Who’s the daddy?

I’ve recently discussed the importance and influence of polyandry for honey bee colonies. Briefly, polyandry – the mating of the queen with multiple (~12-18) drones – is critical for colony fitness e.g. ability to resist disease, forage efficiently or overwinter successfully.

Hyperpolyandry, for example resulting from instrumental insemination of the queen with sperm from 30+ drones, further increases colony fitness and disease resistance.

How do you measure polyandry?

Essentially, you genetically analyse the worker bees in the colony to determine the range of patrilines present. Patrilines are genetically distinct offspring fathered by different drones. Essentially they are subfamilies within the colony.

With a finite number of patrilines – which there must be, because the queen does not mate with an infinite number of drones – there will be a point at which the more workers you screen the fewer new patrilines will be detected.

Search and ye shall find – detecting rare patrilines

The more you screen, the more you are likely to have detected all the patrilines present.

However, the queen uses sperm randomly when fertilising worker eggs. This compounds the difficulty in determining the full range of different patrilines present in a population. In particular, it makes detecting very rare patrilines difficult.

For example, if 20% of workers belong to one patriline you don’t need to sample many bees to detect it. In contrast, if another patriline is represented by 0.0001% of randomly selected workers you would probably have to screen thousands to be sure of detecting it.

Consequently, rare patrilines in the honey bee worker population are very difficult to detect. Inevitably this means that the number of drones the queen mates (~12-18) with is probably an underestimate of the actual number 1.

Half-sisters and super-sisters

Worker bees are often described as ‘half sisters’ to each other. They share the same mother (the queen), but different fathers.

Actually, as you should now realise, that’s an oversimplification because – with only ~12-18 different fathers contributing to the genetics of the colony – some workers are going to be more related to each other because they share the same father and mother.

Half-sisters share the same mother but have different fathers and share about 25% of their genes.

Super-sisters share the same mother and father and so share about 75% of their genes (25% from the queen and 50% from the drone).

Super-sisters are more likely to help each other in the colony 2.

Emergency queens and nepotism

What’s the most important decision a colony makes?

If the queen is killed (or removed) the workers rear new queens under the so-called ’emergency response’. They feed selected young larvae copious amounts of Royal Jelly to rear a replacement queen.

Arguably, the most important decision the workers make is the selection of the day-old larvae to rear as new queens.

If they get it wrong the colony is doomed. If they get it right the colony will flourish 3.

But as described above, workers are more or less related to each other genetically.

To ensure the continued propagation of at least some of their genes it might be expected that the nurse bees making this selection 4 would choose larvae more closely related to themselves.

Do worker bees exhibit nepotism when rearing emergency queens?

If workers were nepotistic you’d expect the most common patrilines in the nurse/worker bee population would also predominate in the queens reared.

However, for at least 20 years evidence has been accumulating that indicates bees are not nepotistic. On the contrary, emergency queens appear to be reared from some of the rare patrilines in the colony.

A recent paper from James Withrow and David Tarpy has provided some of the best evidence for the existence of these so-called royal patrilines in honey bee colonies 5.

Royal patrilines

Evidence for these goes back to at least 1997 6, with about half a dozen publications in the intervening period. Essentially all used broadly the same approach; they genetically screened worker bees and the emergency queens they reared to determine which patrilines were present in the two groups. 

With certain caveats (size of study, number of microsatellites screened, colony numbers etc.) all concluded that colonies rear emergency queens from some of the rarest patrilines in the colony.

The recent study by Withrow and Tarpy is well explained and probably the most comprehensive, so I’ll use that to flesh out the details.

Experimental details

Six double-brood colonies were each split into a three separate colonies; a queenright single-brood colony and two five-frame nucs. The latter contained eggs and young larvae and so reared emergency queens.

Seven days later the developing emergency queens were all harvested for future analysis. One or two frames from the nucs were then exchanged with frames containing eggs and day-old larvae from the matched queenright colony.

