eHarmony go around

About me

First thing – I’m a honey bee researcher, writer, and invited speaker.  If you’ve got an issue with honey bees, I’m unfortunately not for you.

I was a devoted and loving husband to my long-time ex-wife.  She was younger than me, but aged more quickly, and as her energy wound down she became more and more withdrawn and introverted.  Wanting a quieter and more isolated life, she decided that that she’d be happier following her mother’s footsteps, and live alone.  So I helped her buy a house, and we separated last June, remaining close and amicable friends.  I’m finally getting over my crying and heartbreak (since I was very much in love with her) and with her encouragement, am looking for a new partner and soul mate with whom to share a new life together.

My previous relationships have been based upon love, attractiveness, and intimacy, but after signing up for eharmony as an experiment, I realize that I’d be wise to find someone whose energy and personality traits match mine.  I’m a likeable guy, so I’m not desperate or trying to sell myself, seduce or mislead you, so I will be honest and straightforward.  I do not want to waste your time or mine.

I’m a high-energy, high IQ, scientifically-minded guy, biologically about 10 years younger than my chronological age, lean and very active, and I work nonstop — so am not the right partner for a low self-esteem or low-energy lady.  Although I’m an intense and focused hard worker, I’m also very humorous, laughing and joking all the time.

My friends would describe me as the busiest guy they know, and the guy who gets things done. The answer man to any question. Helpful, loving, sharing. Widely loved by many.

My joy is sharing with others – I’ve always been a teacher and giver.

Disclaimer: My eharmony photo is from 2018 (so I’ve now got a few more wrinkles), but it best captures my essence.  Here are a few more (all but the first two are recent):

I’m likeable, happy, and appreciated by others.  I’ve got no issues or traumas, and am responsible and caring.  I exemplify the Scout Laws (I’m the inverse of our ex-president).  You will never hear an untruth cross my lips.

I’m an ex hippy child of the ’60s, now a responsible, environmental, fiscally-conservative, socially-liberal adult.  I was raised to honor and respect women, and have no desire to dominate a relationship.

I’ve got many skills, and am highly competent, capable, and accomplished.  I’m always happy, free of stress, loving, generous, and looking for a soul mate and partner to share the everyday joys of life with — like a new flower blooming, or the taste of the first fruits from my orchard, or a beautiful sunset.

I live on a dream property with garden, orchard, and a view, am in good health, and financially stable.  I’ve handed my sons our beekeeping business, so am now free to follow my passion of performing honey bee research and providing scientific information to beekeepers worldwide (at ScientificBeekeeping.com).

I lead a simple, frugal, rustic life – nothing fancy (other than traveling to speak).  I’m early to bed, early to rise.  In my office before sunrise, cranking data, writing, email correspondence, and reading scientific papers.  After breakfast, generally working outside for the rest of the day.  Other than the news, watch very little TV.  Back in for dinner, which I enjoy cooking.  Enjoy watching a movie together afterwards.

I’m home or nearby most days, and don’t go to town much, but do travel to speaking engagements, which I try to limit to no more than once a month.

I respect and honor women, and have worked with ladies most of my life.  I’m well liked and loved – any of my ex’s or female coworkers will give me a thumbs up.

The partner I’m looking for

I’m not the right person for someone who is intimidated by intelligence, or put off by me being so engaged in my research, writing, shop work, and property maintenance.  I can also be kinda messy, since I always have so many projects in the works.

As far as a partner, I’d prefer an energetic, physically active, preferably lean, and affectionate woman who enjoys a simple rustic, eco-friendly lifestyle.  You’d need to be OK with being around honey bees.  I’m more of a cat person than a dog person.  I follow the Golden Rule, but have no interest in organized religion.

As far as relationship, since I’m a brainy, strong guy, I want an equally strong, self-assured partner who can stand up for herself, and communicate and articulate her feelings and wants.  I don’t argue or criticize, and have zero desire to dominate or always get my way.

Although I love doing things together, I need a partner who has her own interests and hobbies to happily entertain herself while I’m working on my own projects.  I’m far more interested in an active tomboy in dirty jeans than a beauty queen in high heels.  I want a gal who doesn’t mind getting dirt on her hands, and will enjoy working in the garden and orchard with me.

I ain’t perfect, and don’t expect you to be either.  I want my partner to accept me in full as I am, and I will do the same in return.  I don’t want to merely love my partner, I want to be in love with her every moment of my life.

Thymol — A new application method? Part 2

Contents

Extended-release thymol 1

The optimal release rate and placement of thymol 1

What’s The Optimal Dose?. 2

Our Field Testing. 3

The Dose-Response Curve. 12

Discussion. 12

Citations and notes 13

Thymol — A new application method?

