dufour’s gland is a marker of “queen fertility”, as these waxy compounds are tied to her ovarian development (Katzav-Gozansky et al., 1997). The laying workers of A.m. capensis produce a strong mimic of these queen esters, reinforcing the idea of Dufour’s secretions as a fertility signal (Sole et al., 2002). Like the queen mandibular pheromone, extracts of Dufour’s gland caused the formation of a retinue around the source, with a higher rate of attraction in mated versus virgin queen extracts (Richard et al., 2011).
Queen Tergal Gland Pheromone
“Tergites” are the openings between abdominal segments on the upper insect exoskeleton. Queens have six tergites, and her glands are located between tergals 3-5. Tergal gland are thought to be complementary to queen mandibular pheromone, assisting in worker bee attraction. Each compound alone can form a retinue, with attraction being strongest in combination. In the A.m scutellata Queens (African savanna bees), the glands both attract workers (releaser) and inhibit worker ovaries (primer; Wossler & Crewe, 1999a).
Tergal Glands in Cape Honey Bees
The retinue pheromone of A.m. capensis is a 50-50 blend of QMP and tergal gland pheromones (Wossler & Crewe, 1999b). This could explain why these workers readily become “false queens” – they lack the QMP production, and compensate with tergal secretions. Further, if their queens’ tergal secretions are equally potent to QMP, it could be why Cape honey bees become social parasites of non-Cape bee colonies. A.m. capensis worker bees could be used to higher levels of tergal & queen mandibular pheromones to suppress their ovaries (Wossler & Crewe, 1999c). Neighbouring colonies of A.m. scutellata do not share this potency, leading to laying Cape workers.
Queen Footprint Pheromone
The tarsal gland (as oppose to “tergal”) refers to a single layer of cells on the base of the bee’s foot (“tarsus”). Queens secrete pheromones as she walks on comb. Along with the mandibular pheromone, they signal her presence to the hive and stops the construction of queen cups (Lensky & Slabezki, 1981).
Recall that swarm cells are built on the bottom edges. In crowded colonies, the queen is restricted to the center of the frames, never reaching the bottom bar. Can you see the link between the lack of queen footprints, and the production of swarm cells? Due to high colony densities, these bottom areas go untouched by the queens, triggering swarm cell construction by worker bees.
Virgin Queen Rectal Gland Pheromone
Didn’t expect this one did you? During the swarming season, multiple queens emerge and fight for colony dominance. Page and Erickson noted that when these virgin queens defecated, causing the workers to move away from them (Page & Erickson 1986). Follow up studies showed that the rectal content of virgin queens contains a worker repellent. What this means is unknown – perhaps it stops workers from interfering with the fighting (“the queen is injured!”)? (Post et al., 1987). Note that it doesn’t always happen, as in the screenshot below.
Worker Bee Pheromones
Worker Mandibular Gland Pheromone
Unlike queens, workers mandibular glands produce s10-hydroxy-2(E)decenoic acid (10-HDA) & 10-hydroxydecanoic acid (10-HDAA). Workers can produce small amounts of the queen pheromone 9-ODA, as evident in queenless colonies; their laying workers have developed queen-like characteristics. An exception is the Cape honey bee, whose workers are able to lay both fertilized and unfertilized eggs in the presence of the queen, and parasitise hives of other subspecies.
In general, the worker mandibular gland pheromone levels increase as the rate of worker egg laying increases (Robinson and Huang 1998), peaking in hopelessly queenless colonies.
2-Heptanone as a Defensive Pheromone
Worker mandibles produce 2-Heptanone, a component of the alarm pheromone. Despite having a lesser potency compared to the sting gland compounds, it could serve as an immobilising agent that accompanies biting behaviour. When Varroa mites or wax moth larvae are bitten, the 2-heptanone paralyses this too-small-to-sting pest (Papachristoforou et al. 2012).
Worker Nasonov Gland Pheromone
If you see workers bees standing with an elevated, pumping abdomen followed by fanning, then that’s the Nasonov gland in action. It’s located between the sixth and seventh abdominal tergites. The gland releases a blend of seven chemicals: eraniol, nerolic acid, geranic acid, (E)-citral, (Z)-citral2, (E-E)-farnesol, and nerol (Pickett et al., 1980). It has multiple functions, from swarming, hive entrance marking, identifying foraging sources, and helping the queen find her way back home.
