Bigger queens, better queens - part 2

I think I'm more excited by the potential created by the identification of the maternal effect in honey bees than anything beekeeping-related in the last decade.

Perhaps I need to get out more?

I first mentioned the maternal effect in a post titled 'Bigger queens, better queens - part 1'. I'm not going to rehash the science here. You should go back and read that post for the gory details scientific evidence. And - while you're at it - don't forget the prequel, which explains why bigger queens are better queens.

More research is needed, but the key observation is that queens lay bigger eggs in queen cups than they do in worker cells. These develop into bigger queens. Furthermore, these bigger queens 'perform' better in terms of brood production, and the trait appears to be inherited.

Bigger than what?

Bigger than queens reared from day-old grafted worker larvae.

So what?

Beginners and the grafting-averse

In a nutshell this means that beginners or beekeepers who cannot graft (lack of confidence, poor eyesight, unsteady hands etc.) should still be able to rear perfectly good queens.

Of course, they can already.

Splits, using swarm cells, or simply adding a frame of eggs/larvae to a terminally queenless colony will all yield queen cells.

But, if you want several, or you want them in an 'easy to handle' format (e.g. no attached comb), or you want a little more control over the entire process (e.g. of timing or number), then grafting larvae into plastic cell cups is the way to go.

Except it involves grafting 😞.

I've lost count of the number of beekeepers I've talked to who have tried grafting, achieved only limited success, and then given up in favour of 'easier' approaches.

Two comments on grafting before I move on ... firstly, even those who now use it regularly were less successful when they started. Unsurprisingly, practice makes perfect ... or at least improves things to the point where it can be routinely depended upon.

This was my experience, and my first grafts of the season are usually less successful than subsequent ones.

Secondly, grafting is so widely used, I'd be surprised if less than 99% of commercially reared queens were produced using the method.

If you want 200, or 2,000, queens, grafting is the way to go.

But what if you want just a few?

Assuming good eyesight - or have help achieving it - and steady hands, grafting is easy. Or, to qualify that, it's easier than some other aspects of queen rearing, like preparing and managing mini-nucs for mating.

But, if you attend a queen rearing course you will almost certainly be encouraged to graft day-old larvae and use mini-nucs for queen mating.

There are easier ways to get queens mated and - with the recent identification of the maternal effect - excellent justification to not graft day-old larvae.

And, there's the added bonus of potentially creating bigger and better queens.

So, why the enthusiasm?

If beginners or the grafting-averse (for whatever reason) do not need to graft to produce small numbers of queen cells, then it may increase the proportion of beekeepers who actively rear queens.

'Actively', to distinguish the process from passive methods in which you rely upon the colony to produce queen cells when (and if) they want e.g. during splits, or when swarming.

I'd be surprised if more than 10-15% of beekeepers actively rear queens each season. That's one reason such huge numbers of queens imported to the UK every year.

Whilst I'm broadly against imports, I think the greater significance is that queen rearing improves both the understanding and enjoyment of beekeeping (at least, it does mine). I've previously argued that cheap, readily available, imported queens reduces the quality of our beekeeping ... why learn how to do something if you can order online and fix it for £25?

If you rear your own queens (successfully) you need to learn how to prepare the colony, judge when it is ready, understand the timing of all the events, and manage the queen mating process.

All of these things will improve your beekeeping, and pay handsome dividends for other aspects of our fascinating pastime.

And, all that becomes more achievable if there's no need to graft day-old larvae with your shaky hands, dodgy eyesight or lack of confidence.

So, let's revisit the demonstration of the maternal effect, specifically looking at the methods used.

Then, let's consider how beekeepers (rather than scientists) can recapitulate those methods.

And, excitingly, I'll discuss some practical studies conducted by two readers of The Apiarist.

Artificial queen cups

A queen cup is an enlarged, rather rounded, cell, usually facing wholly or partly downwards. In contrast, worker cells are angled slightly upwards, hexagonal, very regular in appearance and size.

Although it is known that workers will - or can - move eggs to queen cups, it is also known that the queen does lay eggs in them, and that as these eggs hatch, the cell is elongated into a sculpted queen cell, the larvae are nurtured by the colony and eventually emerge as new queens.

In the original paper on the maternal effect (Wei et al., 2019) the authors used an artificial queen- or worker-cell system and referenced an ABJ article from 2013 (Pan et al., 2013). This consisted of a sheet of plastic foundation, embossed with a standard comb pattern and containing a regular pattern of holes through which 'pegs' fit from the rear to hold a cell. My reading of the ABJ article suggests that the plastic sheet is coated with wax and drawn out as comb before the queen is introduced.

