# Hygienic test: UBO (unhealthy brood odor) assay



## Jonathan Bennett (Jul 19, 2021)

Right now ubo isn't available to just buy right now in the form of some spray. Word on the street though is that it's in development. It would indeed make assay test much easier and quicker. Not only can you do the nitrogen test but some breeders will uncap purple eye pupae and count reproductive mites. Look up harbo assay for more info on that vsh test.


----------



## crofter (May 5, 2011)

Below is a clip from a post [email protected] A method to see if your bees are active uncapper/recappers. Having a spray or paint on solution to cover a marked area of capped brood and see what the bees do with it, would certainly be simpler.

*{While certainly more 'quick-and-dirty' than the precise protocol you have developed, Dr. Seeley sent me this idea, which I thought was pretty good:

"I do not know if there is a standard protocol for this assay, except that researchers like to sample ca. 100 capped cells from the frames of capped brood in a colony. This can be done by pressing a patch or two of duct tape down onto a frame of capped brood in a colony, then pulling it up and studying the undersides of the cells’ cappings. By sampling 100+ cell cappings, one can get a good estimate of the % of cells that have been capped and recapped. 

A cell capping that has been capped and recapped is recognized as follows: "The recapping behaviour can be easily detected as a hole in the spun cocoon of the pupated larva ranging in size from one mm to the entire area of the cap. The hole is subsequently covered over with wax by the adult bees. This hole can be seen as a dark, matte spot on the underside of the cell cap distinct from the glossy coating of the cocoon.” 

This is a quote from the paper that I have attached. Fig. 1 in this paper contains excellent photos that show more clearly than the above words about how to recognized cells of mite infested brood that have been uncapped and recapped. I hope you find that your bees are controlling the Varroa mites in this way."} *


----------



## Litsinger (Jun 14, 2018)

crofter said:


> A method to see if your bees are active uncapper/recappers.


Another thing that works really well is waxing strips. They have the built in advantage of being consistently sized so you pull off approximately the same number of caps every time:



Litsinger said:


> Regarding this assay I was recently in an e-mail exchange with Dr. Büchler and I asked him about the specific product they employ for uncapping/recapping assays. He indicated that after trying several options, they settled on the Isana brand depilatory strips as the best solution.
> 
> It does not appear that we have the Isana brand strips available domestically, but it looks like Rossmann also makes the Veet brand.
> 
> A search for 'Veet Cold Wax Strips' produces a lot of results.


----------



## SeaCucumber (Jun 5, 2014)

Hygiene-Eliciting Brood Semiochemicals as a Tool for Assaying Honey Bee (Hymenoptera: Apidae) Colony Resistance to Varroa (Mesostigmata: Varroidae)


Despite numerous interventions, the ectoparasitic mite Varroa (Varroa destructor Anderson and Trueman [Mesostigmata: Varroidae]) and the pathogens it vectors remain a primary threat to honey bee (Apis mellifera Linnaeus [Hymenoptera: Apidae]) health. ...




www.ncbi.nlm.nih.gov




UBO and Freeze-killed brood (FKB) assays were compared


----------



## Michael Palmer (Dec 29, 2006)

I should say something.
I've been given permission to use the UBO assay. Last summer we started with 64 colonies that had performed well over the previous few years. Wintering, disease's present, temper, honey produced, frozen brood assay, etc...just typical categories in a breeding program that I've been running since 1998. We chose the 5 highest scorers in all categories to be the breeders for 2022.
In 2022, we raised 100 queens...50 from the program queens and 50 from 5 breeders of my own choice as controls. So, two apiaries of 50 nucleus each...separated by 20 miles. 5 nucs from each program queen and 5 controls from my own stock selection. This summer we added the UBO assay. Understand that UBO assay and frozen brood assay are measuring different things. Frozen brood assay is selecting for brood death. UBO is selecting for unhealthy brood...brood with high varroa population or brood with low varroa population but high virus titre. I'm working with the UVM bee lab and some grant money. Took two days with two lab techs and 4 of us to do the UBO sampling. The variation in uncapping was something to see.
Next season, we continue. Selecting the best from 2022 sampling to raise the next generations of queens.
Very excited about this program. Pretty much what will keep me in bees.


----------



## CiCi (Dec 5, 2016)

Mike, have you or someone else tried taking colonies with excellent uncapping percentage from the UBO spray and performing Harbo assays to see how they rank? ( I'll email Kaira Wagoner on this, too...) 

It seems that there are two ways to determine Varroa Sensitive Hygiene behavior, and if that is the case, your top UBO-performing queens should rank as 4's on the Harbo scale.

Concerning recapping behavior:
I find myself wondering if hygienic colonies that rank high on freeze assays but low on Harbo or USO assays would show any recapping behavior. If NOT, then this might show that recapping is a good indicator of VSH traits. If they DO, then recapping may be an indicator for a wide variety of traits, which might not be as useful as we hope. (I understand that hygenic stock typically do well spotting disease, while VSH do better spotting mites. Although disease-detection is great, I'd rather focus on getting the elephant out of the hive before the mice.)

It would be wonderful if checking for uncapping/recapping behavior would give insight on VSH status of colony. What a great quick tool that would be for queen breeders to have out in the field, with minimal time and cost. Not to replace the Harbo or UBO assays, but to supplement them.

Thoughts?


----------



## Kaira Wagoner (2 mo ago)

CiCi said:


> Mike, have you or someone else tried taking colonies with excellent uncapping percentage from the UBO spray and performing Harbo assays to see how they rank? ( I'll email Kaira Wagoner on this, too...)
> 
> It seems that there are two ways to determine Varroa Sensitive Hygiene behavior, and if that is the case, your top UBO-performing queens should rank as 4's on the Harbo scale.
> 
> ...