Part 2

First published in ABJ in December 2022

Randy Oliver
ScientificBeekeeping.com

I do a lot of experimentation in beekeeping, often triggered by questions or ideas from other beekeepers.   This spring a beekeeper showed me a homemade thymol concoction, which reminded me to pick back up on some previous thymol research of my own. 

Extended-release thymol

Last month I raised the question of whether we could use a ½” thick cellulose matrix (Homasote [[1]]) in order to throttle the evaporation rate of a thymol treatment over a longer period of time, thus allowing us obtain efficacious mite reduction with only a single application.

A scientific note:  Before investing time in large-scale controlled trials, I generally first perform some preliminary “quick and dirty” experimentation, as detailed here [[2]].  Not needing to follow a rigid protocol, this opens the door for ad hoc experimentation and unanticipated discoveries, which may then lead me in new directions.

The optimal release rate and placement of thymol

I’ve mentioned that bees don’t care for the odor of thymol, and if a treatment is placed above the cluster, they may build a wall of propolis around it (Figure 1).

Fig. 1. If the bees are unable to remove a substrate with a strong odor of propolis, they will attempt to cover or wall it off with a layer of propolis.  Of interest, I did not observe any colony doing this with the Homasote blocks.

Practical note: A strong thymol treatment applied within the cluster can be very disruptive to the colony, causing the bees to noisily attempt to fan the odor away, uncap and remove brood (with lots of white pupae showing up on the bottom board), or in the worst case, the colony may completely abscond from the hive. 

I found that it’s not just the dose, but rather the amount of surface area from which the thymol (with its low vapor pressure) is able to evaporate.  Thus, application by thymol-saturated shop towels, with their large surface area relative to volume, results in too rapid release of the thymol (especially when applied between the brood chambers, due to the bees intensely fanning air over the towel surfaces).

Thymol on a shop towel would be a “flash” treatment.  On the other hand, the registered thymol formulated products apply thymol in an extended-release manner.

Practical application: But even those products require at least two applications.  So I wondered whether in order to save the labor involved in reapplication, is there a way to prolong the release of a larger dose?

What’s The Optimal Dose?

Thymol can be applied as a flash treatment to broodless colonies, with most mites dropping within a few hours of application.  Chiesa [[3]] sprinkled 1 gram of powdered thymol over the tops bars of broodless colonies, four times, at 2-day intervals, and obtained over 95% mite kill, especially if the powdered thymol was mixed with powdered sugar.  So 4 g of thymol appears to be enough to kill the mites in a colony not containing brood.

But when colonies contain brood, it apparently takes a larger dose, delivered over a period of weeks rather than days.  Let’s take a look at the number of grams of thymol per dose of the registered products (the red numbers in Table 1).

It looks as though a colony with brood can handle 6-12 grams of slow-release thymol per week. Since I’d already determined that 5.1 square inches of Homasote will hold 12 g of thymol (after absorbing a saturated solution of thymol in ethanol), it was a simple matter to precut blocks of Homasote to hold any dose of thymol that I wanted to test. I decided to apply four blocks of Homasote per hive, so I calculated how large to cut each block to attain each desired dose per hive (Table 2).

Our Field Testing

It was late August when we started this project, so unfortunately when I asked my sons as to the availability of colonies with high mite counts in which I could test these experimental thymol treatments, they said that all their 1500 colonies had the mites well under control, with mite wash counts (per half cup of bees) in the 0-2 range.  This was good for them, but bad for me.

So the only colonies with high enough mite levels to use for testing were the hives left in three yards in which Rose and I had previously tested formic and oxalic treatments, some of which still had measureable mite infestations.  So we started testing to see how the colonies reacted, in hot weather, to having a 12-gram dose of thymol placed right between the brood chambers (Figure 2).

Fig. 2. My assistant Rose Pasetes (wearing proper protective gear against the sun) showing the placement of four blocks (this photo is of a moderate dose).  The bees initially moved away from the fumes, so it’s not obvious in this photo that the hive was full of bees and brood.

We applied the 12-g dose to six hives.  To our great surprise, within a day the bees acted as though the thymol wasn’t even there.  We could smell the fumes when we opened the hives, but there was no fanning, and no apparent effect upon broodrearing (Figure 3).

Fig. 3.  Again, a higher dose, but showing the lack of disturbance to the bees in this freshly-opened hive.

Surprised and emboldened by the lack of bee response to the 12-g dose, we applied 24-g doses to seven colonies, some with the blocks applied between the brood chambers, some in a rim above the second brood chamber (Figure 4).