However, not so fast ... the photo in the Wei et al., (2019) paper does not look much like that in the ABJ article 🙁.

In that 2019 paper, they simply show a picture of large and small artificial cells, and a Pan-like peg system for subsequently holding them vertically on a cell bar frame.

In the Wei et al., paper the queen was caged on queen- or worker-cell comb for 6 hours, during which time she laid eggs. The queen was then removed.

Vague or missing details

Although not explicitly stated, I presume that the foundation was presented vertically in the hive with the caged queen. That being the case, it means that the queen will lay eggs in a horizontally-orientated queen-cup {{1}}.

That is important.

It was these eggs that were shown to be different in size when subsequently analysed ... and that produced bigger queens.

Better queens.

It's not clear what happened between 6 hours and hatching. Where were the eggs maintained? Were they transferred to the cell raising colony immediately, or after a delay?

All the paper explains is:

Eggs sampled from queen cells and worker cells were transplanted into standard plastic queen cells. Queen cell bars were placed into a strong queenless hive with 8 frames for queen rearing.

I'm pretty certain that the first sentence does not mean the eggs/larvae were directly handled. Instead, the peg system I mentioned earlier was used to transfer the cells containing eggs (or hatched larvae?) onto the cell bar frame.

Details we do have

Although there is a little vagueness about the timing of events, what is clearly stated is the size of the worker- or queen-cups used in the study.

Worker cells were 4.9 mm in diameter {{2}} .

Queen cups were 9.7 mm in diameter.

This prompted several people to submit comments on using Nicot cups - though generally without stating how. I didn't post these comments at the time (I will now ... ) as I didn't want to get into a discussion about cell size and what works, or does not work {{3}}.

And that's because I'd already measured the plastic cell cups that are routinely used when grafting larvae.

We have the technology

It is the internal diameter of the cell that is probably important.

Probably as we don't know how the queen measures it, assuming she does measure it {{4}}.

It's known that a queen can measure drone cells (where she lays unfertilised eggs) using her forelegs. I expect that queens identify queen cups in the same way ... but that's what's known in the trade as an 'informed guess'.

Or, more accurately, 'guess'.

I have regularly used JzBz and Nicot plastic cups for queen rearing, so dug them out of the shed. I also went out and spent a ridiculous proportion of my honey profits on a Jenter kit, a German variant that appears to be less popular {{5}} than the Nicot system.

These three types of cell cups all differ slightly in diameter and depth. For comparison, I measured the internal diameter of the rim (some are flush-sided, others curved) using a pair of digital calipers that may or may not be accurate {{6}}.

Because I was a pedantic scientist before I became a pedantic writer, I measured a few of each cell cup type and calculated the average. Who knows what the quality control is on this sort of equipment?

JzBz cups are 8.1 mm in diameter

Nicot are 8.5 mm

Jenter are 9.3 mm

Therefore, of the three, the Jenter cups are the closest to those used by Wei et al., to demonstrate the maternal effect.

Does this matter?

Who knows?

Cups or systems?

JzBz cups are 'standalone'. You graft larvae into them, present them vertically in a cell raiser and voilà, queen cells.

No cup holders, no hair-roller cages, nothing else. Simples.

In contrast, both the Jenter and Nicot systems are designed to:

1. allow the queen to lay directly into the cup (i.e. no grafting)
2. protect the cell before emergence using a hair-roller cage

I'll discuss the hair-roller cages some other time; suffice to say, the Nicot system is much better {{7}}.

More importantly, the first point is achieved by caging the queen in a small(ish) plastic cage where she is offered ~100 cell cups to lay in.

However, in both systems, the cups are presented behind a grille with small holes in it, effectively reducing the aperture size of the cell cup to one closer to worker size (or smaller!). The queen lays through this grille into the underlying cell cup.

The circular holes in the Nicot grille are 5.4 mm in diameter.

The hexagonal holes in the Jenter system are 4.2 mm 'flat-to-flat'.

It's not been tested, but I'd bet the price of a Jenter kit (or my mortgage ... about the same value) that the queen lays worker-sized eggs in these worker-sized - or smaller - holes.

So, if you want to recapitulate the maternal effect you might use Nicot or Jenter cell cups, but you should not use their systems for caging the queen or presenting the cups horizontally {{8}}.

Which creates a bit of a problem ...

It gives me great pleasure to introduce ...

... Rebecca Noll from upstate New York, and Adrian Tonks from Northland, New Zealand.

