Laying Workers, Worker Policing & Worker Relatedness

How Apis mellifera Maintains Genetic Harmony in the Hive

Workers laying eggs could either be due to queen failure, random genetic mutations or a recent swarm. The foremost sign of a laying worker is the presence of multiple scattered eggs per cell, as their abdomens are too short to reach the bottom. If these eggs develop, they will emerge into male drones, as workers lay unfertilized eggs. Under normal circumstances, other worker bees will remove these non-queen eggs (hence “worker policing”) to maintain consistent colony genetics (all births performed by the queen), and thus go unnoticed by the beekeeper.

To manage a laying worker hive, consider a newspaper combine or a “shakeout” followed by requeening. Requeening right away is likely to lead to queen death, as the colony will continue treating introduced queens as intruders right until the colony collapses. This article describes solutions to laying worker hives and the genetic basis of worker policing, followed by descriptions on the genetic basis of worker policing, honey bee ovary development and anarchism in bees. It end with a brief comparison to Cape honey bees (Apis mellifera capensis), whose diploid worker-laid eggs avoid policing altogether.

Fixing Worker-Laying Hives

Fixing a hive with laying workers is more complicated than “requeening your hive“. As far as the colony is concerned, Her Majesty is still there, she’s just laying drone eggs. The colony will not have an issue with this until the last worker dies (which will never happen, since the colony collapses sooner than that). Any introduced queen will be rejected and killed.

Depending on the extent of worker-laid egg combs, it may be best to replace them with fresh frames. For the removed frames, freeze them for one or two days to kill the eggs, then return them to the hive for the workers to clean out. This approach is combined with one of these three options:

• Take the brood box a short distance (~50-100 meter), shake the bees off the frames, and return the box to the original location. Then, add a brood frame from another hive.
  • The foragers will find their way back to the hive, along with some of the nurse and middle-aged bees. The rest will drift to other colonies.
    • If the laying worker drifts into a queenright colony, the presence of queen and brood pheromones will suppress her ovaries and she will cease egg laying, resuming normal worker functions (Reviewed in Smith et al., 2013).
  • The colony will raise new a queen from the larvae in the added brood frame, who will mature and (hopefully) mate successfully, restoring order. Alternatively, you can introduce a mated queen.
  • There is probably not enough space for urban beekeepers to successfully do this method, sorry.
• Perform a newspaper combine. The queenless colony is going to be weaker, so combine it with a hive that already contains a laying queen. Take two pages of newspaper, poke some small holes (with a toothpick) into the papers to help bees chew through the material, and place the sheets atop the queened hive. Then, place the queenless hive box on top, and wait for around two weeks.
  • The pheromones from the queenright colony will spread throughout the queenless hive, and by the time the workers chew through, they would have adjusted to her scent and unite as one colony.
• A prolonged approach suggested by a member of my beekeeping club was to add a brood frame every few days for about a month. The logic here is that over time, the brood pheromones suppress the activation of additional worker ovaries while the old ones die out. After a month, requeen the hive with a caged queen
  • A very time-intensive and slow method, listed here for example’s sake. However, it has merit in maintaining the colony population until it is requeened.
    • Personally, I would destroy the colony and start over. I also don’t understand why the colony wouldn’t be able to raise a new queen from the supplied brood frames.

Identifying Worker-Laid Eggs

If you see clusters of eggs scattered around the cell, then you’ve got yourself a laying worker. This is the tell-tale sign; if you are uncertain, check the entire frame after a week or two – you’ll see capped drone brood. When workers lay unpoliced eggs, it is usually a sign of a “hopelessly” queenless colony, in that they lost the queen and have no brood available to rear a placement. That colony will keep producing drones until it collapses from a dwindling workforce, or you intervene.

Laying Worker or a Newly-Mated Queen?

Worker abdomens are shorter than queens and so cannot reach the bottom of the cell. As a result, worker-placed eggs tend to be off-centre as the glue to the cell wall, eventually falling to the bottom.

How do you tell if it’s a rogue worker, or a new queen learning how to lay eggs?¹ Look at the egg number and their positions- new queens lay less eggs per cell, and are relatively orderly; when multiple eggs are laid in a single comb, workers will place the excess eggs into an empty cell. After one or two weeks, these larvae will be capped into worker brood, and only single eggs will be observed in comb. By contrast, more drones and drone comb will be present in laying worker colonies, with more chaos in the cells.

Genetic Basis of Worker Policing

Non-technical readers may need to re-read this section

Workers are related to their own sons by 50%, but to sons of the queen (“brothers”) by 25% (math explained below). In evolutionary terms, the individual worker benefits more from selfish egg laying and having the colony raise her offspring. Despite this, Visscher notes that only 0.12%, or one in a thousand drones are born from worker-laid eggs (Visscher, 1989). One possible reason for the suppression of worker-laid eggs is that although a colony’s workers are more related to their own sons than to those of the queen, they are even less related to their half-sister’s drones (half-nephews?), at 12.5% – hang in there, math will come soon.