Hi CiCi, 
Great question about recapping. We have clear evidence that UBO is correlated with pin-killed and FKB, and growing evidence that it is correlated with mite non-reproduction. We are already making plans to look next into the relationship between UBO and recapping, and should know more after the 2023 field season. Despite being correlated with other hygienic parameters, though, the UBO assay is measuring something different, as mentioned by Michael Palmer above. From my experiments, including a few new ones focused on antennae that I have not published or talked about publicly yet, it looks like colony UBO response is a reliable measure of nurse bee sensitivity to and memory of specific odors associated with mite-parasitized or otherwise _unhealthy_ (but living) brood. Our data suggests UBO high colonies can detect a range of sublethal issues such as Varroa and high virus loads (including viruses NOT transmitted by Varroa). Folks above are correct that we are working to bring it to market, with launch in the US projected for spring of 2024. To help get a sense of where interest in UBO lies and how many tests we will need to produce, we are accepting pre-orders now, with priority given at launch to those who placed a pre-order. If you're interested, you can check out our new website opterabees.com (still under construction so please forgive updates and pages still in progress!) Thanks for your interest, we are eager to get UBOs out into the hands of breeders and beekeepers so they can try it out themselves!


----------



## ruthiesbees (Aug 27, 2013)

Kaira Wagoner said:


> Hi CiCi,
> Great question about recapping. We have clear evidence that UBO is correlated with pin-killed and FKB, and growing evidence that it is correlated with mite non-reproduction. We are already making plans to look next into the relationship between UBO and recapping, and should know more after the 2023 field season. Despite being correlated with other hygienic parameters, though, the UBO assay is measuring something different, as mentioned by Michael Palmer above. From my experiments, including a few new ones focused on antennae that I have not published or talked about publicly yet, it looks like colony UBO response is a reliable measure of nurse bee sensitivity to and memory of specific odors associated with mite-parasitized or otherwise _unhealthy_ (but living) brood. Our data suggests UBO high colonies can detect a range of sublethal issues such as Varroa and high virus loads (including viruses NOT transmitted by Varroa). Folks above are correct that we are working to bring it to market, with launch in the US projected for spring of 2024. To help get a sense of where interest in UBO lies and how many tests we will need to produce, we are accepting pre-orders now, with priority given at launch to those who placed a pre-order. If you're interested, you can check out our new website opterabees.com (still under construction so please forgive updates and pages still in progress!) Thanks for your interest, we are eager to get UBOs out into the hands of breeders and beekeepers so they can try it out themselves!


Thank you for joining the discussion! I've seen a few of your presentations to local bee clubs. Do you have a Youtube channel or another avenue for people to watch your presentations?


----------



## Kaira Wagoner (2 mo ago)

ruthiesbees said:


> Thank you for joining the discussion! I've seen a few of your presentations to local bee clubs. Do you have a Youtube channel or another avenue for people to watch your presentations?


Hi Ruth, 
Thank you for your interest! I don't have a youtube channel but we do plan to start sending out some project updates soon (you can email us through the website opterabees.com if you want to be added to the list to receive these) and the UBO work has been featured on Inside the Hive TV here: 



 and here: 



, with another update planned for this December (date TBD).


----------



## CiCi (Dec 5, 2016)

Thank you for your response, Kaira... looking forward to this summer's research results!

Crofter or Litsinger, could you share the discussion link you refer to in the third & forth response of this discussion? 

I've been digging on the web, and I want to personally thank *Litsinger *for mentioning Dr. Büchler's recapping studies... however, what I could find out there was incomplete. The most current item of interest in the recapping (SMR) phenomenon is this link that provides recapping assay details in Sept 2017: NEW SMR Protocol – RNSBB Unless I misread, it looked like that assay protocol was for a study being launched, but I could not find results of that study.

If anyone could point me to more recent (after 2017) discussions or studies done specifically with recapping and Dr. Buchler, I would appreciate it. I have found only general studies, like the 2022 Jan ABJ article about study of commercial beeks using mite-resistant stock in Europe, but not specific studies on recapping.

For folks needing an explanation on the Harbo assay, he kindly provides details here: harbobeeco - Measure VSH The amount of detail says a lot to this gentleman's dedication to sharing with colleagues, instead of keeping 'trade secrets' that helps only the insiders. Thank you, Dr. Harbo.


----------



## crofter (May 5, 2011)

CiCi said:


> Thank you for your response, Kaira... looking forward to this summer's research results!
> 
> Crofter or Litsinger, could you share the discussion link you refer to in the third & forth response of this discussion?
> 
> ...


Here is a search link to a number of mentions by @Litsinger of uncapping recapping. What you are looking for might be in there.









Search results for query: uncapping recapping







www.beesource.com


----------



## Litsinger (Jun 14, 2018)

crofter said:


> Here is a search link to a number of mentions by @Litsinger of uncapping recapping. What you are looking for might be in there.


Thanks for the assist, @crofter. I sincerely appreciate it!

All the information I have read concerning uncapping/recapping involving Dr. Buchler has been by personal correspondence.

That said, if you are interested in SMR/VSH here is a great recent study if you haven't seen it already:



https://www.researchgate.net/publication/344079335_Evaluation_of_Suppressed_Mite_Reproduction_SMR_Reveals_Potential_for_Varroa_Resistance_in_European_Honey_Bees_Apis_mellifera_L


----------



## CiCi (Dec 5, 2016)

Eureka! That's the study that was eluding me... I knew the Sept 2017: NEW SMR Protocol – RNSBB had to have resulted in some kind of paper. I have some Black Friday reading to do... looking forward to it.

Thank you, Crofter, for helping me dice out search parameters for Beesource. I've searched this awesome forum for years during frequent information-seeking binges, but hadn't thought to link a user name with keywords. I can think of several other possible combos I'll need to check out while I'm playing 'catch-up' on this topic.


----------



## CiCi (Dec 5, 2016)

I had quite a fun time wading through tons of threads & studies, including the one listed above. 

The 2020 SMR/VSH study shows pockets of resistant honey bee colonies in Europe using the 2017 NEW SMR Protocol – RNSBB. It is great reading, but what stood out the most for me is the following:

"Obtaining a reliable estimate of the ability of a given colony to suppress mite reproduction is difficult 
and labor intensive. In addition, the score can be influenced by a number of different factors, such as
the amount of worker and drone brood available [49] or the mite load of the colonies. The number of 
offspring per mite tends to decrease with high infestation levels [50]. {removed text for succinctness}
*Together, these challenges may lay behind the reluctance of breeders to integrate and use the SMR 
trait in selection programs* [58]. "

There is no doubt that the time invested + the variables listed in above quote make scoring for SMR/VSH through examination of infertile and fertile foundress mites difficult for small-time queen breeders. The process of determining VSH through harbobeeco - Measure VSH seems to be more clear-cut than what was used in the above study, but unfortunately, evidence of recapping was left out of the Harbo VSH scoring equation.