The nucs then started rearing new queens … again.

And again … and again.

This process was repeated until the nucs failed.

In total over 500 queens were reared (to 7 days old) from these six original colonies. These queens were analysed genetically by microsatellite analysis, as were over 500 workers from colonies.

Within the 6 experimental colonies the authors identified a total of 327 patrilines (or subfamilies as Withrow and Tarpy describe them), ranging from 34-77 per colony. 108 patrilines (4-40 per colony) were exclusively detected in worker bees and 130 patrilines (5-55/colony) were exclusively detected in queens.

Cryptic “royal” subfamilies

Over 40% of queens raised per colony were produced from the patrilines exclusively detected in the queen population.

Subfamily distribution per colony.

As shown in the figure above, many queens (black bars) were reared from subfamilies (patrilines) not represented in the worker bee population (grey bars, sorted left to right by abundance).

Since there were different numbers of patrilines per colony (34-77), the bias towards the rarer patrilines is more apparent if you instead split them into tertiles (thirds) based upon worker abundance.

Are the queens predominantly reared from the most common tertile, the intermediate tertile or the rarest tertile?

Frequency distribution of subfamilies.

It’s very clear from this graph that workers select queens from the rarest patrilines within the colony.

It is therefore very clear that worker bees do not exhibit nepotism when choosing which larvae to rear emergency queen from.

Implications for our understanding of honey bee reproduction

Two points are immediately apparent:

  • there is a cryptic population of queen-biased patrilines that have largely been overlooked in genetic studies of honey bee polyandry
  • honey bee queens mate with more drones than conventional studies of worker bee patrilines indicate

Colony 5 had at least 77 distinct subfamilies (there might have been more detected had they screened more than the 94 workers and 135 queens from this colony). By extrapolation it is possible to determine that the effective queen mating frequency (me; the number of drones the queen had mated with) was ~32 if all the samples (worker and queen) were taken into account. If only the worker or queen samples were used for this calculation the effective queen mating frequency would be ~12 or ~65 respectively.

The average effective queen mating frequency over the six colonies was ~33 (total), significantly higher than the oft-quoted (including at the top of this page) me of ~12-18.

So perhaps honey bees really are hyperpolyandrous … or even extremely hyperpolyandrous as the authors suggest.

It’s worth noting in passing that routine mating frequencies over 30 are almost never quoted for honey bees 7, but that the ‘normal’ me ~12-18 is rather low when compared with other species within the genus Apis. The giant honey bee, Apis dorsata, exhibits mating frequencies of greater than 60.

Who’s the daddy?

So, when it comes to emergency queens , although we might not know precisely who the daddy is, we can be pretty certain the particular patriline selected by the workers is most likely to be one of the rare ones in the colony.

Mechanistically, what accounts for this?

Are these larvae selected solely because they are rare?

That seems unlikely, not least because it would require some sort of surveying or screening by nurse bees. Not impossible perhaps, though I’m not sure how this would be achieved.

Perhaps it is not even worker selection?

An alternative way to view it is larval competition. A better competing larvae would be fed Royal Jelly and would be much more likely to pass on her genes to the next generation.

We don’t know the answers to these questions … yet.

Or whether they’re the wrong questions entirely.

Swarming and supercedure

The colony rears a new queen under three conditions; enforced queenlessness (as described above) which induces emergency queen rearing, prior to swarming and during supersedure.

These are fundamentally different processes in terms of the larvae used for queen rearing.

During swarming and supersedure 8 the queen lays the egg in a ‘play cup’ which is subsequently engineered into a queen cell in which the new queen develops.

Play cups

However, it is known that the patrilines of queens reared during the swarming response are similar to those of workers in the same colony 9, implying that there is no overt selection by the workers (or the parental queen).

Queen rearing

Does this insight into how bees rear new queens have any implications for how beekeepers rear new queens?