Since Her Majesty mates with multiple drones, the majority of nestmates in a colony are half sisters, rather than full sisters. This makes workers more related to queen-laid drones rather than worker-laid drones (25% vs 12.5%). From this point, Ratnieks defines worker policing as when workers kill other worker-laid eggs to maintain “reproductive harmony” in the colony (Ratnieks, 1988).

How do workers distinguish between worker- and queen-laid eggs? It is possible that queen eggs are marked with a queen-produced pheromone from her Dufour gland as it gets laid, and workers lack this scent? Or is it as Visscher & Dukas suggest, that workers recognise when the ovaries of other workers are activated, and are hence more aggressive towards them (Visscher & Dukas, 1995)?

Relatedness Within the Colony

Worker bees are born from a fertilized egg, meaning that the worker has a mother, and a father, each contributing 50% of their genes to their daughter worker. As such, each daughter is 50% related to the queen, on account of receiving 50% of their genes from her. Drone bees are born from unfertilized eggs, from their mother queen, meaning that they only contain genetics from the mother, making them 25% related to other workers (their “sisters”)².

Remember that queens mate with multiple drones. As a result, when comparing two workers, they are more likely to be half sisters, as their eggs were fertilized with sperm from different fathers. Workers are more related to their full sisters (other workers from the same father) than to their half-sisters (workers from different fathers).

The Math Thickens

As queens mate with multiple drones, each worker bee’s father will be different despite having the same mother queen. When two worker bees have the same mother and the same father, they are considered full (or in social bees, “super”) sisters. Each worker receives half of her genes from her mother and half from her father. The queen passes on a random half of her genes to each egg she lays. Since the drones are haploid (providing only the set of genes they received from their mother queen), the sperm from that drone is genetically identical across all its sperm cells. Therefore, for two sisters:

• From the mother (queen), there is a 50% chance of sharing the same allele (“gene variant”) for any gene because they receive a randomly selected half of her genes.
• From the father (drone), they always receive the same alleles because all his sperm are genetically identical.
• This adds up to an average of 0.5×0.5=0.25 (25% from the mother’s side) + 0.5 (50% from the father’s side since the sperm are identical) = 75% relatedness.

Half-sisters share the same mother but have different fathers (since the queen mates with multiple drones). They will still share about 50% of their genes through their mother, as mentioned above. However, because they have different fathers, it is only the mother’s genes that are shared, and the father’s genes are excluded from the calculation. Thus, the relatedness among half-sisters is 0.5×0.5=0.25; 25% from the mother’s side only.

What if that half sister was to lay eggs that hatch into a drone? Since the egg is unfertilized, it will emerge as 50% of the worker bee, so the relatedness between a worker and her half-sister’s son is 0.25 * 0.5 = 12.5%. There you have it, workers police the eggs of one another since those drones are less genetically related than those laid by their mother queen.

Pheromone Composition of Laying Workers

Worker ovaries are suppressed by brood and queen mandibular pheromones. Should the queen pheromones disappear, then physical changes emerge in the potential laying worker, leading to the emergence of “false” or “pseudo” queen. These workers have queen-like characteristics, do not work, and attract a retinue as a normal queen would… oh, to be royalty (Crewe et al., 1980). Note that in an Asian honey bee colony, A. cerana workers have higher rates of ovariole activation than A. mellifera (Oldroyd et al., 2001).

In general terms, the two main queen and worker mandibular gland secretions are 9-ODA & 10-HDA, respectively (Plettner et al., 1996). Measuring the ratios between the two chemicals can determine the presence and activity of the queen: the more 9-ODA circulating in a colony, the less likely it is for workers to activate their ovaries. However, in laying worker colonies, Tan and colleagues found higher levels of 9-ODA, suggesting that these workers developed queen-like pheromones to suppress other workers from laying eggs; “false queens” (Tan et al., 2012). They suggest that the higher ratios in A. cerana compared to A. mellifera, resulted from competition with other workers for reproductive dominance in queenless hives.

Laying Workers are Physiologically Similar to Nurse Bees

Nakaoka and colleagues analysed the hypopharyngeal glands of laying workers in queenless colonies and found their composition to be similar to nurse bees in queenright colonies: High vitellogenin levels and well-developed hypopharyngeal glands (Nakaoka et al., 2012). It is possible that vitellogenin elevation is a result of being the egg yolk precursor protein, and is also used by the hypopharyngeal gland to produce royal jelly.

Further, vitellogenin suppresses juvenile hormone levels, which controls the workers’ division of labour (Sullivan et al., 2000). Newly-emerged workers have low levels of this hormone, increasing as they age. Foraging bees having the highest concentration of juvenile hormone.

Ovarian Development in Honey Bees

The adult queen has large ovaries, consisting of 120–200 units termed “ovarioles”, whereas workers have small ovaries each made up of 2–12 ovarioles (reviewed in Hartfelder et al., 2017). The sperm storage vessel, the spermatheca, is fully developed in queens, and nonfunctional in workers. During the larval phase, the two ovaries are indistinct until the fifth instar prior to cell capping.