Both are regular readers and both contacted me about their efforts to get queens to lay in plastic cell cups after the first of the 'Bigger queens, better queens' posts

Rebecca used Nicot cups pressed into the comb, held in place with a 3D-printed facia plate that provided the correct aperture size for the underlying cups. She isolated the queen using another 3D printed cage that allowed worker access, but kept the queen trapped.

Adrian took a different approach. He used JzBz cups mounted in comb, and then caged the queen on the entire frame using an ingenious cage constructed from queen excluder.

I've exchanged several emails with both Adrian and Rebecca; they have patiently answered my questions and, in several cases, repeated experiments.

I am very grateful to them both 😄.

Rebecca's use of Nicot cups

Rebecca caged the queen for 24 hours over an array of 25 Nicot cups behind a facia that had 9 mm holes in it. The queen laid in the cell cups, but workers subsequently removed the majority of them and attempted to reduce the aperture of the hole in the facia plate with a wax collar.

The two eggs that were not removed were transferred to a cell raiser (as less than 24 hour larvae). However, possibly due to technical problems, only one of the resulting queen cells looks potentially promising.

This is a proof of concept, and Rebecca is repeating things 'as I write'. It was interesting to note - though, considering the small numbers, possibly not statistically relevant - the cell cups in which eggs were not removed were waxed before use.

Rebecca has now designed a fiendishly clever 3D printed box to hold the Nicot cups better - causing less damage to the underlying comb. The design is being finalised and printed, so we won't know the results for a bit.

If the box works Rebecca will be happy to share the design for anyone with a 3D printer.

Adrian's use of JzBz cell cups

The approach Adrian took was elegantly simple. He melted holes in fresh comb and pushed the JzBz cups in, flush with the surface of the comb. The warmed wax helped seal the cell cups in place. Then - after acclimatising the frame in the hive - introduced the queen and allowed her to lay.

This was achieved by either caging the queen on the frame, or placing the frame in a 3-frame nuc (with a frame of sealed brood and one of stores).

You can purchase cages for queen trapping. They are often used for Varroa control, but the ones I've and purchased are not inexpensive (c. £70).

Adrian built a cage from queen excluder, using it to encompass a frame into which the JzBz cups were pushed. He used thin ply sheet to blank off the reverse face of the comb, and additional space at the sides, so restricting the queen to the area containing the JzBz cups.

The queen laid in the JzBz cups and several were moved to a cell bar frame (as late-stage eggs or very young larvae) in a queenless cell starter, from which 3 queen cells were produced. Conditions were sub-optimal (it was raining and near the end of the season, so credit to Adrian for persevering) and this, or the state of the cell-raiser, may have accounted for the limited number of queens produced.

The season in New Zealand is winding down, but Adrian also tested JzBz cups pressed into comb vertically orientated. The queen did not lay in these.

That is also an important observation, and tallies with my interpretation of the sketchy description of the methods in Wei et al., 2019.

What could be easier than just pushing a few cell cups into a frame of comb?

Summary

If you assume that it was the queen who placed the eggs into the Nicot or JzBz cups used by Rebecca and Adrian (and it's easy to test if they did not ... think about it, how would you confirm this?) then these results are very encouraging.

It shows that a queen will lay in an oversized, horizontal, plastic cell cup and that the resulting larva can be reared as queens.

No grafting 😄.

In addition, it may not be necessary to handle the queen or trap her in a restricted cage {{9}} ... either the entire frame can be caged, or she could be placed in a dummied-down nuc together with a frame of comb containing suitable cell cups {{10}}.

The assumption here is that the queen, laying under these conditions, will produce larger eggs which will subsequently develop into bigger queens.

Numbers are small, and I'm sure things can be improved and made more efficient. In particular, the timing of egg/larval transfer to the cell raising colony needs to be determined.

Once the season has warmed up here, when the permafrost thaws, the polar bears leave the sea ice and the sun never sets, I'll be attempting to rear queens using a variant of the approaches outlined above.

In reality, that's probably still the best part of a month away ... the 'springroses' are only now out in force, the cuckoos have just arrived and that usually means that queen rearing is still 2-3 weeks away.

Important considerations

I'll use Jenter cell cups.

Firstly, they are the closest in diameter to the cell cups used in the Wei et al., paper. I've no idea whether the fraction of a millimetre difference from the Nicot/JzBz cups is significant or not ... but since nobody else does either, it seems like the sensible thing to do.

Secondly, the two-part construction of the Jenter cups means they have a short 'stalk' that can be easily pushed into the comb, and which should hold them securely in place.

Thirdly, I sold a kidney to buy the Jenter kit, so I'm sure as heck going to use it 😉.