I have hope that someone experienced in using that assay in their current operation might be able to improve on the original 'formula' that shrinks the time commitment per colony, and share it with the rest of us. Cory S. said that he has been using Dr. Harbo's assay for the last three years in his VSH operation, and has made no changes. He also invites his friends over with the lure of beer to help out on the microscopes... apparently, it works well! If he leaks word out WHEN his next 'party' is going to be, lots of us small-time Q breeders might show up on his doorstep with a case in hand. Sounds like a great time.

Quite frankly, the UBO spray seems like it would be LOTS quicker than the VSH mite-counting assay to perform, and possibly much more reliable; I'm really hoping the pricepoint allows the small-time breeders to utilize this tool to confirm top breeder candidates. I know Kaira is working hard to keep the price as low as possible, but it will be hard to do so during the start-up phase. She was talking about how long each batch takes to prepare in the lab, and of course the pricepoint will have to reflect that.


----------



## CiCi (Dec 5, 2016)

If you haven't found it already, there's a 2021 study on recapping that interested parties should read:

Oddie, M.A.Y., Burke, A., Dahle, B. _et al._ Reproductive success of the parasitic mite (_Varroa destructor_) is lower in honeybee colonies that target infested cells with recapping. _Sci Rep_ *11*, 9133 (2021). https://doi.org/10.1038/s41598-021-88592-y

If the title didn't give it away, this golden nugget quote sums it up perfectly... "_The results show that colonies with a *higher recapping efficacy* also have a lower average mite reproductive success." _The authors are quick to point out that recapping IN ITSELF may not be the actual mechanism but seems to be a clear indicator of mite-surviving populations_._

Even better, this 2021 recapping study gave clear markers to assess potential breeder populations: _"The recapping efficacy in mite-susceptible populations was between 22 and 31% and between 35 and 55% in mite-surviving populations." _To me, this says that the marker to look for is at least 45% recapping sign (or roughly half out of 50 cells)_._ 

What do you say? More? Less?


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> The authors are quick to point out that recapping IN ITSELF may not be the actual mechanism but seems to be a clear indicator of mite-surviving populations_._


If you haven't already, I encourage you to read-up on Dr. Stephen Martin (here on BS and elsewhere). He has studied resistant populations around the globe and has found that uncapping/recapping is ubiquitous in every one he has evaluated. In my mind, it is the clearest evidence yet that we have of a common thread woven between these populations.


----------



## CiCi (Dec 5, 2016)

Yes, funny you said that... I actually have a tab currently open on his 2019 _Varroa destructor reproduction and cell re-capping in mite-resistant Apis mellifera populations_ at Varroa destructor reproduction and cell re-capping in mite-resistant Apis mellifera populations - Apidologie. It was a fascinating read, but when it comes to usefulness, I am particularly frustrated at the import of this graph posted below... basically, it tells me that you need a high mite presence to be able to judge recapping traits in a colony at a higher rate of 40-75%. The previous study I referred to earlier showed 25-35% recapping rate in susceptible populations, while this study shows 10-18% recapping rate. If you have a fairly mite-clean apiary already, what rate should you go off of? Or am I on the wrong track entirely? GRRR... I love research and overviews, but am specifically looking for tools to improve my stock.

What did you find that worked for checking recapping in your apiary? I read you were experimenting with wax strips and bale tape...


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> If you have a fairly mite-clean apiary already, what rate should you go off of?


@CiCi:

Good question- I frankly don't know. I did however send Dr. Buchler, Dr. Martin and Dr. Seeley an email to get their input on this and I will report back should I learn anything. What I suspect is that there is likely no consensus on this factor given it likely depends not only on infestation rate in the cells but also upon brood-initiated cues that are not yet well-understood.

I expect this is why the Harbo Assay has remained so important- while it is tedious it plots the best curve fit in predicting reduced mite population growth:



Litsinger said:


> The most reliable method is to select for a high infertility rate in the mite population (Fig. 2). We are not certain how it happens, but somehow VSH increases mite infertility. Generally, 15-25% of mites in non-resistant colonies do not lay eggs. Infertility increases to 80-100% in colonies with pure VSH queens. Mite populations eventually decline in these colonies because so few mites lay eggs.'


When Dr. Buchler assisted a community resistance breeding program, he suggested the following:



Litsinger said:


> '... identifying traits of varroa resistance from bees that have already demonstrated uncapping and low mite loads. We also like to see good bee health, evidenced by strong foraging and brood development in the spring, plus gentle temperament.'


So I'd say the easy answer is that more uncapping at a given brood infestation rate is better all other things equal, but this likely should not serve as the sole metric for resistance breeding- but I could easily be wrong.

I've not really figured out how to fit uncapping/recapping assays into my program, but I can attest that the waxing strips work great.

As I've considered working it in, I've convinced myself that these assays probably only have to be done once per year, and at the same period of seasonal development- thinking about the Harbo Assay it might be best to test during peak brood rearing but before swarm season is in full swing.


----------



## Gray Goose (Sep 4, 2018)

so in uncapping do the bees take the mite eggs out and destroy, OR do the mites when Interrupted lay less eggs?
possible, either, both or something else. do they spit somethin into the cell to prevent egg laying.
any studies around this?

most logical is to open steal the mite eggs, or young mites and close.
groom a few , bite a few, steal the babies.

GG


----------



## CiCi (Dec 5, 2016)

Gray Goose, 

One study found different results under the recapped mite-infested cells, namely: no male, delayed procreation, infertile foundress. The quantity of each result varied depending on colony country, interestingly enough. This might indicate a different mechanism at play with the different bee populations, or mite populations. _Evaluation of Suppressed Mite Reproduction (SMR) Reveals Potential for Varroa Resistance in European Honey Bees (Apis mellifera L.)_

Another study indicates that mite-invested cells tend to have a larger recapping hole then non-infested cells. I can't lay my hands on that source right now, but it hints that extra air flow may cause lack of mite progeny in those particular recapped cells. Some larger openings had no mite present, creating speculation that the mother mite might have crawled out to find another larvae about to be capped.

