Rob Stone's Re-Queening Protocol

Orange County Beekeeping Supply By Rob Stone May 16, 2018


Or, Why did I spend big bucks on queens and none worked?!

Re-queening colonies is always a challenge, even the best beekeepers report success rates of at most 90%, with beginners typically around 50%. There are many factors that affect success, including colony size, genetics, honeyflow, weather, stress factors, etc. The biggest factor by far is colony size. You will not be able to requeen colonies of 2 and 3 boxes with any direct introduction technique, but sometimes it can be accomplished with more elaborate efforts and will take quite a bit longer. One other major prerequisites is the colony you are trying to requeen must be queenless. And it  is much better if there are no active queen cells being raised. If a virgin emerges after you have  introduced your desired queen and she is laying, she will be unsuccessful at fighting a feral virgin queen and most often the nurse bees will not let the introduced queen destroy the queen cells before their ‘home’ raised queens emerge, so you must intervene.  Of course, a colony cannot be queenless long enough to have the dreaded ‘laying workers’ syndrome, but that often can be rectified by introducing a frame of brood with all stages of eggs and larvae each week for 3 weeks, then trying the queen introduction as described here. Colonies need to be of normal mix, meaning you cannot wait till dark, pull a honey super full of foragers from a large colony to get your 5 (or 4 or 6) frames of bees and requeen that ‘colony’ because foragers are the least likely to accept your ‘foreign’ queen.   

Back to what is correct, the optimum colony size for requeening is of 4 to 6 frames of bees. 3 frames will still work if they have food and pollen. Even two frames, but expect a very slow buildup following introduction.  If you have bigger colonies, split them and requeen both halves or as many splits as you wind up with, but each should have at minimum 1 frame of brood. Two is better, three is best. And some of the brood should be unsealed, this is important as it provides the colony the brood pheromone which suppresses laying workers. You can use a nuc or a regular deep, each are OK but if you had a choice the fuller the box, the better, so the Nuc is preferable. Give them a frame of honey or two of honey (in the outside positions, thermal barrier) if you have it. Of course, if you working with 5 or 6 frames of bees, use the full size 10 frame hive body.   

Make the colony queenless. Yes, find her and remove her, untold thousands of queens have tried to be introduced to queenright colonies and they will be rejected. Without exception. I know, I have unknowingly tried this many times and it always has the same outcome, bad. A colony knows it queenless in about 8-12  hours, and they begin building emergency queen cells within 24 hours. Wait for about 3 days after you removed her and inspect. If you didn’t find queen cells being built, they think they have a queen. Or another queen is. So keep looking, you must confirm this colony is queenless by finding queen cells. Many colonies have multiple queens, most often mother/daughter combos that can persist like that for months. The colony must be in desperate need for a queen before it will accept a foreign one. Verify they need a queen by seeing the evidence of queen cells. 

Ok, you have waited 3 days and there are queen cells under construction, destroy them. Put your new queen (in her queen cage!) with corks in both ends in between brood frames or next to the one, but still be jammed between two frame top bars. You want nurse bees to come in contact with her. Before hand,  smear a tiny amount of honey on the screen of the cage. This will keep her fed for a few hours and hopefully the bees will begin to feed after that.  If you put the cage with the screen up between two frames you can check the next day how they are acting towards her and smear honey on the screen again in case they aren’t friendly to her yet. But be SURE they bees can get to screen, so they can come in contact with her when they are ready.  If they cant access her (but through the protection of the screen), she will die. Its OK to put the cage in screen down or up or whatever. She is going to remain confined like this for 3-5 days. She will be accepted within this time period, or not and she will be dead.  