There are about as many queen rearing methods as there are adult workers in a double-brood colony in late June. Many  exploit the emergency queen rearing response by a colony rendered temporarily or permanently queenless.

Beekeepers often comment on the differential ‘take’ of grafted larvae presented to queenless cell raising colonies.

Sometimes you get very good acceptance of the grafted larvae, other times less so.

Of course, we only show the ones that worked well!

3 day old QCs ...

3 day old QCs …

Differential ‘take’ is often put down to the state of the cell raising colony or the nectar flow (or the cackhandedness of the grafter, or the phase of the moon, or about 100 other things).

I have never heard of beekeepers comparing the ‘take’ of larvae originating from the cell raising colony with those from another colony. The latter are always going to be ‘rare’ if you consider the patrilines present in the cell raising colony. However, grafts taken from the same colony as used for cell raising 10 are likely to reflect the predominant patrilines.

Are these accepted less well by the nurse bees?

I suspect not … but it is testable should anyone want to try.

My expectation would be that the presentation of larvae in a vertically oriented cell bar frame would likely override any genetic selectivity by the colony. They’re desperate to raise a new queen and – thank goodness – here’s a few that might do.

Alternatively, differential acceptance is more likely to reflect use of larvae of an unsuitable age, or that have been damaged during grafting.

As I listen to the wind howling outside it seems like a very long time until I can test any of these ideas … 🙁


Colophon

Ray Winstone (as Carlin) 1979

Who’s the daddy? is British slang for who, or what, is the best. It originated in a line by Ray Winstone’s character Carlin from the 1979 film Scum. This was not a romantic comedy and I’m certainly not recommending viewing it. Nevertheless, the phrase became widely used over the subsequent couple of decades and seemed appropriate here because the colony is dependent on selecting high-quality larvae for colony survival.

Footnotes

  1. Though the difficulty in detecting underrepresented patrilines is at least partially taken into account in the way the mating number is determined.
  2. I’ll return to this sometime in the future as it’s otherwise going to drag us kicking and screaming into a discussion of kin selection and altruism.
  3. No pressure then!
  4. This is a presumption on my part that it is the nurse bees which decide the larvae to feed … there are other ways in which it could be determined.
  5. Withrow JM, Tarpy DR (2018) Cryptic “royal” subfamilies in honey bee (Apis mellifera) colonies. PLoS ONE 13: e0199124. https://doi.org/10.1371/journal.pone.0199124.
  6. Tilley CA, Oldroyd BP. (1997) Unequal subfamily proportions among honey bee queen and worker brood. Animal Behaviour 54:1483–90.
  7. As discussed recently.
  8. At least probably during supersedure … I’m not sure it’s definitely known that the workers do not have a role in selection here. Perhaps they move selected eggs to the preformed play cup?
  9. Lattorff HMG, Moritz RFA. (2016) Context dependent bias in honeybee queen selection: Swarm versus emergency queens. Behav Ecol Sociobiol. 70:1411–7.
  10. Essentially recapitulating the Withrow and Tarpy paper described above.

26 thoughts on “Who’s the daddy?

  1. Christopher Harris

    It would make sense for workers to promote the most distant relative (i.e. the cell with the least common patriline) as the probability is that this one is least likely to be genetically close to the lost queen.

    Reply
    1. David Post author

      Hello Christopher

      I’m not sure this makes sense to me. If the colony is strong and successful and the queen – for whatever reason – is lost, why gamble on something distantly related? Perhaps I need to do some more reading?

      Cheers
      David

      Reply
  2. Dave Stokes

    A thought provoking article, as always. When I first started bee keeping, a queen mated several times, the number slowly crept up to 20 and then started reducing slightly in recent years. If I read this correctly, you are exploding the number up to 50 which begs the question of how she has time for such an extravagant sex life. I would make the perceived number of 20 colonies in a confined area needed to avoid inbreeding and gives me much more confidence that Andrew Abrahams in his venture on Colonsay is able to maintain an adequate long term biodiversity with 50 colonies.