Fig. 4.  Typical placement of thymol blocks in a rim.

I opened the colonies regularly to inspect for the sort of brood disruption I was accustomed to seeing during a thymol treatment, but even with the 24-gram dose I didn’t see anywhere as much as I expected.  So what the heck — we made up some more blocks in order to apply 36-g doses to seven additional colonies.

During this ongoing experimentation, I was able to observe what the bees did as the intensity of the thymol fumes wore off in the first hives.  As I’d noted in my experiments in 2017, most colonies began chewing away at the blocks, rather than propolizing them over (Figures 5 & 6).

Fig. 5.  The blocks still smell of thymol after three weeks, but the bees don’t appear to be bothered by it by that time.  Once the thymol fume intensity diminished, the bees in most of the hives started chewing away at the blocks.  There were of course large colony-to-colony differences in the amounts of propolization and chewing removal, but this photo is typical.

Fig. 6. Note how where evaporation was inhibited by the blocks being squeezed between the top and bottom bars, that there was chewing only on the edges.

What surprised me most about this experiment was how little the colonies appeared to be bothered by these high-dose treatments.  There was no noticeable fanning, and after an initial suppression of the rearing of young larvae, most colonies quickly resumed the rearing of all stages of brood in the center frames — no matter whether the blocks were placed between or above the brood chambers.

I was even bold enough to apply 48 grams of thymol, in a rim, applying the treatment to a strong double on 27 July, with temperatures often in the high 90sF (Figures 7 & 8).

Fig. 7.  Photo on 17 August — 21 days after application of four 12-gram thymol blocks — of healthy brood of all ages in the lower brood chamber in the heat of summer drought. The solid sealed brood indicated that the larvae had been started and reared during the most intense period of thymol evaporation. I could hardly believe my eyes!  The mite wash count had gone from 14 mites to zero.

Fig. 8.  Same colony on Oct 9, 74 days after application of blocks (total 48g of thymol), which by then had no noticeable thymol odor.  The mite wash count was still zero, with the colony containing plenty of good-looking brood and honey.

Surprised and emboldened by how well this colony did with 48 grams of thymol placed in a rim (and needing more data for a dose-response curve), in late September I identified two (unfortunately) weak colonies with measureable mite counts, and placed four 12-g blocks between the brood chambers (Figure 9). Weather was in the 70sF, but soon rose again to the 90s.  Unfortunately the nectar and pollen flows were over, and there was little broodrearing taking place before or during this intense treatment, but I wanted to see (1) whether a 48-gram dose would zero out the mite counts as it did with the colony above and (2) to see how weak colonies would respond to this extremely high dosage of thymol.

Fig. 9. A weak colony four days after application of four 12-g thymol blocks late in the season (an intentionally intense test).  Due to dearth there wasn’t much brood when I applied the blocks, but even after four days of this intense exposure, there were still patches of brood of all ages on three frames in the upper box.  Other than their immediate buzz when I had first applied the strips, the bees did not appear to be disrupted by the obvious odor of thymol – the bees appeared to be acting “normally.”

I’m not about to say that there was no adverse effect from this strong dose upon the colonies, but I was surprised by how the bees appeared to quickly acclimate to the odor of thymol in the hive (it was easy for me to smell when I opened them).  These two weak 48-gram colonies continued to rear some brood of all ages throughout the treatment, and their mite wash counts had dropped to zero by 17 days after application (one going from a count of 44 mites to zero).

The Dose-Response Curve

I was only able to find 23 colonies with mite counts high enough on which to test this method of thymol application (having starting mite wash counts ranging from 5 to 44; mean 20, median 18), so this data is only preliminary and suggestive.  In order to estimate a dose-response curve, I calculated percent reduction of the counts vs. the applied dose of thymol, so that I could get an idea as to the optimal dose to test next season (Figure 10).

Fig. 10.  The higher the data points, the better the mite reduction — 100% indicates and ending count of zero.  A 12-gram dose (divided between 4 blocks) gave inconsistent mite reduction, but 48 grams zeroed the counts in all three hives.  The less-intense 36-gram dose may be the optimal target dose, but it’s not yet clear whether it is best applied between the brood chambers or in a rim above (I tested both).

Discussion

As a long-time user of Apiguard, I’m pretty stoked by the convenience of this application method of thymol in Homasote blocks.  I was dumbfounded by how gentle the 12- and 24-gram doses applied in this manner were on the bees and brood, and pleasantly surprised by how well the colonies handled the 36- and 48-gram doses.  I plan on obtaining a permit to expand my testing of this method next season.