When the first workers depart as part of swarm, those bees fan their Nasonov and direct the rest of the swarm to their temporary location. Later, the same scouts waggle dance to communicate the location of their new permanent home. They then return to that location and start fanning their Nasonov to lead the bees home.
In the field, forager bees use their Nasonov gland to attract other foragers to a source of water, or a plant with abundant forage. In experiments, a profitable forage was defined as one with the relatively highest sugar concentrations, making it the best source of nectar (Fernandez et al., 2003). For further discussions on nectar quality vs quantity, see “Nectar Chemistry Part One” & “Nectar Chemistry Part Two“.
Alarm Pheromones: Worker Sting & Koschevnikov Glands
During hive defence, guard bees at the hive entrance raise their abdomen and expose the sting chamber along with alarm pheromones; they fan their wings to dispersing these volatile components. These compounds signal actual or perceived dangers to the bee hive, and are released not just when exposing the stinger, but also during stinging, and when the barb is left in your (unfortunately- exposed) skin.
Alarm pheromones are produced by the Koschevnikov gland and gland-containing areas around the sting sheath. The active component of the sting alarm pheromone is isopentyal acetate (“IPA”, not one you’d want to drink). Its release alerts other workers to commence defensive behaviours. The concentrations of IPA are nil in queens, and slowly develop in worker bees, peaking at 2-3 weeks of age when they begin guard duties (Allan et al., 1987). IPA is the main active component, but not the only one – others like 2-heptanol or 2-nonnal can trigger defensive behaviour, and the complete alarm pheromone is a blend of over 40 compounds.
The composition of the alarm pheromone is subspecies specific – Africanised bees have higher levels of its component chemicals, and more IPA. This could be why they’re so aggressive (“defensive”) when triggered.
Bees Attack Bananas: Isopentyl Acetate
Every once in a while, a social media post goes viral showing honey bee aggression towards bananas, or the unfortunate consumer of one. IPA is also a component of banana oil, and when exposed at the hive entrance, triggers defensive bee-haviour in line with their alarm response (Shearer & Boch, 1965). On the contrary, if you want bees to avoid a certain water source (say, your bird bath) – perhaps add in some whole or mashed bananas, and see if it’ll repel water foragers in the same way it repels flower foragers (Free et al., 1985).
Worker Tarsal Pheromone
Like queens, the workers have a footprint pheromone. Williams and colleagues suggest that it is a “close proximity” equivalent of the Nasonov gland, serving as a method of marking comb, the hive entrance and rich forage for nearby bees (Williams et al., 1981). The Nasonov is for long-distance marking, as demonstrated by its ability to guide swarms to their new home.
Worker Tergal Gland Pheromones
In queenright colonies, the worker tergal glands are inactive, as the queen controls worker fertility. Once queenless, they are activated and used for establishing reproductive dominance amongst other bees. Once again A.m. capensis is the model worker to investigate this (Okosun et al., 2015 & 2019).
False queens use the tergal gland pheromones to complete with her sisters, producing the compounds palmitic acid, oleic acid, n-heneicosene and n-tricosene, n-pentacosene, n-heptacosene and n-nonacosene. They also produce brood pheromone compounds like ethyl palmitate, ethyl oleate and ethyl stearate; perhaps part of the reason why their retinue feeds and tends the false queens more often?
Drone Pheromones?
The male drones don’t contribute much by way of pheromones. The drone’s mandibular gland secrete a pheromone that aids other drones in finding (and forming?) the congregation areas (Villar et al., 2018). Drones also have a different tarsal gland structure, the purposes of which are (still) unknown (Lenksy et al., 1985). Given how little economic value there is in drones, I don’t think we’ll see funding for “drone tarsal gland research” anytime soon.
The drone mandibular gland produces pheromones until 7-8 days of age, when their mating flights commence. The drones release pheromones as they fly to the congregation areas, attracting other drones over long distances. Note that some of the compounds found in the drone mandibular glands overlap with those found in QMP (hexadecanoic, & octadecenoic acids).