    Reply
    1. David Post author

      Hi Dave

      I’m just the messenger … hyperpolyandry in honey bees (evidenced by genetic analysis of emergency queens) is pretty well established. 50+ probably isn’t routinely out of the question. Of course, there my be other explanations – for example, two queens in the hive – but the authors of the paper went out of their way to exclude all the obvious ones.

      Mating itself doesn’t take long and the queen may go on two or more mating flights. She clearly finds the time for this critically important event in the future success of the colony.

      I’m pretty sure that Andrew has looked at the diversity of his colonies on Colonsay. They were originally sourced from a number of different locations if I remember correctly. There are people much more expert on this than me in the Scottish Native Honey Bee Society.

      Cheers
      David

      Reply
  3. Jim

    Another great post as usual. How do you feel about the most recent BBKA News printing a letter of anti-5G nonsense? While you’re doing such sterling work, giving sensible advice, based on actual evidence and promoting scientifically literate thinking and yet the same publication allows Joe-conspiracy to spout off such unintelligible ramblings, without even a hint of a rebuttal.

    Reply
    1. David Post author

      Hi Jim

      Although I write the BBKA Q&A page(s) I don’t actually get to see the Newsletter as I’m not a member (Scotland has a separate insurance scheme). If it was a letter they’d argue that beekeeping is a broad church and they need to represent the breadth of views etc etc. There’s less excuse if it was an article.

      I’d encourage you to write a rebuttal. All of the beekeeping publications I read (or have read; BBKA Newsletter, SBA’s The Scottish Beekeeper or Bee Improvement from BIBBA … and others) have contained some arrant nonsense at times. Phenomenology dressed up as science. Uncontrolled and biased subjective views etc. They should be challenged.

      Beekeeping is confusing enough for beginners without worrying them unnecessarily.
      Cheers
      David

      Reply
  4. Jan Uitdewilligen

    Super-sisters share the same mother and father and so share about 75% of their genes (50% from the queen and 100% from the drone)

    Sagili, R.R., Metz, B.N., Lucas, H.M. et al. Honey bees consider larval nutritional status rather than genetic relatedness when selecting larvae for emergency queen rearing. Sci Rep 8, 7679 (2018). https://doi.org/10.1038/s41598-018-25976-7

    Answers your last question?

    Thank you for writing your blog and this post, it triggers me to dive deeper into the current subject and beekeeping in general.

    Reply
    1. David Post author

      Thanks Jan

      Yes, that’s probably part of the story and I was going to cover it in a future post. However, doesn’t it simply raise the question … Why are genetically distantly related larvae better fed?

      This again brings us back to nepotism and kin selection theory. I’d better go and do my homework 😉

      Cheers
      David

      Reply
  5. Jackie Elliott

    It would be interesting to know through what mechanism they recognise their super-sisters. They do use antennation to interrogate in general so maybe taste? Just a guess. Thanks for another interesting Saturday morning read. 😊

    Reply
    1. David Post author

      Hi Jackie

      There’s a long and confusing body of literature on super-sisters, nepotism and kin selection. It’s an uncomfortable distance away from my own specialities (pretty hardcore molecular biology of RNA viruses) so I’m definitely no expert. I’ll probably cover some of it in future posts. For starters perhaps look at some of the work by Robin Moritz. Although there’s clear recognition of super-sisters there is a body of theoretical and practical evidence that suggests this breaks down with the level of genetic diversity seen in the hive (as opposed to in the laboratory). Inevitably recognition involves pheromones … doesn’t it always?

      Cheers
      David

      Reply
  6. Neil

    David,
    This reminded me of an article that I read somewhere (possibly BIBBA mag) that said natural selection favours the rarer sex alleles. It was concluded (if I remember correctly) that this was due to the subsequent performance of the daughter colonies due to having fewer diploid drones etc etc. No consideration was given to the “success” of these rarer alleles being due to worker selection.

