Undertaker Bees, Hygienic Behaviour and Pheromones
Bring out your dead… Undertaker bees are middle-aged workers who specialize in adult corpse removal to prevent pathogen buildup. This behaviour is triggered by a reduction of cuticular hydrocarbon emissions, as a result of decreasing body temperature from the deceased. For dead brood, odour cues (β-ocimene and oleic acid) are used to determine if the larva or pupa should be uncapped and removed from the colony. If however, there are large numbers of dead bees at the hive entrance, this could indicate pesticide poisoning.
Undertaking is an evolutionary adaption to maintain a “social” immune system. This article explores the pheromone cues used by undertaker bees (cuticular hydrocarbons; oleic acid) and the role of hygienic behaviour in undertaking and disease management. It then examines the diseases found in dead honey bees, as well as the disadvantages of hygienic behaviour.
Role of the Undertaker Bees
The hive is a crowded, dark place (pun intended). When a worker bee steps on her nestmates, the sensation of doing so is indistinguishable from a recently dead bee. So chemical cues come into play. A death cue is a postmortem change in the compounds found within the body that cause the living to react in specific ways.
To perform undertaking behaviours, the undertaker bee touches the corpse and walks around it several times, presumably to detect the cues that confirm death. Once confirmed, the undertaker’s mandibles grab onto the bee and fly away to dispose the corpse away from the colony. In one experiment, freeze-killed bees were placed in a hive, and of the 77 interactions the cadavers had with living workers, only five workers had moved the corpse a significant distance. The remaining bees either moved it a small distance, examined it with their legs or proboscis, or ignored it (Visscher 1983).
Visscher noted that dead bees are removed from the hive within an hour of detection. Other objects, like wood chips or plant material may remain inside the hive for extended periods of time (Visscher 1983). The undertakers who cleaned up the bodies were made up of middle-aged bees, whereas the general nest cleaners consisted of bees of all ages.
How is Undertaking Different to General Cleaning?
Consider the newly-hatched bee. She cannot fly or sting, so she is confined to mature inside the hive. Here, her tasks are largely related to cleaning cells. Eventually she will become a nurse bee, and then a middle aged bee. It is in this group we see the specialisation of undertaking, amongst many other tasks (15 tasks have been identified – see Trumbo et al., 1997 or Johnson, 2003).
Cleaning can be done by any bee at any stage in her life, and the tasks are plentiful. Undertaking as you’ve read above, is a specific task. Even if bees see a corpse, which can be viewed as an unwanted object inside the hive, they will not remove it unless they specialise in this task.
Self-removal of Infected Individuals: Self-undertaking?
Rueppel & colleagues found that in order to reduce the spread of disease, infected bees will leave the hive (Rueppel et al., 2010). This voluntary self‐removal of diseased or parasitized individuals decreases the risk of infection for their healthy kin. In their study, artificially weakening or infecting bees caused them to leave hives more often, and for younger bees to commence foraging earlier. Both are strategies to reduce their time inside the crowded hive, reducing the risk of transmitting disease.
This adaption can backfire – if high numbers of individuals are infected, whether by pesticides or other disease, then a large number of adults may leave the colony, causing colony collapse. Nonetheless, this shows that in addition to recognizing the need to remove dead nestmates, the infected individual bee can remove herself from the hive to reduce the prevalence of disease.
Oleic Acid & β-ocimene are Associated with Dead Brood
The presence of oleic acid triggers the hygienic behaviour in bees, namely the removal of the dead nestmates1 from the hive. Oleic acid and beta-ocimene2 (“β-ocimene”) are released from freeze-killed brood, with the former serving as the actual death cue (McAfee et al., 2018). Hygienic bees are more sensitive to changes in pheromone compositions, and will uncap diseased brood for inspection.
In the freeze-kill assay conducted by McAfee and colleagues, they found that a combination of the two chemicals synergised in triggering hygienic brood removal, rather than either compound alone. Chemically, the two compounds are very different, and it is possible that the release of two in a blend acts as follows:
The highly-volatile β-ocimene attracts worker bees to the brood cell in order to feed it,
Once the cell is inspected, oleic acid is detected and serves as the death cue for brood removal.
On a molecular level, the expression of two proteins, OBP16 & OBP18 (“Odourant binding proteins“) are associated with hygienic behaviour (Guarna et al., 2015). Honey bees with higher OBP16/18 levels are more hygienic, and the work by McAfee and colleagues showed that both β-ocimene and oleic acid bind strongly to either of the proteins, triggering hygienic behaviour (McAfee et al., 2018).
Oleic acid is known to be a death pheromone in many insects (reviewed in Yao et al., 2009). In A. mellifera, it is the combination of oleic acid and β-ocimene that triggers undertaking and hygienic behaviour. This provides an answer to the question of social immunity and colony hygiene – removal of dead and diseased individuals after sensing oleic acid (Yao et al., 2009).