It seems doubtful that the nurse bees would be able to manually remove mites or their eggs without pulling out and killing the bee pupae.


----------



## CiCi (Dec 5, 2016)

Litsinger said:


> I've not really figured out how to fit uncapping/recapping assays into my program, but I can attest that the waxing strips work great.


This begs the question... with both the UBO spray and uncapping manually with tape/strips, WILL THE BEES RECAP THOSE CELLS and the brood survive with no damage?

I know its not crucial to the survival of the hive, as 50 to 100 brood is nothing in the whole scheme of things, but I wonder how much air exposure is too much for bee brood to recover from.

Kiara, Mike P, could you comment on this, too? How quickly after the UBO assay are they recapped, or does the solution cause the nurse bees pull out the larvae so you don't know for sure?


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> ... creating speculation that the mother mite might have crawled out to find another larvae about to be capped.


I believe this is the general consensus thus far- the uncapping interrupts successful reproduction.

Randy Oliver also recently made an interesting observation along these lines in that a foundress might may only receive a few dozen sperm during a successful mating and so if her reproductive effort is interrupted a few times she will be unable to successfully produce progeny.


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> ... the brood survive with no damage?


While I can't speak to what happens with the UBO spray, I do know that bald brood in my colonies tends to develop and emerge just fine.

As I understand it, the capping is only critical during the early instar stages- beyond that the pupae are sufficiently tough to continue successful development in the fairly well maintained environment of the brood nest.


----------



## Litsinger (Jun 14, 2018)

Litsinger said:


> Dr. Seeley...


Here is Dr. Seeley's input- I expect this is likely to represent the consensus answer:

_'I can’t give you a precise answer based on either personal experience or papers on this topic.
There is also the challenge of not knowing how many of the capped brood cells that one sees contain (or contained) Varroa.
So, all in all, I think the best answer I can offer is that, the best thing is to select based on colony survival and having consistently low mite counts.
The proof is in colony survival (and low mite counts).'_


----------



## crofter (May 5, 2011)

Litsinger said:


> I believe this is the general consensus thus far- the uncapping interrupts successful reproduction.
> 
> Randy Oliver also recently made an interesting observation along these lines in that a foundress might may only receive a few dozen sperm during a successful mating and so if her reproductive effort is interrupted a few times she will be unable to successfully produce progeny.


Only the first few eggs laid have time enough to reach maturity before emergence. What is it- just over 2 successful offspring from a worker cell and approaching 3 in the extra time in a drone cell. 

It would not take much of an interruption to effect a large mathematical difference. It would have to occur at the crucial time though. How soon would the uncapping bees detect and uncap?


----------



## CiCi (Dec 5, 2016)

crofter said:


> just over 2 successful offspring from a worker cell and approaching 3 in the extra time in a drone cell.


Here's a rather revolting New Zealand video of mites in with a drone larvae... never have I seen so many in one cell...gag... only a couple are slightly pale. I counted 12 on the larvae and one at the bottom of the cell.
Varroa mites feeding on bee larvae | By Ecrotek Beekeeping Supplies NZ | Facebook

Since the cappings are porous, maybe forcing high levels of oxygen through the colony from the bottom (like OAV) would create an artificially 'open cap' environment... of course, that would be yet another treatment instead of a solution of breeding better bees.


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> never have I seen so many in one cell...gag... only a couple are slightly pale. I counted 12 on the larvae and one at the bottom of the cell.


That is one mited-up colony!


----------



## CiCi (Dec 5, 2016)

Litsinger said:


> Here is Dr. Seeley's input- I expect this is likely to represent the consensus answer:
> 
> _'...The proof is in colony survival (and low mite counts).'_


Yes, that is what is frustrating, and simultaneously is what's exciting about trailblazing the VSH path... I desire a highlighted roadmap, but there are only vague landmarks with no clear directions so far. We have hints and teases, but in the end there is a clear danger: the slippery slope of S's Bond Theory. I rejected that long ago after debunking it for myself, but find that in some ways, VSH research is pointing a similar path that requires some faith and risk to our livestock's wellbeing. 

Have you run across the 2022 article called, _Give Varroa a Chance!“I Destroy my Enemies When I Make Them my Friends” Aleksandar Uzunov, Cecilia Costa & Marin Kovačić_ https://doi.org/10.1080/0005772X.2022.2113714

They summarize several studies nicely, and discuss Dr. Büchler's belief that high levels of varroa are necessary to reshape bee genetics to combat infestations. It makes sense, but only with tools at our disposal to help along our livestock's genetic pool. One potential solution offered is to only treat mid-season AFTER selecting breeding stock, and definitely don't treat going into winter. 

Its easy to imagine new beekeepers reading the above-quoted article and 'going Bond,' as the VSH breeding path is still being developed in a lot of ways. That's way it is SO important to get an already highlighted roadmap into the hands of small breeders ASAP, so we can sell our VSH queens locally, en mass.


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> They summarize several studies nicely, and discuss Dr. Büchler's belief that high levels of varroa are necessary to reshape bee genetics to combat infestations.


I had not seen this study- this is great. It reminds me of Kirk Webster's ethos:






Feral Bees | Kirk Webster







kirkwebster.com





As the paper alludes to, the key seems to be a laser focus on local-adaptation:

_In short, this holistic concept advocates the use of locally adapted honey bee stock and management of the colonies with limited or absent chemical treatments, thus allowing survival of the fittest genotypes and their identification for further breeding and propagation._

And then there is this, which is very thought-provoking:

_One of the basic conditions in Büchler’s concept is avoiding the winter treatment. This way colonies winter with higher number of mites.

This enables relatively high selection pressure and colonies that possess resistance mechanisms are able to produce more vital drones that are capable to mate with queens. Alternatively, when winter treatments are performed, mites that could create high selection pressure are removed and opportunities for natural selection are diminished, making it almost impossible to breed bees resistant to varroa._

@Snarge- have you been following this thread? Lots of good stuff to consider for your situation IMHO.


----------



## Snarge (May 4, 2015)

Litsinger said:


> I had not seen this study- this is great. It reminds me of Kirk Webster's ethos:
> 
> 
> 
> ...


I hadn’t Russ. Thank you. 

It is being noted in the nick of time, too!


----------



## CiCi (Dec 5, 2016)

Interesting, I hadn't seen that peice before. Thank you for posting.