I think a critical thing to determine is the timing of the transfer from the queen cage to the cell raising colony. Both Adrian and Rebecca noted that eggs that initially appeared in cell cups were subsequently removed, presumably by workers.

Therefore, if the queen lays in the cell cups, there's a risk those eggs will be removed before they hatch into larvae.

Perhaps it would be better to transfer them - or, rather, the cell cups they are in - as eggs?

It is known that grafting eggs is problematic. It can be done, but acceptance rates by the cell raiser are often ~50% or less. My understanding (from the very limited amount of literature in this esoteric and cobwebby corner of queen rearing) is that eggs are accepted best by queenless colonies with no open brood, but that maximal acceptance requires 3 day old eggs (Gąbka et al., 2011).

I'll be writing more about this in due course ... lots to do before then.

Part of the puzzle solved?

Queen rearing involves multiple factors, all of which contribute to the overall success - both in number and quality - of the resulting queens.

• good genetics of the donor colony
• well-nourished larvae
• strong cell starter colony
• grafting (or transfer) of larvae of the right age
• ample feeding of both pollen and nectar
• a suitable colony to support the virgin queen
• good drones, and lots of them
• good weather for queen mating

The problem for beginners to queen rearing is that if just one of these stages (and there are others I have not listed) fails, the entire procedure fails.

That's a lot of wasted effort and very disappointing (I speak from experience).

If each of the stages is just 10-20% sub-optimal - slightly questionable genetics, grafting 'elderly' larvae, poor 'take', underwhelming cell starter, inadequate feeding, insufficient drones or dodgy weather - then the resulting queens will almost certainly be disappointing.

But, not all of the stages are equally problematic; even a relatively inexperienced beekeeper (who keeps good records!) can discriminate good from bad colonies, and you don't need a lot of experience to produce a perfectly acceptable cell starter.

But grafting is different ... in a way it's the crux of the entire process. If you only achieve a 10-20% acceptance rate, then disappointment beckons.

Therefore, anything that makes grafting easier, or that replaces grafting entirely, has the potential to increase the success of the entire process.

Which may increase the numbers who try to rear their own queens 😄.

And, if the method also has the potential to generate bigger queens and better queens, then - as trendy youngsters have been heard to say - What's not to like?

It's not a quick fix, it's not a shortcut to success (you still need to pay attention to the rest of that list of 'factors' above), and it's not guaranteed to work ... but I'm excited by the potential benefits, both for my own queen rearing {{11}} and for those who would like to rear their own queens but are put off by the apparent need to graft.

Particularly if they turn out to be bigger, better queens.

Informative? Useful? Entertaining? ... choose any three.
Please support further articles by becoming a sponsor or funding the caffeine that fuels my late night writing ...


Thank you

References

Gąbka, J., Ochnio, M., Kaminski, Z., and Madras-Majewska, B. (2011) Effect of age of eggs used for rearing honey bee queens on the number of received queen cells. Journal of Apicultural Science 55: 47–53.

Pan, Q-Z, Wu, X-B, Guan, C. and Zeng, Z-J (2013) A new method of queen rearing without grafting larvae ABJ 153:1279.

Wei, H., He, X.J., Liao, C.H., Wu, X.B., Jiang, W.J., Zhang, B., et al. (2019) A Maternal Effect on Queen Production in Honeybees. Current Biology 29: 2208-2213.e3 https://www.cell.com/current-biology/abstract/S0960-9822(19)30673-6.

{{1}}: Formally, Wei et al., did not prove that the queen laid the eggs in the plastic queen cups provided. There remains the possibility that workers moved them there, though I think this is unlikely.

{{2}}: Hmmm ... about the size of small-cell foundation, interesting. Standard worker foundation has a cell size of ~5.3 mm. Significant?

{{3}}: I did email those who left comments to explain the situation. If you leave comments here I usually get to them within 48 hours assuming I'm not off-grid, beekeeping or frantically trying to beat the deadline for a Friday post.

{{4}}: Can you think of another way the queen could identify a queen cup?

{{5}}: From discussions with other beekeepers and the supplier I used (Thorne's), and from the number of knock-off looky-likey Nicot kits available that are similar but incompatible. You have been warned.

{{6}}: Let's trust them.

{{7}}: In my opinion ... convince me I'm wrong!

{{8}}: You can buy the cups separately. That's saved you a few quid for starters!

{{9}}: I think they get quite stressed in these - apologies for the anthropomorphism - and often lay rather poorly.

{{10}}: Or a brood box with a QE divider ... something else on my list of things to butcher build.

{{11}}: And I can graft larvae reasonably successfully.