In this phase, programmed cell death occurs, and is largely inhibited by presence juvenile hormone (Hartfelder, & Steinbruck, 1997). The concentrations of juvenile hormone are ten times higher in queens than workers, leading to large sparing of her ovaries, amongst other tissue. Hormone levels drop to baseline in both castes after cell capping, and rises again in queens during the prepupal phase. The resulting structural differences generated are part of differences in ovary function, resulting in sterile workers and fertile queens.

Anarchistic Bees Lay Eggs in Queenright Colonies

In some cases, genetic mutations can cause workers to begin laying eggs in queenright colonies. Oldroyd and colleagues found these workers to develop and behave as other workers, with the added task of laying eggs (Oldroyd et al., 1999). They were not larger and did not produce queen-like pheromones as in the false queens, nor were they attacked by other workers for having their ovarioles activated, nor did their eggs get policed.

Barron and colleagues observe that these bees undertake normal worker tasks like foraging, brood rearing and queen care, all while laying eggs that have reduced policing (reviewed in Barron et al., 2001). The authors suggest a desensitisation to brood and queen pheromones, and that these laying workers produce queen egg-marking pheromone mimics, reducing policing activity. All in all, anarchist bees likely impede colony activity – whether by dedicating resources (energy) to egg laying, or taking filling up brood comb with drone rather than worker eggs.

Short-lived Anarchy Post-Swarming?

Woyciechowski & Kuszewska found that after swarming, there is a period of time when the colony members are unrelated as the departed queen’s eggs and larvae remain (Woyciechowski & Kuszewska, 2012). This older generation is only half as related to their new sister queen’s offspring (12.5% rather than the usual 25% for half sisters). The mother queen had produced their sisters and brothers, whereas this new sister queen produces nieces and nephews.

The authors found that worker larvae reared in the absence of their mother had more ovarioles, smaller hypopharyngeal glands and larger mandibular glands, giving them a more queen-like physique. These “rebels” laid unfertilized eggs even in the presence of a queen, and despite the presence of sister brood (from the departed queen). They did not tend to brood or conduct general maintenance. Fortunately, these workers only live for a sort time, so their disruption is minimal. After two months, the new queen continues laying as normal.

Are Worker-Laid Eggs Weaker?

Considering how few worker-laid eggs reach maturity due to policing, it is hard to determine if worker eggs are weaker. Should the eggs hatch, they will emerge into drones whose one job is to mate with the queen, not clean or forage or defend. According to Martin & colleagues, the survivability of worker-laid drones in queenless colonies ic consistent across subspecies, with no occurence of “nepotistic cannibalism” by other workers (Martin et al., 2004).

Is it farfetched to assume that if a worker-laid drone mated with a queen, the sperm from that drone will give rise to a new generation of laying workers? I do not know, and think that there are more complex factors at play than sperm (pheromones, policing, etc…)

So perhaps worker eggs are not weaker, but their presence indicates a weak hive, one whose queen is failing or is absent. In this case, the extent and presence of worker eggs could mean that the worker population is too low to conduct policing activity, and it could collapse if not properly requeened. Exceptions exist, like the anarchistic bees described above, or the parasitic Cape honey bees, described below.

Parasitic Worker Egg Laying in Cape Honey Bees

We conclude by describing how A.m. capensis workers lay eggs that avoid policing. A genetic mutation in the “thelytoky locus” (gene) caused A.m. capensis workers to lay eggs in hives of other subspecies, namely A.m. scutellata (Aumer et al., 2019). As these eggs are subjected to thelytokous parthenogenesis (“female birth”), they develop into female clones of the Cape worker, and these parasites are reared into adulthood by the host hive.

Brood cells containing diploid Cape bee eggs are ignored by the host colony and no worker policing occurs (Martin et al., 2002). Martin and colleagues, found that when A.m. capensis workers laid eggs in A.m. scutellata colonies, the eggs were allowed to hatch and develop into workers. In contrast, when an A.m. scutellata worker laid eggs in her colony, they were policed and removed within 24 hours. The authors suggests that Cape workers could produce a chemical mimic to queen-laid eggs, hence sparing their own eggs from policing.

Thus begins the host colony’s death spiral: the Cape adults become laying workers, resulting in a non-productive colony. The A.m. capensis pseudo-queens do not forage or clean, unlike the anarchistic workers above. Eventually, the number of Cape clones exceed the colony’s sustainability threshold, and the lack of “working” workers cause the colony to dwindle and die out.

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

1. A colony takes around 30 days to go from queenless to visible worker brood. 16 days for the queen to emerge from her cell, another 7 days for her to sexually mature, and then complete her mating flight. Larvae should be present 4 days after the mating flight, assuming she is successful. ↩︎
2. Why are drones not 100% related to the queen, or 50% related to their worker sisters? The former would mean the drone is an exact clone of the queen, i.e. another queen, which is not the case. The latter would be true if drones were diploid and had the same father as the worker – 50% of genes from her mother, 50% of the genes from the father. ↩︎