However, Villar and colleagues do not suggest that the drone mandibular secretions are a “pheromone” on the basis of insufficient information: “[C]ontexts in which these compounds are secreted are still unknown and will need to be elucidated before a function can be ascribed. Identifying whether drones secrete the contents of the DMG [Drone Mandibular Gland] on their way to and/or at mating sites will be valuable in understanding whether they are using chemical cues in addition to landscape cues to arrive at and maintain a presence at congregation areas” – (Villar et al., 2018).
Colony Pheromone – Ethyl Oleate
Ethyl oleate delays the transition from nest bee to forager and is produced by the foragers as they ferment nectar (Leoncini et al., 2004). Rather than getting bees drunk, the compound is secreted from their exoskeleton to form a negative feedback loop:
When there are high number of foragers in a colony, the high ethyl oleate concentrations inhibit the maturation of young bees into foragers, maintaining a population of nurses or middle-aged workers. As foragers die, the pheromone concentration decreases and so the young bees become foragers. Note that both brood and queen pheromone blends contain this compound, so it becomes a pheromone of the “colony” rather than an individual bee caste (Keeling & Slessor, 2005).
Wax Comb Pheromone
Wax comb is a “reservoir” rather than “emitter” of pheromones. Aged comb will contain a variety of brood and adult pheromones, making for attractive swarm bait (Berry & Delapine 2001). On the other hand, older comb has a buildup of shed bee pupa, feces and worse, diseases. Prolonged use leads leads to smaller adult bees (decreases in cell size) or a brood infection, as well as pest infestation (wax moths and small hive beetles). Preventing these issues is why beekeepers are encouraged to recycle brood comb every two-three seasons.
Cuticular Hydrocarbons & Death “Pheromones”
Extracted from “What do bees do with their dead?“
In death, it is the absence of chemical substances that signals a corpse, rather than the production of “death pheromones”.
The loss of cuticular hydrocarbons signals a corpse. In life, these carbon-based compounds are constantly being produced and as part of the chemical signatures used in nestmate recognition. In the Asian honey bee Apis cerana, Wen and colleagues isolated two cuticular hydrocarbons, heptacosane and nonacosane, and found that reduced emissions of these compounds signals to undertakers that this was a dead bee (Wen et al., 2023).
Honey bees on the other hand, rely on a combination of esters and polar compounds to classify nestmates (Frohlich et al., 2001). Workers assigned to different tasks inside the hive, whether undertaking or cleaning, had similar but distinct cuticular hydrocarbon signatures, serving as a marker of their role in the hive (Vernier et al., 2019).
How Do Diseases Impact Pheromone Production?
Now that you’ve read about the effects of pheromones in a healthy colony, I’ll conclude by discussing how these compounds change in a diseased state.
In Nosema disease, the parasite increases ethyl oleate production, with a higher pathogen load leading to higher pheromone concentrations (Dussaubat et al., 2010). As stated above, ethyl oleate controls the maturation of nurses into forager. As its presence increases, the number of foragers in a colony decreases. The implications of this are twofold:
- Hive supplies dwindle, as there are less foragers to replenish them.
- Nurse bees remain in the colony for longer, leading to increased concentrations of Nosema in brood and nestmates as they are not leaving the hive, and
- On the contrary, if more bees stay inside they are likely to have a longer lifespan as they do not conduct risky/demanding foraging tasks (Dussaubat et al., 2013).
Whether this increased ethyl oleate is as an adaption by Nosema to increase its virulence, or an adaption by Apis mellifera to withstand infections, that is currently unknown. I’m sure that other diseases will impact pheromone concentrations, they just haven’t been studied yet.
Read the whole post and found puns, typos mistakes? Voice your displeasure with a strongly-worded message to admin@tgbee.com.au, or by filling this form.
Footnotes
- Queen mandibular pheromone affects many genes in the brain, impacting foraging and nursing behaviour in a similar manner to brood pheromone (Grozinger et al. 2003). ↩︎
- Citral is a compound found in lemongrass oil, and forms the basis of swarm lures during spring. Making a bait hive is easy: On a high point (trees or a rooftop), place a langstroth box containing a brood frame (or old brood comb) and cotton balls soaked in lemongrass essential oil. It can take between a few hours to a few days for scouts to show up, and once they do, it’s a high likelihood of the colony moving in. ↩︎