Practical application: An easy-to-apply thymol treatment that requires only a single application would be great to use in rotation with oxalic and formic acid.

Note:  This application method is not approved in the U.S, but could be used by beekeepers in New Zealand or other countries where it is legal.

Addition:  Checking the Safety Data Sheet  for Homasote, I find that it contains a tenth of a percent (1000 ppm) copper metaborate (likely to inhibit mold and termites).

So this raises the question as to whether that amount of copper metaborate is of concern as far as adverse effects upon the bees, or contamination of honey (unlikely, since we don’t normally apply thymol treatments during a honey flow).

The EPA concluded in 2009 that “Available data from a honey bee acute toxicity study indicated that copper is practically nontoxic to honey bees.” (https://www3.epa.gov/pesticides/chem_search/reg_actions/reregistration/red_G-26_26-May-09.pdf), apparently based upon a single study:

Hoxter, K. (1988) Copper Hydroxide: An Acute Contact Toxicity Study with the Honey Bee: Wildlife International Ltd. Project No. 190- 140. Unpublished study prepared by Wildlife International Ltd. 14 p.

A point to keep in mind is that it’s unlikely that a bee would consume any of the Homasote.  I did not observe any indication of adverse effects after treatment, but will run some cage trials.

Hladun, K.R., Di, N., Liu, T.-X. and Trumble, J.T. (2016), Metal contaminant accumulation in the hive: Consequences for whole-colony health and brood production in the honey bee (Apis mellifera L.). Environ Toxicol Chem, 35: 322-329. https://doi.org/10.1002/etc.3273

Citations and notes

[1] http://www.homasote.com/assets/files/homasote-440-sds.pdf

[2] For this off-label experimentation with thymol, I obtained a Pesticide Research Authorization from the California Department of Pesticide Regulation, since unlike with my previous research that did not involve using thymol as a miticide, in this case I was using it for that purpose.

[3] Chiesa, F & M D’agaro (1991) Effective control of varroatosis using powdered thymol. Apidologie 22(2): 135-145.

Thymol — a New Application Method? Part 1

Contents

“Off-Label” use of thymol 4

Thymol products for varroa control 5

Understanding Thymol 6

Reaction to the delivery matrix by the bees 6

Thymol in Shop Towels. 7

Removal and dispersion of thymol matrices by the Bees 9

Extended-Release Thymol?. 11

A Thicker Matrix. 11

moving ahead to 2022. 15

Citations and notes 15

Thymol — a New Application Method?

Part 1

First published in ABJ in November 2022

Randy Oliver
ScientificBeekeeping.com

As our summers get hotter, some of the “natural” treatments against varroa have issues, since the adverse effects of both formic acid and thymol increase at high temperatures. Thus I’ve been experimenting with reducing and extending the release rate of oxalic acid and formic acid. But how about thymol — could we extend its release rate too? This summer I resumed some research that I’d initiated back in 2017.

Thymol is a well-proven “natural” miticide, fairly well tolerated by the bees. My sons and I use thymol in rotation with oxalic acid and formic acid, with each miticide being appropriate for use during different “treatment windows” (Figure 1).

Fig. 1. I created the above generic chart (adjust the dates for your area) to point out the best treatment windows for varroa management, as well as the most appropriate active ingredients to apply during those windows. We’ve found that in the California climate, it typically requires at least four treatments per season to maintain low varroa levels (in non-resistant stock). To avoid the evolution of miticide-resistant varroa, we all must rotate different active ingredients.

Thymol, due to its temporary suppression of broodrearing, is appropriate for use if you can get harvestable honey off the hive before mid-August. It can also be used to both control mites and suppress colony buildup prior to swarm season.

In my state of California, there is only a single registered thymol product — Apiguard, with specific instructions as to how it can be applied. We’ve been happy with Apiguard, but that’s not to say that there might not be a more bee- and beekeeper-friendly way to apply this proven miticide. Some limitations of Apiguard are that:

1. A space must be provided below the hive cover, which means that for those of us who use migratory covers, a spacer rim of some sort must be provided (Figure 2).

Fig. 2. The only thymol product approved for use in California is Apiguard “gel,” here applied in a spacer rim. My sons and I have successfully used it for many years in rotation with other treatments, mainly during hot weather in August, when we don’t mind its temporary suppression of broodrearing during our dearth period.