Cuticular Hydrocarbons in Death, Development and Signalling
The loss of cuticular hydrocarbons signals a corpse. In life, these carbon-based compounds are constantly produced and are 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 signaled to undertakers that this was a dead bee (Wen et al., 2023).
Outside of death, the hydrocarbons have other functions- In the social stingless bees of Brazil Melipona marginata, cuticular hydrocarbon differences exist between newly-emerged workers, nurses and foragers (Ferreira-Caliman et al., 2010); this helps the colony distinguish the age and role of the bees. 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).
During the pupal stage, the larval undergoes extreme changes to form the adult bee shape. In this stage of the bees’ life cycle, the cuticle’s chemical profile varies throughout the moult, leading to differing signals between pupa, the developing- and newly-emerged adults (Falcon et al., 2014). Foragers had a higher proportion of alkane and alkene hydrocarbons compared to the newly merged bees, who had a mix of compounds. The technical reader is referred to the aforementioned paper for further reading.
Cuticular Hydrocarbons and the Honey Bee Immune System
When a bee falls ill and fights infection, her cuticular hydrocarbon profile changes, and she is treated differently by her nestmates (Richard et al., 2008). When the authors artificially stimulated a worker bee’s immune system, she was groomed and examined more frequently by her nestmates. This could serve as a form of social immunity; the infected bee is either isolated from the nest, or intensively cared for by her nestmates in order to treat her.
The authors mention “social vaccination”, in which the grooming of infected individuals results in low doses of the pathogen spreading to nestmates, stimulating their immune system to develop resistance to the infection. However, this strategy may be ineffective in the case of lethal conditions like high dose pesticide poisoning.
As the infected bee produces more antimicrobial peptides into the haemolymph (“bloodstream”), her cuticular makeup shifts. In insects, the haemolymph and cuticule are connected via transport channels (Schal et al., 1998); compounds in the blood are transported to the external and internal tissues. Richard and colleagues note that no new cuticular hydrocarbon compounds are produced during infection (Richard et al., 2008). Instead, their ratios change in an infected bee, which enables nestmates identify her both as a colony member and an infected individual.
Hygienic Bees and Diseased Brood Removal
Hygienic behaviour is defined in two steps:
Worker bees that detect and uncap diseased brood,
Removal of diseased brood in a timely manner by worker bees.
The keyword here is “timely”. If workers were just to uncap brood (for inspection, rather than disease monitoring), and not remove it, then an early stage disease like European Foulbrood progresses and leads to the production of infective bacterial spores. If the brood was removed early (“a timely manner”), then this limits pathogen transmission and keeps the colony health (reviewed in Evans & Spivak, 2009).
The two steps can be performed by separate bees. Scannapieco and colleagues showed that while some workers perform both uncapping and removal, others are solely focused on uncapping, or solely focused on removal (Scannapieco et al., 2016). The more bees allocated to these tasks, the more hygienic a colony.
A worker bee is not performing hygienic tasks all day round. The intensity and requirements for hygienic behaviour shifts in response to pathogen loads. In times of lower workloads, that bee may perform other tasks, or stay idle. In my opinion, I do not think these bees remain idle for long – if the pathogen load is reduced, then they may turn to other tasks, like grooming nestmates (allogrooming), or even undertaking.
I hope you disagree with this, because I’m about to show you why I’m wrong.
Hygienic Behaviour is a Form of Worker Bee Specialisation
Pereaz & Johnson showed that under lower pathogen presence, the hygienic bees turn to undertakers; rather than removing dead brood, they removed dead adults (Perez & Johnson, 2019). Most generalist bees in this study spent their time conducting hive inspections and working with wax. (Recall that in Visscher’s study, most bees ignored the corpse, and continued with other duties).
The specialisation of hygienic bees was described as being focused on corpse removal or patrolling. When the demands for the (rare) tasks of hygienic behaviour or undertaking are present, they performed it. If not, they are patrolling and inspecting the hive until their services are required (Perez & Johnson, 2019).
Is it possible that specialisation in dead and diseased nestmates is an adaption to limit the chances of disease to certain individuals? If all bees conducted hygienic behaviour, then all have a chance to be infected from that one corpse. By distributing the workload to a low number of specialised (yet otherwise regular middle-aged bees), they confine the pathogen transmission to those select individuals.
Dead Bees at the Hive Entrance is a Sign of Pesticide Poisoning
Adapted from “Pesticides that kill bees“
Pesticides applied at high concentrations lead to a mass of dead bees at the hive entrance. This is one of the reasons why they recommend pesticide application for commercially-pollinated crops (almonds, apples etc…) to occur when after the bees finish pollination; the chemicals will be present in the gathered nectar and pollen. Once high concentrations are inside the hive, it does not take long for the colony to succumb to poisoning. Examples of pesticide poisonings include:
- The neonicotinoid imidcloprid reducing honey bee survival by disrupting the immune and circulatory systems,
- Pyrethrins like fenpropathrin decreasing the homing abilities and overall survival of young bees in a dose-dependent manner,
- Tau-fluvalinate, the miticide used for Varroa control, accumulates in wax comb. At higher doses it contaminates larval food, and larva fed high doses of tau-fluvalinate have a reduced lifespan,
- Inert ingredients such as adjuvants, surfactants, and carrier solvents are added to improve the chemical properties of a pesticide. They causer various sublethal-turned-lethal effects on bees, depending on the product used.