The below quote from Kirk Webster is an interesting way to kickstart TF in a responsible way, perhaps integrating an outside VSH breeder queen into an operation with otherwise good traits.



> "...here’s the method I have found to be most efficient and effective:
> 
> Even before this new queen has overwintered as a part of your apiary, graft immediately from her and raise at least 30 daughter queens (50 is better, and 100 is probably the best). At the same time raise queens from your other best sources of stock, and get them all mated in the same, best possible situation. Establish the new queens in small nucs to begin with and allow them to grow onto 4-10 combs by the end of warm weather. Everything works better when the entire apiary has been untreated for 3-4 years, but during the transition away from treatments, you can get a good test by making up the nucs initially with brood from colonies that have not been treated for at least 14 months. Overwinter all the nucs in a similar situation as best you can, and in the following spring you will usually have a very good idea of the new stock’s potential for your location.
> 
> The fastest and best way to test a new source of stock coming into your apiary is to raise this series of daughter queens. Raising the new queens in mid-summer and keeping them over the first winter in a nuc gives the fastest, definitive results with the smallest investment of time, equipment and money. A breeder queen with strong potential for the long term will have a really good showing among the daughters after the first winter. The basic strength and resilience of the line is shown by the survival percentage and comparative size of the clusters in early spring."


This method makes sense for the larger operations, as it may be a disaster to just jump on whatever the current bandwagon is and requeen all colonies at once. This way, full production colonies could continue to be treated while the nucs are tested for successful TF breeder queens. I'm not saying this is the way to do it, just saying it's good food for thought.


----------



## Litsinger (Jun 14, 2018)

CiCi said:


> This way, full production colonies could continue to be treated while the nucs are tested for successful TF breeder queens.


I'd say overwintered nucs are the secret of Kirk's operation.

Looks like the theme tonight is that winter is the true TF proving ground.


----------



## Gray Goose (Sep 4, 2018)

from Kirk
"Everything works better when the entire apiary has been untreated for 3-4 years, but during the transition away from treatments, you can get a good test by making up the nucs initially with brood from colonies that have not been treated for* at least 14 months*. Overwinter all the nucs in a similar situation as best you can, and in the following spring you will usually have a very good idea of the new stock’s potential for your location."

CiCi have you actually done this?
I and I believe @GregB have done this and it did not work well.

Brood with mites will hatch and reinfect for a couple cycles, before the so called VSH bees hatch and start their Magic.
good bees can be overwhelmed, IF the initial mite count is high enough.

Greg and I are on the same page, we put "queen to be tested" into clean bees, then the bees roll over to the new genetics with out the over whelm, and stand a chance to see what they truly can do.
never worry here the mites will come.

My experience is every thing works worse with out clean starts.

so to followers of this thread. consider both options.

my experience shows this to not be the best way to test new stock.

GG


----------



## GregB (Dec 26, 2017)

Gray Goose said:


> I and I believe @GregB have done this and it did not work well.


Confirmed.

I have killed a number of promising queens because dropping them into the middle of the mite-pile is a *bad idea.*
It is rather a good idea to provide a *clean start.*

Even proven resistant queens have poor chances if dropped in the middle of a mite-infested colony.
Don't expect them to magically turn things about.


----------



## Gray Goose (Sep 4, 2018)

Greg
I have seen this and did recall you had the same experience.

thanks for the confirm.

GG


----------



## Litsinger (Jun 14, 2018)

Gray Goose said:


> good bees can be overwhelmed, IF the initial mite count is high enough.


Dr. Martin speaks often of this and suggests one must be in a position to exert some genetic influence over the population - search 'halo effect' here on BS.

I think the key with Kirk's advice are bees that have already survived a season w/o treatments. 

If one can't get bees to make it through at least one winter w/o chemical intervention in their locale I'd say Kirk's program would be non sequitur.


----------



## Michael Palmer (Dec 29, 2006)

Litsinger said:


> I'd say overwintered nucs are the secret of Kirk's operation.


I'd say the same. He showed how nucleus colonies were the key for apiary survival and overcome the Tracheal infestation. Following that lead, TM became a minor pest while I was able to avoid the huge losses. Same is now true with varroa...although not as successful. I haven't seen the same results as with TM, but without the mid-summer nuclei I wouldn't be supporting my apiary without buying in bees. Same with Kirk. He loses massive numbers every winter, but can restock his apiaries. 

Same here. For instance, I have two apiaries in a breeding program. Each has 50 nucleus colonies...established in June of 2022...all daughters of the 10 program breeder queens. We run tests on them once the little colonies are strong enough and the worker bees are all daughters of each breeder. The mite counts in August...one apiary had 5 nucs with mite loads above treatment threshold of 2% and the other had 3 nucs with mite counts above 2%. That's 8 nucs of 100. Next summer, I expect many of those nucs will have mite counts well over the TS. But that's how it works...some will maintain low mite counts. Every year I expect more of these colonies to maintain low mite numbers. But, it's just not that easy. Kirk's results mirror this. Many losses every year. You would expect that after so many years as a TF beekeeper working on a serious breeding program, the results would be more encouraging.

What I'm getting at...while you're working on a breeding program to crack the varroa nut, continue to raise mid-summer nuclei as a way to support your apiary. Kirk and I have found it's the only path forward.


----------



## Michael Palmer (Dec 29, 2006)

Litsinger said:


> Looks like the theme tonight is that winter is the true TF proving ground.


Winter...the great selector of good breeding stock.


----------



## Snarge (May 4, 2015)

Michael Palmer said:


> He loses massive numbers every winter…Many losses every year. You would expect that after so many years as a TF beekeeper working on a serious breeding program, the results would be more encouraging.


Michael

It is mind-boggling how this pest has proven itself to be impervious to most of these efforts over a significant period of time.

(I was at the E.A.S conference in Virginia Beach a few years back and had the pleasure of attending one of your lectures. I believe Randy was sitting in the front row. Felt like I was in the presence of royalty).


----------



## CiCi (Dec 5, 2016)

Gray Goose said:


> CiCi have you actually done this?
> I and I believe @GregB have done this and it did not end well


Heck, no! I have no interest in sorting out everything from ground zero. I don't have the bee numbers or time for such a fiasco. Bless those that do!