    Reply
    1. David Post author

      Hello Neil

      Diploid drones are often taken as a sign of inbreeding – they originate from fertilized eggs that are homozygous (the same) for the sex locus allele. There are 18-20 variants of these and heterozygosity (mismatches) result in females, whereas homozygosity leads to the production of diploid drones. These don’t last long … under normal conditions the workers eat the young larvae. In an inbred colony there are a much smaller range of variants of the sex allele, so more chance of them matching in the diploid progeny.

      Avoiding inbreeding would certainly help the performance of the daughter colony, but it’s still not clear how any selection occurs.

      Cheers
      David

      Reply
  7. DaveM

    An extremely interesting and thoughtful blog, as always.
    On your note 8; I and a number of others will have observed late supersedure cells in the supers above the queen excluder. So this shows that workers do have a direct role in egg or larval selection during supersedure. The question is can they recognise the genetic family of the egg/ larvae?
    As you say pheromones always play a role.
    Or perhaps they are chosen on size, vitality, growth rate and the rest is just statistics.

    Reply
    1. David Post author

      Hello Dave

      The statement about workers not selecting which eggs to rear as supersedure Q’s was from the paper. I’m aware that there are sometimes eggs above the QE. I’ve often thought eggs above the QE were from rare laying workers and don’t remember any queen cells. Formally, to be sure that workers have had a role in their selection – assuming they were laid by the Q – you’d need evidence that they originated from below the QE. If they were, then genotyping them would indicate whether they were chosen at random, or actively selected. As with all these types of studies, the numbers of samples are important. This paper genotypes over 500 queens and is probably the most statistically convincing study of its type … we’d have to look through a lot of colonies to find 500 QC’s above the QE 😎

      Cheers
      David

      Reply
  8. Steve Donohoe

    HI David. Midblowing stuff, at least for a humble Odobenus 😀

    Given the massive clouds of drones at DCAs I don’t see any reason why a queen can’t be mated with 30+ drones on one flight. I suppose the first few dominate in terms of sperm volume that gets to the spermateca (?), but I don’t know.

    So much of what honey bees do seems to perpetuate/accentuate genetic diversity. Perhaps this special treatment for rare patrilines is part of that?

    Reply
    1. David Post author

      Hi Steve

      I think there’s some work on sperm competition in honey bees, and there’s certainly studies of sperm mixing. I’m pretty sure it’s not the case that the first drone sires the largest proportion of the workers. The ‘order’ of fertilised egg production is also known and is mixed if I remember correctly.

      The selection of rare patrilines might well be to further reinforce diversity. Since there’s recognition of worker patrilines – and some suggestion of cooperative activity between them – this suggests that the selective pressure might come from the rare larvae being more ‘attractive’, rather than the workers actively seeking out the rarities (which would ‘contradict’ their altruism).

      More work is needed 😉
      Cheers
      David

      Reply
  9. TB

    Clearly the next step is to genotype the drones in a colony to see how closely drones match their sisters. I suspect they’ll find some oddities.

    Reply
    1. David Post author

      Drones are haploid and arise from unfertilised eggs … these will have 16 of the Q’s 32 chromosomes. Since drones have no father a significant proportion of the diversity present in the workers will be absent.

      Of course, if you genotype adult (rather than emerging) drones in a colony there will be significant genetic diversity as at least a third have drifted in from other colonies.

      Cheers
      David

      Reply
    1. David Post author

      Hello Greg

      Jan Uitdewilligen (above) picked out the same reference as likely being relevant … I don’t disagree, but note that it still leaves some questions. I’ll tackle these sometime in the future.