Honey bees are exposed to these pesticides through direct contact with treated plants, ingestion of contaminated nectar and pollen, or contact through pesticide drift. The presence of these chemicals in increases the risk of acute poisoning, leading to the mass deaths observed at hive entrances.
Why is the Proboscis Extended in Pesticide-Killed Bees?
The proboscis extension observed in pesticide-killed bees may be a sign of neurological impairment, or the bees’ attempt to cool down as their normal thermoregulation processes are disrupted, causing overheating (reviewed in Belzunces et al., 2012).
Pesticides like neonicotinoids and organophosphates target the nervous system of bees, impairing their ability to navigate. Disoriented bees may be able to find their way to the general hive area, but be unable to enter (deterioration of the homing ability), whether by flying or crawling back inside. The symptoms often rapidly progress and prove fatal, leading bees to die at the entrance or nearby.
What Diseases are Found in Dead Honey Bee?
All this talk of honey bee diseases, infections and colony collapse, yet none address the question of “What diseases are found on a honey bee corpse?” For larvae, there are plenty:
- Sacbrood diseases,
- American & European foulbrood disease,
- Chalkbrood disease, and many more.
Failure to remove brood in the early stages stage will lead to the replication and emergence of the pathogen, which can infect other larvae. If uncontrolled, the entire colony can weaken and collapse from the disease burden.
Parasitiod Fly, Apocephalus borealis is an Emerging Pest of Honey Bee Colonies
What about adult bees? We know that Nosema infects adults, and that these infected adults can pass the disease onto their nestmates via oral trophallaxis or faecal contact. In North America, the native parasitic Phorid fly, Apocephalus borealis has been shown to infect and kill A. mellifera.(Core et al., 2012). The adult fly lays eggs inside the honey bee abdomen, where the larvae grows within. The host dies, and the larvae emerges to feed until it grows to adulthood, restarting the cycle.
The authors mention that the North American honey bees do not possess the mechanisms to deal with A. borealis. If undertaker bees did not remove this infested individual, then the hive would be overwhelmed by the feeding larvae.
Serratia marcescens is a Bacterial Pathogen
Serratia marcescens is an opportunistic bacterial pathogen that is found in low numbers within the honey bee gut community . In times of severe Varroa mite infections or gut microflora imbalance, it can overwhelm the normal microbial populations and become infectious (Raymann et al., 2018). When bees were infected by S. marcescens, they did not show increased immune activity, rather there was a decrease in two antimicrobial peptide genes, suggesting that the pathogen evades the immune system.
Death occurs via septicemia, as the bacteria is found in high numbers inside dead bees, spreading to other bees if the corpse is unmanaged. Consider that bees constantly groom themselves and others. In a weakened colony, an infected groomer will spread S. marcescens into the adult bee gut, where it can infect and kill other bees (Raymann et al., 2018).
What are the Disadvantages of Hygienic Behaviour?
I conclude by sharing some downsides of hygienic behaviour. The enlightened reader may think “if bees spend so much time checking for diseases, then this will affect other areas, like honey production”. You might be right, as I do not know of any literature comparing the honey yields between hygienic and non-hygienic strains – research is done in the context of disease management.
Common wisdom states that hygienic bees will remove both dead and healthy brood, impairing the colony’s worker populations. Bigio & colleagues found that in a freeze-killed brood assay, both hygienic and non-hygienic bee strains and found that even in hygienic populations, the bee-haviour is rare (Bigio et al., 2014). Their results showed no correlation between the removal of the dead brood and untreated, healthy brood brood at four stages (eggs, small and large uncapped larvae, capped cells).
However, just because a colony is hygienic in laboratory settings (freeze-killed brood assays), it does not mean the same in real-world conditions. Gerdts & colleagues showed that bees with hygienic traits in freeze-killed brood tests did not show the same behaviour under infection by chalkbrood (Gerdts et al., 2018).
So a major disadvantage is that beekeepers would be sold ‘hygienic’ bees, thinking that all their disease management issues are solved, only to have them succumb to a different infection. Being hygienic is not an all-in-one remedy, different diseases have different markers and producing an omnipotent bee strain is not quite possible… yet.
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Footnotes
- “Necrophoresis” is the term used to describe the removal of dead individuals away from the colony (Wilson et al., 1958). In bees, this takes the form of hygienic or undertaking behaviour. ↩︎
- Under normal conditions, β-ocimene is released when larvae beg for food, prompting nurse bees to feed them. The queen also produces β-ocimene, which contributes to the suppression of the worker bees’ ovaries and egg laying behaviour (Gilley et al., 2006). ↩︎