Before I ever got bees, I read up on MB and followed TF forums closely, then rejected what didn't make sense to me, and kept other elements that did when I got bees. MP and RO became my gurus in the early years, and I still follow RO's trials very closely. EOA and good genetics has keep mites from ever being an issue in my apiary.

As a small local queen PRODUCER (30 full, 10-50 nucs), I'm trying to chart a sustainable path to become a small Q BREEDER. The difference between producer and breeder is planning & caring for the genetic outcome of Qs, IMHO.

I believe a better path for a small Q breeder would be in supporting serious TF breeding programs by buying their queens, then building on that with proven localized stock that preferably has other mite resistant traits. 

Hoping to score a couple AI VSH Q's this spring to open mate with my existing four types of mite mauler stock and two 'mostly' TF survival stock that was ranked the highest in a statewide study. Luckily, my neighbors have my bees already. (I introduced open-mated VSH stock from CA last spring, and they averaged 0-3 mites per 300 with the rest of my stock)

Anyhow, knowing my background might explain why there is appeal in figuring out how to ensure the mite resistance is sustained in local breeder stock over time with tools that don't require tons of ongoing effort, ie, recapping counts, Harbo VSH assay & the potential for UBO spray assay.

If those three tools require higher counts then my current average of 0-3 mites per 300, then perhaps eliminating treatment in nucs containing potential breeder queens but NOT in full production colonies is the way to go. 

Treating once and HARD midseason, if needed, after testing tools have been implemented and breeding is done seems to be a sustainable path that allows fat winter bees and good traits to continue to be traced the following spring. I'm not going Bond. If mite levels are low midseason in the breeder nucs, then hallelujah, that is what we all are looking for, after all.

I'm all for baby steps. Gotta start somewhere.


----------



## CiCi (Dec 5, 2016)

Michael P, I had a question or two about the UBO... 

How quickly after the UBO assay are the cells recapped, or does the solution intensity cause the nurse bees to pull out the larvae?

Have you experimented with the Harbo VSH assay at all? If so, do you think the UBO assay might replace it eventually (price notwithstanding)?


----------



## CiCi (Dec 5, 2016)

CiCi said:


> I'm not going Bond. If mite levels are low midseason in the breeder nucs, then hallelujah, that is what we all are looking for, after all.


I should add, my Q buyers are told that good genetics are only a PART of a successful mite treatment plan, not an alternative to treating.

The danger of discussions about 'TF bees' are new beeks that dont understand that a one-time purchase of a very expensive solitary bug won't exclude them from mite problems. It is not commonly understood how quickly mite resistant traits can be naturally removed from stock.


----------



## ruthiesbees (Aug 27, 2013)

Snarge said:


> Michael
> 
> (I was at the E.A.S conference in Virginia Beach a few years back and had the pleasure of attending one of your lectures. I believe Randy was sitting in the front row. Felt like I was in the presence of royalty).


EAS in Hampton, Virginia 2018 maybe? Both Michael and Randy were at that one. I was there as well since I'm from that area and it was a real treat to hear them speak in person. I had the different observation hive out in the apiary if you wandered out there.


----------



## Snarge (May 4, 2015)

ruthiesbees said:


> EAS in Hampton, Virginia 2018 maybe? Both Michael and Randy were at that one. I was there as well since I'm from that area and it was a real treat to hear them speak in person. I had the different observation hive out in the apiary if you wandered out there.
> 
> View attachment 72001


No kidding Ruthie~we were both there and probably passed right by each other.

What a beautiful observation hive!

Another favorite lecture I attended was by Jamie Ellis on “the colony as a super-organism”.


----------



## Michael Palmer (Dec 29, 2006)

CiCi said:


> Michael P, I had a question or two about the UBO...
> 
> How quickly after the UBO assay are the cells recapped, or does the solution intensity cause the nurse bees to pull out the larvae?
> 
> Have you experimented with the Harbo VSH assay at all? If so, do you think the UBO assay might replace it eventually (price notwithstanding)?


I haven't looked at recapping so I can't answer. As the treated comb is placed back in the hive for only two hours, it's not possible to see recapping. Also didn't see any pupae pulled out. We didn't go back to check any of this. Keira has promised to look into recapping. 

No, I haven't done the Harbo assay. It is quite possible that the UBO assay will be sensitive enough so the time consuming Harbo assay is necessary. Time will tell.


----------



## AR1 (Feb 5, 2017)

CiCi said:


> View attachment 71957


Interesting that the tolerant populations seem to more-accurately target actually infested cells and more ignore uninfested cells. This suggests two avenues that the bees are using. One is recapping behavior itself, either it is present or not present, like an on-off switch, or maybe on-off with a dimmer switch. The second is detecting mites in the cells, or anyway in infected brood.

The naïve bees do almost no recapping. The more-experienced but susceptible populations do more recapping, but it is less targeted, less efficient, while the tolerant populations do lots more recapping, targeting infested cells. 

The African sub-species do the most recapping. Not surprising given the severe selection they faced when mites first hit. They were never restocked for decades with naïve bees from breeders. Also interesting is that the capensis bees do little recapping, and very inefficient recapping of drone cells. Are they practicing their own version of Darwinian black box Bond on just the drones?


----------



## AR1 (Feb 5, 2017)

GregB said:


> Confirmed.
> 
> I have killed a number of promising queens because dropping them into the middle of the mite-pile is a *bad idea.*
> It is rather a good idea to provide a *clean start.*
> ...


Also, don't put a badly infested hive near a clean hive. Done that.


----------



## Adam Foster Collins (Nov 4, 2009)

Michael Palmer said:


> ...Also didn't see any pupae pulled out....


A Reminder Mike: Bianca's got video of bees removing pupae at Rainville during the second UBO test. That's just at the 2 hour point, so it's likely they remove more than we saw. I think we saw removal happening in two colonies at the 2 hour check at Rainville. 

None that I remember at the first yard.

Adam


----------



## Michael Palmer (Dec 29, 2006)

Thanks Adam. I didn't remember seeing that.


----------



## CiCi (Dec 5, 2016)

I appreciate your comment, Adam. Makes me wonder about % dilution for UBO spray, on minimal amount to keep effectivity. Guess that doesn't matter for me, as I'm not testing it. Still, my mental wheels turn regardless.