      Cheers
      David

      Reply
  10. Edward Lewis

    Hi David,
    A most intriguing post. There might be a necessity to this queen selection policy for honey bees. The resulting queen would be less closely related to the local population of drones she would be mating with a few short weeks later, if we assume a broad scale similarity of colonies in a particular area.
    If workers just selected queens at random, let alone choosing larvae closely related to themselves, it would be very difficult for new genetic material to enter an area and stay there if they were not activity selected for in queens. Any mechanism that tends to preclude genetic diversity could lead to a spiral of reducing diversity which would be bad for any population…
    As honey bee colonies are essentially single (admittedly super) organisms, which do not naturally have many colonies to the square mile, maintaining genetic diversity is a fairly fundamental problem.
    Keep up the excellent work.
    Ed

    Reply
    1. David Post author

      Hello Ed

      Whatever the mechanistic explanation it’s certain that evolution will have achieved a process that ‘works’. As you suggest, a process that reduced diversity in the colony would be selected against.

      Seeley has looked at patrilines in feral colonies in the Arnot Forest (discussed recently). His results suggest – at least from worker analysis – that the queen mating number is similar to that of managed colonies.

      Cheers
      David

      Reply
  11. Janet Wilson

    Another fascinating blog post! Thanks David. There is another point to consider in terms of patriline selection. In their excellent “Mating Biology of Honey Bees”, Koeniger and Koeniger discuss the fact that the semen the queen receives on her mating flights is not uniformly mixed in the spermatheca, and appears to be stored in somewhat discrete layers/bundles. This would mean the queen accesses stored sperm in such a way that all the workers laid this week may come from sperm in a different layer = different drones than the workers who will be laid next week. That might make the phenomenon of queen cells representing rare patrilines merely a perception, not a reality, ie. this week’s queen larvae are from a different layer of sperm than the workers being sampled at the same time. Depending on the age of the workers analyzed, they may be from sperm the queen was using a month ago…and from a very different subsection of the drones she mated with. Something to look into further!

    Reply
    1. David Post author

      Hello Janet

      Apologies for the delay in responding … there seem to be lots of non-beekeeping jobs at the moment.

      This is one of the many topics that; a) I’m not up-to-speed on, and b) probably needs more work.

      I agree that for true comparison they should have perhaps screened late-stage worker pupae of the same age as the emergency queen pupae.

      Early studies suggested ‘sperm clumping’ or successive use of sperm from one drone before another. However, more recently there are also publications that report that patriline composition is broadly equal (suggesting mixing) and that successively laid eggs are fertilized by different drones.

      There must be some mixing before the spermatheca are filled as the queen only stores about 5% of the sperm received on mating flights. It’s difficult to see how this could be achieved without some mixing.

      The more we know, the more we don’t!
      David

      Reply
      1. Janet Wilson

        Koeniger and Koeniger paraphrase a 1999 study by Franck and associates that showed mixing happens but not immediately. The sperm “donations” do stay somewhat discrete, even after moving to the spermatheca. Think layers of jello rather than a fluid solution. But mixing is complete after 3 months (ie by the end of the season). In spite of that, mixing was not entirely mixed even one year after mating. So that suggests the age of the queen may be something to consider: patrilines of worker bees may not match patrillines of larval bees when looking at the work of fresh queens.

        Reply
        1. David Post author

          I’ll look into this in a bit more detail …

          The year upon year patriline composition of the colony was a single queen study over four years by Robert Brodschneider and colleagues in 2012. The relevant quote from the abstract is: “No significant changes in patriline distribution occurred within each of two foraging seasons, with samples taken one and five months apart, respectively. Overall and pair-wise comparisons between the three analyzed years reached significant levels. Over the three-year period we found a trend for patrilines to become more equally represented with time.”

          In the introduction they also make the following statement about the Franck (1999) study “On a somewhat larger time scale, Franck et al. studied the first three months of offspring production by an artificially inseminated queen: samples were taken three times and only samples taken more than two months apart give significant differences in patriline representation.”

          I’ll do some reading, think up a snappy title (perhaps avoiding the word Jello 😉 ) and write something in the future when I understand it a little better.

          Cheers
          David

          Reply

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