So, the five confined days have passed, open up the colony and see how the bees are clinging to the screen of the queen cage. Gently coax them away from the screen. If they are trying to sting her, something is still wrong and you should remove her, and I bet you will find a queen, a virgin or queen cells. Correct and start over. Otherwise, put her aside and go through the colony very carefully to make sure you haven’t missed any queen cells, which should be large and sealed by now. If you find any destroy them. Pull one of the frames of brood and shake the bees off. The best selection is sealed brood ready to hatch or hatching now with a little corner of honey too. Bees just emerging will accept any queen.  Now we are going to let her out of her wood  cage into a push-on wire cage over some sealed brood (and some honey, if sealed poke a couple of cells so she can feed herself). This is the trickiest part of this whole procedure. An all wire queen cage is just a wire cage open on one side that your press down onto the comb. Yes, it will kill some brood where the cage knifes into the comb. I now cut a small door the size of the queen cage into the side of wire cage and flip it up (open).  Pull the cork  from the wood cage (not the candy end) and stick your finger over it (no gloves for this part of the procedure). Lay the brood frame down on something and lay the wood cage down on the frame. Place the wire cage up to the door you have cut into the push on cage. You now have your finger off and the queen can come out, and it can take a long time for her to find her way. Be patient.  When she (and attendants too if included) comes out pull away the wood cage and imprison her in the wire cage on the comb above honey and brood. I have seen her run straight to the honey and drink so it’s not true about queens not being able to feed themselves. When she is in the cage and on the other end, flip down the door and push it into the comb. She just have enough room to crawl around on the comb underneath the cage. Replace the frame with her on it and leave them alone for 5 to 7 days. She will have  bees hatch with her and she will start laying in the comb she can access. You pull the cage off after the five to seven days and now you have done it. Smile and congratulations.   

So its complete and all your stress is over. Not quite. Many times, in particular with aggressive feral colonies, they will accept your foreign queen, let her lay eggs for somewhere between a week and month,and then she is gone and you find a full set of queen cells. The only action you can take to prevent this is to look each week at your newly requeened colony and if they build queen cells, destroy them. But always verify there are eggs in worker cells before you destroy their chances of replacing your new foreign queen who has now somehow  failed and has stopped laying or is laying drone. 

Additional thoughts: Other factors influence your success rate. One is honeyflow. If there is no honeyflow things are more complicated, the bees are cranky and wont tolerate your interventions as well, and the small colony you are working with will be more  susceptible to Robbing. If your working with one colony in your backyard, robbing is a much smaller issue, but if it’s a small colony in a beeyard, you have to be so very careful. Without a honeyflow, you should put in a feeder and provide syrup, like to 1 to 1 (water weight to sugar weight) to stimulate the colony and get them to want to grow. You dont need to feed, they should have a couple of frames of nectar or honey already. You just need the stimulation a honeyflow provides. Be extra careful with the syrup (no spills, open the colony the minimum time necessary) so the small colony doesn’t get robbed out, that will ruin your chances of a happy outcome. And just put in an inch or two at a time and do it again in a couple of days. If they didn’t drink it there is a reason. Figure out why.  With feeding the big issue is robbing, so already have reduced the entrance to one bee space before you begin the requeening protocol, and use a robbing screen if the time year warrants it.  

If your working with nicer bees and can go gloveless, you can add newly hatched bees into the cage with the queen before you mash it into the comb. Find these fuzzy bees on a brood frame and pick them up from behind or by the wings and insert. They wont sting you. There are a couple of these bees on the picture above close to the queen. Again, newly hatched bees accept any queen.  

Queen pheromone strength.  If you buy a batch of queens they come in individual cages inside a large box that has few hundred attendants with it. The attendants care for all queens in the box, but you will see some queens have much larger groups of bees surrounding them. Those queens have stronger pheromone and will be easier to introduce. This is a factor for introduction. Use the more powerful queens for the larger colonies you are attempting to requeen, and the less powerful queens for the smaller colonies. 

1. Get colony to proper size, 3 to 6 frames.  Entrance reduced, correct size box, etc. Honey, brood, pollen. 
2. Make queenless, and verify queen cell construction. Destroy cells
3. Put in queen, feed her first. Let them get used to her. Feed her again.
4. Destroy cells again and transfer queen to screen comb cage. 
5. Brood hatches and accepts her, she begins to lay in comb inside cage. 
6. Remove cage.
7. Inspect weekly for queen cells for the next month.

[Note: Rob Stone, owner of Orange County Beekeeping Supply, was our featured speaker at the May 6, 2019 monthly meeting of the Los Angeles County Beekeepers Association. Whether or not you were one of the fortunate attendees at this meeting, you’ll remember Rob’s highly informative, engrossing, and entertaining talk about bees and beekeeping, you’ll want to check out his blog at: Thank you to Rob Stone - your talk was awesome!]

Improved Regulation Needed As Pesticides Found to Affect Genes in Bees

EurekAlert From: Queen Mary University of London March 6, 2019

Bumblebee Colony Credit: TJ Colgan

Bumblebee Colony Credit: TJ Colgan

Scientists are urging for improved regulation on pesticides after finding that they affect genes in bumblebees, according to research led by Queen Mary University of London in collaboration with Imperial College London.