Hmmm.... if the mite population is low in an apiary, I wonder if drones could be used for the recapping (size difference of uncapped area) or the Harbo VSH assay. I suppose with the UBO assay, it wouldn't matter which brood type was used, if an average of 45 cells was sprayed (as per MP's earlier image of the UBO uncapping test).

Now that I'm thinking about it, I seem to remember Cory S saying something about resorting to testing drone brood when mites were scarce... wish I recall specifics better. Someone else in my readings said drone brood was unreliable for mite scoring, but perhaps that is when a breeder is still trying to narrow down the desirable traits, not when those traits are just being maintained.


----------



## crofter (May 5, 2011)

Forgive me if it has been covered. Does the UBO spray kill the cells it is applied to? and do any of them get recapped?


----------



## ruthiesbees (Aug 27, 2013)

crofter said:


> Forgive me if it has been covered. Does the UBO spray kill the cells it is applied to? and do any of them get recapped?


My understanding from Dr. Wagoner's lectures is that the spray only mimics the unhealthy brood odor, but does not in any way kill the larvae underneath the cappings. So the hygienic workers that think there is a "problem" and go investigate by uncapping the cells in that area would find no issue and at some point recap the cells. (I have not heard her speak about if she is observing that recapping after the UBO test. We will hear another lecture from her in January and I hope we can ask her that)


----------



## ruthiesbees (Aug 27, 2013)

CiCi said:


> I appreciate your comment, Adam. Makes me wonder about % dilution for UBO spray, on minimal amount to keep effectivity. Guess that doesn't matter for me, as I'm not testing it. Still, my mental wheels turn regardless.
> 
> Hmmm.... if the mite population is low in an apiary, I wonder if drones could be used for the recapping (size difference of uncapped area) or the Harbo VSH assay. I suppose with the UBO assay, it wouldn't matter which brood type was used, if an average of 45 cells was sprayed (as per MP's earlier image of the UBO uncapping test).
> 
> Now that I'm thinking about it, I seem to remember Cory S saying something about resorting to testing drone brood when mites were scarce... wish I recall specifics better. Someone else in my readings said drone brood was unreliable for mite scoring, but perhaps that is when a breeder is still trying to narrow down the desirable traits, not when those traits are just being maintained.


I'm not sure if you have heard yet about the 2023 study that Dr. Zac Lamas out of the University of Maryland is doing with Virginia Tech Extension Dr. J. Wilson but it will be sampling immature drones on the comb (so drones that have not yet flown outside the hive) to measure their varroa levels. Zac is proposing that varroa preferentially feed on drone larvae as well as adult drones in the hive and that we might be better to sample as a measure in the hive vs. nurse bees from the brood nest. This is an excerpt from one of the Virginia Club's pages:
𝐂𝐢𝐭𝐢𝐳𝐞𝐧 𝐒𝐜𝐢𝐞𝐧𝐜𝐞: 𝐏𝐫𝐞𝐝𝐢𝐜𝐭𝐢𝐧𝐠 𝐕𝐚𝐫𝐫𝐨𝐚 𝐈𝐧𝐟𝐞𝐬𝐭𝐚𝐭𝐢𝐨𝐧𝐬 𝐢𝐧 𝐕𝐢𝐫𝐠𝐢𝐧𝐢𝐚
Dr. Zac Lamas and Dr. James Wilson are working together and with Virginia beekeepers in 2023 to see if a new sampling methodology can predict mite infestations months before they actually occur. The project is simple, easily adoptable, and can be done by beekeepers of all skill levels. They need hobbyist beekeepers across Virginia to join in.
More information and a signup form below






















Varroa is the leading cause of death of honey bee colonies. Despite years of research there is much we still don't know about mites. New findings from the University of Maryland and USDA-ARS suggest an alternative sampling method may allow beekeepers to detect Varroa earlier than current sampling methods, nor does the new method require beekeepers to sacrifice handfuls of worker bees in the process. But we need your help!
Here is your chance to be a scientist! Beekeepers across Virginia are invited to join a citizen science project in 2023. Your participation will provide valuable data. We want to know if our new method works in Virginia, and if it in fact, can predict fall mite infestations months beforehand.
You do not need to provide much to participate. All you need is to sample a few of your own colonies each month, and then submit the results on a monthly questionnaire. Our team at Virginia Tech will provide instruction material, monthly zoom sessions, and Q&A support via e-mail. google doc link Predicting Mite Infestations 2023


----------



## CiCi (Dec 5, 2016)

crofter said:


> Forgive me if it has been covered. Does the UBO spray kill the cells it is applied to? and do any of them get recapped?


On post #49, Adam was saying that larvae were observed being pulled out on some UBO test frames... so, while the spray is designed to not kill the brood (testing for VSH vs general hygienic behavior), the nurse bees may end up killing them anyways after uncapping the sprayed cells.

One of the reasons this interests me is that at least two studies that discuss recapping suggest that uncapping/recapping is potentially a shared mechanism of different types of Varroa resistance. In other words, there might be different genetics in play that end up with the same uncap/recap response. Might the intensity of response to the Brood Odor (pulling larvae out vs disrupting mite mating cycle with temporary large uncapping) be a genetic difference, or simply the dilution percentage for the UBO spray? An answer will probably become clear with time, and widespread consumer availability of the UBO spray.


----------



## CiCi (Dec 5, 2016)

ruthiesbees said:


> sampling immature drones on the comb (so drones that have not yet flown outside the hive) to measure their varroa levels.


Thank you for sharing. I was unaware of this upcoming study. I'll have to search those names for past mite studies with drones.

Lots of beeks break open capped drone larvae cells to check for mites as an informal check, but I'm confused about how testing immature drones for mite levels will be beneficial and easy to do. Unless feeding wounds are detectable with the naked eye? Perhaps I just have that gag-worthy FB video still on my mind, with over a dozen dark mites in one drone cell. 

I wish them luck and hope their new sampling method is a success.


----------



## Michael Palmer (Dec 29, 2006)

CiCi said:


> Lots of beeks break open capped drone larvae cells to check for mites as an informal check, but I'm confused about how testing immature drones for mite levels will be beneficial and easy to do.


Zac began his sampling of mites on young drones in several of my apiaries last summer. His theory is that early season mite counts can be flawed as we usually sample worker bees from the broodnest. He says in the early season, mites are on young drones, <3 days old. For instance, last April, we washed bees in a sampling of colonies...enough to get a handle on what's going on. 
We found zero mites in every wash. By late May/early June the mite counts were over threshold. Some significantly.


----------



## CiCi (Dec 5, 2016)

I would give my last top bar to get a copy of Zac's instructions to his study participants. I'm curious about what he decided the protocol should be for sampling drones... surely you couldn't scoop 1/2 cup drones as easily as nurse bees... Hmm. Perhaps knock off a couple of drone frames in a bin, then cover that bin with a queen excluder so workers could escape (maybe flipping bin over w/ excluder so any nurse bees can crawl down into colony), then sample captive drones at your leisure. Alcohol/soap wash or sugar roll as usual, maybe 3/4 cup due to the larger size of the drones? That would be straightforward enough...

Sorry, I think we're officially off-topic.


----------



## ruthiesbees (Aug 27, 2013)

When I heard Dr. Lamas speak at the Virginia state meeting in Nov, he mentioned that we would be doing a regular alcohol wash with nurse bees, reporting those numbers and than plucking off a fixed number of drone bees on the next frame over in the brood nest (not the ones on the outside frames). My notes don't say if we will be putting them in a new wash of alcohol to measure the mite load or if it will be a visual look. The one he had a photo of in his presentation was really covered with mites. I also know that he will be taking 5 frames of capped drone brood from our coastal Virginia bee clubs in early March to test the capped drone larvae for mites. I have his gmail address and I will private message that to CiCi. Just send him an email to see if he has something that he can send to you, or even to put you on the list of "participants".


----------



## CiCi (Dec 5, 2016)

Thank you, Ruth.


----------



## Michael Palmer (Dec 29, 2006)

CiCi said:


> I'm curious about what he decided the protocol should be for sampling drones...


While Zac was sampling drones last summer, he claimed that very young drones, <3 days old if I remember correctly, buzz differently when you pick them up. Zac and his parents spent days and days searching for young drones.


----------



## Adam Foster Collins (Nov 4, 2009)

CiCi said:


> ...Makes me wonder about % dilution for UBO spray, on minimal amount to keep effectivity...


I have no idea on that. As I understand it, the fluid comes ready to apply. We do not mix it or have specific information on percentages of different constituents.

Adam


----------



## CiCi (Dec 5, 2016)

Michael Palmer said:


> very young drones, <3 days old if I remember correctly, buzz differently when you pick them up


I was practicing eversion this summer, and noticed the different tenors, but never associated it with age. Little nuggets like this just makes my evening! Who'd a thunk voice changes happen to adolescent drones, too?


----------



## ruthiesbees (Aug 27, 2013)

Here is the latest interview with Kaira about the UBO assay


----------



## Litsinger (Jun 14, 2018)

ruthiesbees said:


> Here is the latest interview with Kaira about the UBO assay


Great update, @ruthiesbees. A handful of things I found interesting:

1. UBO is not varroa-specific. This might help explain why hygienic behavior is helpful against a range of brood diseases.

2. UBO is present on all bees and the response to elevated UBO is context-dependent.

3. Uncapping/recapping and hygienic behavior are related but not analogous. 

4. The freeze-killed brood assay is not selecting for hygienic behavior but rather for necrophoresis.

5. Hygiene performers have higher virus loads, but the colony as a whole have lower virus loads.

6. Maternal effects are most important to hygienic behavior to daughter queens, but paternal effects also contribute.


----------



## Michael Palmer (Dec 29, 2006)

Litsinger said:


> Great update, @ruthiesbees. A handful of things I found interesting:
> 
> 1. UBO is not varroa-specific. This might help explain why hygienic behavior is helpful against a range of brood diseases.
> 
> ...


----------



## Litsinger (Jun 14, 2018)

Thanks, Michael. Your operation and some of the testing are mentioned in passing near the end of the video.

On the hygiene bit- I understood Dr. Wagner to be making a further distinction between hygienic behavior that is elicited by dead brood and that which is elicited by unhealthy brood. She spends quite a bit of time describing how while these two factors are related, they are not the same- and that colonies can express high responses to one and not necessarily the other.

So to my mind, she would be suggesting there might be at least 3 hygienic behaviors: VSH, necrophoresis, and UBO.


----------



## Michael Palmer (Dec 29, 2006)

Litsinger said:


> So to my mind, she would be suggesting there might be at least 3 hygienic behaviors: VSH, necrophoresis, and UBO.


*Interesting. I would need more information on this before I could have an opinion. From what I've seen, I'm thinking VSH and necrophoresis are the same thing...removing dead, capped brood. I look forward to additional information on this coming from Kaira.*


----------



## Litsinger (Jun 14, 2018)

Michael Palmer said:


> From what I've seen, I'm thinking VSH and necrophoresis are the same thing...removing dead, capped brood.


Here is a good treatment from Mondet et al. They have suggested resurrecting the term SMR to reflect brood factors associated with resistance:


----------



## Michael Palmer (Dec 29, 2006)

Michael Palmer said:


> *Interesting. I would need more information on this before I could have an opinion. From what I've seen, I'm thinking VSH and necrophoresis are the same thing...removing dead, capped brood. I look forward to additional information on this coming from Kaira.*


Sorry I typed this wrong...I was trying to say that VSH and necrophoresis are NOT the same thing. Hygienic response is the bees alerting to dead brood...necrophoresis


----------



## Litsinger (Jun 14, 2018)

Michael Palmer said:


> I was trying to say that VSH and necrophoresis are NOT the same thing.


I'd go one step further and suggest that the Harbo Assay is in truth an assay for MNR rather than VSH in it's strictest sense- which might be the result of uncapping/recapping, brood factors that delay oviposition, or other factors we've not yet even identified.


----------



## Litsinger (Jun 14, 2018)

In Cory's presentation, Dr. Kaira Wagoner mentioned an upcoming presentation on Inside the Hive TV, which was posted yesterday:






While there is a lot of good stuff in there, I was struck by one thing in particular- namely the clue that helped her stumble upon UBO:










She also notes that a + 60% UBO response was a good approximate selection threshold.


----------