For the first time, researchers applied a biomedically inspired approach to examine changes in the 12,000 genes that make up bumblebee workers and queens after pesticide exposure.

The study, published in Molecular Ecology, shows that genes which may be involved in a broad range of biological processes are affected.

They also found that queens and workers respond differently to pesticide exposure and that one pesticide they tested had much stronger effects than the other did.

Other recent studies, including previous work by the authors, have revealed that exposure even to low doses of these neurotoxic pesticides is detrimental to colony function and survival as it impairs bee behaviours including the ability to obtain pollen and nectar from flowers and the ability to locate their nests.

This new approach provides high-resolution information about what is happening at a molecular level inside the bodies of the bumblebees.

Some of these changes in gene activity may represent the mechanisms that link intoxification to impaired behaviour.

Lead author of the study Dr Yannick Wurm, from Queen Mary University of London, said: "Governments had approved what they thought were 'safe' levels but pesticides intoxicate many pollinators, reducing their dexterity and cognition and ultimately survival. This is a major risk because pollinators are declining worldwide yet are essential for maintaining the stability of the ecosystem and for pollinating crops.

"While newer pesticide evaluation aims to consider the impact on behaviour, our work demonstrates a highly sensitive approach that can dramatically improve how we evaluate the effects of pesticides."

The researchers exposed colonies of bumblebees to either clothianidin or imidacloprid at field-realistic concentrations while controlling for factors including colony social environment and worker age.

They found clothianidin had much stronger effects than imidacloprid - both of which are in the category of 'neonicotinoid' pesticides and both of which are still used worldwide although they were banned in 2018 for outdoor use by the European Union.

For worker bumblebees, the activity levels of 55 genes were changed by exposure to clothianidin with 31 genes showing higher activity levels while the rest showed lower activity levels after exposure.

This could indicate that their bodies are reorienting resources to try to detoxify, which the researchers suspect is what some of the genes are doing. For other genes, the changes could represent the intermediate effects of intoxification that lead to affected behaviour.

The trend differed in queen bumblebees as 17 genes had changed activity levels, with 16 of the 17 having higher activity levels after exposure to the clothianidin pesticide.

Dr Joe Colgan, first author of the study and also from Queen Mary University of London, said: "This shows that worker and queen bumblebees are differently wired and that the pesticides do not affect them in the same way. As workers and queens perform different but complementary activities essential for colony function, improving our understanding of how both types of colony member are affected by pesticides is vital for assessing the risks these chemicals pose."

The researchers believe that the approach they have demonstrated must now be applied more broadly. This will provide detailed information on how pesticides differ in the effects they have on beneficial species, and why species may differ in their susceptibility.

Dr Colgan said: "We examined the effects of two pesticides on one species of bumblebee. But hundreds of pesticides are authorised, and their effects are likely to substantially differ across the 200,000 pollinating insect species which also include other bees, wasps, flies, moths, and butterflies."

Dr Wurm added: "Our work demonstrates that the type of high-resolution molecular approach that has changed the way human diseases are researched and diagnosed, can also be applied to beneficial pollinators. This approach provides an unprecedented view of how bees are being affected by pesticides and works at large scale. It can fundamentally improve how we evaluate the toxicity of chemicals we put into nature."


Research paper: 'Caste- and pesticide-specific effects of neonicotinoid pesticide exposure on gene expression in bumblebees'. Thomas J. Colgan, Isabel K. Fletcher, Andres N. Arce, Richard J. Gill, Ana Ramos Rodrigues, Eckart Stolle, Lars Chittka and Yannick Wurm. Molecular Ecology.

New Laboratory System Allows Researchers To Probe The Secret Lives Of Queen Bees University of Illinois at Urbana-Champaign December 3, 2018

Researchers at the Carl R. Woese Institute for Genomic Biology at the University of Illinois used specially developed 3D-printed plastic honey combs that mimic the hive environment, in order to monitor queen egg-laying behaviors. Credit: Bee Research Facility, University of Illinois

Researchers at the Carl R. Woese Institute for Genomic Biology at the University of Illinois used specially developed 3D-printed plastic honey combs that mimic the hive environment, in order to monitor queen egg-laying behaviors. Credit: Bee Research Facility, University of Illinois

More than a decade after the identification of colony collapse disorder, a phenomenon marked by widespread loss of honey bee colonies, scientists are still working to untangle the ecologically complex problem of how to mitigate ongoing losses of honey bees and other pollinating species. One much-needed aid in this effort is more efficient ways to track specific impacts on bee health. To address this need, a group of Illinois researchers has established a laboratory-based method for tracking the fertility of honey bee queens.

Co-first authors Julia Fine and Hagai Shpigler, both postdoctoral researchers at the University of Illinois, worked with others in the laboratory of Carl R. Woese Institute for Genomic Biology Director and Swanlund Professor of Entomology Gene Robinson to establish a laboratory set-up that would mimic the key aspects of the hive environment and allow detection of egg-laying by honey bee queens living with small groups of worker bees. The resulting system, described in PLOS ONE, allowed them to explore the relationship between worker nutrition and queen fertility.

"The idea that honey bee nutrition influences colony level metrics of reproduction has been demonstrated before, but here, we examined an old story using new tools," Fine said. "We were able to get a clearer picture of how nutrition can affect the relationship between honey bee workers and queens and how this can impact the queen's egg production."

Populations of many pollinator species have been declining in the US and worldwide. Studies of factors influencing wild and managed honey bee hives have identified four main factors influencing health: parasites, pathogens, pesticides, and poor nutrition. These factors can influence one another. For example, parasites may spread pathogens, much as fleas do on people, while poor nutrition might increase the likelihood of foraging on contaminated food sources.

Egg production is a vital aspect of honey bee colony function. Queens lay eggs that hatch into the thousands of worker bees that keep the colony running, as well as males and young queens to allow the colony to propagate. But in the dark, bustling interior of a standard hive, it is challenging to monitor egg laying or to evaluate the impacts of environmental factors.

"Egg laying occurs in the darkness of a hive occupied by thousands of workers and is therefore hard to track," Shpigler said. "Queen egg laying was never studied outside of the colony; the biggest challenge was to give the queens the right conditions for continuous egg laying outside of natural conditions."

To move queen productivity successfully into the lab, the researchers focused on the essentials of their natural environment. They developed a 3-D-printed plastic honey comb that they refined to mimic what a queen would experience in the hive, which ensured that the cage environment could be carefully controlled and kept pesticide free. They also provided each queen with a small group of worker bees to feed and support the queen; this element became the inspiration for their first experiments with the new system.

"Honey bee queens only ingest food in the form of glandular secretions provided to them by their worker caretakers, and queens are not known to lay eggs without the support of their worker bees," Fine said. "The more we worked in this system, the more it became apparent that the easiest way to influence the queen was to first influence the worker bees that care for her. Once we identified this strategy, designing effective experiments became easier."

Fine, Shpigler, and their coauthors provided each group of caged bees with honey, water, and sucrose solution, but varied the source of fat and protein: some bees were fed with a paste of honey and either a low or a high amount of floral pollen, while others were fed with bee bread, a mixture of pollen, honey, and secretions produced by worker honey bees that preserve and ferment the pollen. The researchers monitored how queen egg laying behavior was influenced by the type of diet fed to the workers caring for her.

They found that when a group of workers was fed pollen paste, the queen they attended was likely to increase her egg laying more slowly in the laboratory environment than a queen attended by bee bread-fed workers. This difference was most noticeable when the lower-percentage pollen paste was used, but persisted even in bees fed the richer pollen paste.

The results affirmed the importance of nutrition to queen productivity, as well as demonstrating the potential utility of the laboratory set-up for investigating other factors affecting queen behavior and health.

"The effect of the nutrition . . . was our first successful use of the system, giving us hope for more success in the future," Shpigler said. "The results show very nicely how the honey bee colony functions as one body, with shared digestive and reproductive systems. The workers are the ones that eat the food and the effect is on the queen egg laying—the superorganism in action!"

"It's been exciting to see the kind of quantitative data that we can generate with this system using fewer resources relative to studies that use full size honey bee colonies," Fine said. "Eventually, we hope that this system can be adapted as a risk assessment tool to identify other factors that positively and negatively influence honey bee reproduction . . . there is an immediate need for a laboratory system that can be used to quantitatively assess risks to honey bee queen health and reproduction."

More information: Julia D. Fine et al, Quantifying the effects of pollen nutrition on honey bee queen egg laying with a new laboratory system, PLOS ONE (2018). DOI: 10.1371/journal.pone.0203444

Journal reference: PLoS ONE

Provided by: University of Illinois at Urbana-Champaign