2. For full efficacy, the treatment must be repeated, which takes extra labor. We’d much prefer a treatment that requires only a single application.
3. The label no longer allows for placing the product between the brood chambers, which would be important for application during cool weather.
4. Application above the brood chamber is only efficacious if the colony is strong enough to remove the treatment from the card above the top bars (my own observation). A treatment that could be applied between the brood chambers would be of great benefit.
5. Although Apiguard can be applied at daily highs of up to 105°F, our summers are getting warmer (Figure 3), and during our optimal treatment window in August this year, it got even hotter than that.

Fig. 3. The blue trendline reflects the increase in California’s average August high temperature over my lifetime. It’s not just that I’m getting older — it really is getting hotter! You can plot similar charts for your area at https://www.ncei.noaa.gov/access/monitoring/climate-at-a-glance/statewide/time-series.

6. Perhaps most importantly, when we apply Apiguard as per the label, the broodnest is greatly impacted — the vapor kills the eggs and young larvae directly below the treatment (which the nurses then cannibalize), and then the colony reestablishes a divided and reduced broodnest to either side of the treatment.

Practical application: Apiguard is a great product, but I suspect that there could be even better application methods appropriate for our conditions and colony management. But we beekeepers are legally precluded from tweaking the application methods specified on the label, or from using generic thymol to apply by different methods.

“Off-Label” use of thymol

Practical application: Varroa treatments are registered as pesticides. When a manufacturer goes through the tortuous and expensive process of registering a pesticide, they must provide a specific set of approved application instructions. Any deviation from those instructions is a violation of the law.

The situation is more reasonable with medicines: “From the FDA perspective, once the FDA approves a drug, healthcare providers generally may prescribe the drug for an unapproved use when they judge that it is medically appropriate for their patient” [[1]]. This is referred to as “off label” use, and is very common in medicine.

Doctors and their patients are allowed to experiment and learn about how best to use medicinal drugs under different circumstances, and for not-yet-registered uses. Unfortunately, this freedom does not apply to registered miticides, so we beekeepers (or even our veterinarians) are not allowed to tweak application methods to account for the wide range of situations that we encounter in beekeeping.

EPA’s refusal to consider granting us beekeepers an Own Use Exemption for the organic acids and thymol frustrates me, since it prevents us from learning how to apply these safe and inexpensive generic substances to our colonies, instead forcing us to purchase and follow the rigid label instructions for expensive registered formulated products.

Here’s the rub: As explained by USDA [[2]]: “Relative to miticide uses on agricultural crops, applications of varroacides to bee colonies are considered a minor use that may not allow for registrants to justify the costs to develop varroacides and break-even on such investments, given the small relative market potential.”  So we can’t expect manufacturers to bring a bunch of customized varroa control products to market.

A plea to our national organizations: Registrants are reluctant to go through the tedious process of updating their labels, so we beekeepers are stuck between a rock and a hard place. As far as EPA’s mandate to avoid unreasonable risk to man or the environment, beekeeper use of generic off-the-shelf formic, oxalic, or thymol would pose no such risks. The government of New Zealand wisely recognized this, and gave beekeepers an exemption to use these “natural” products for varroa control however they wish. I suggest that our national organizations approach our senators and congressional representatives to legislate an Own Use Exemption similar to New Zealand’s.

The reality is that due to our limited arsenal of approved formulated varroa-control products, many beekeepers have unfortunately become “pesticide scofflaws.” I gotta walk a fine line with my research, which I do for the benefit of all, rather than for profit. I obtain Pesticide Research Authorizations from my State’s department of pesticide regulation, and publish my findings as scientific research only — I am not in any way suggesting that beekeepers use any unregistered product, or not follow label instructions exactly (I gotta cover my donkey).

Thymol products for varroa control

There are three main thymol products registered in North America for varroa control — Apiguard, ApiLife Var, and Thymovar. Only two are registered in the U.S., and only Apiguard in California. All work by the evaporation of thymol vapors from an inert matrix, and call for placing the treatment a few inches from the brood. Thymol is generally recommended for application during moderate ambient temperatures — typically between 20-25°C (~68-77°F), although some product labels allow application at higher temperatures. All specify not to apply while honey for harvest is being produced.

One widely-used product is ApiLife Var, which is a mixture of thymol, eucalyptol, menthol, and camphor, impregnated into a vermiculite wafer. I tried ApiLife when it was first released in the U.S. (read my report at [[3]]), and found that it was more irritating to the bees than straight thymol, likely due to the additional essential oils. But it may be this additional irritation to the bees — and mites — that gives it greater efficacy at low temperatures, or promotes its distribution throughout the cluster.

Thymovar, also registered in a number of countries, is a cellulose sponge strip impregnated with straight thymol crystals.

Apiguard uses a gel-like matrix of water and polyacrylate (such as is used in diapers) to suspend fine crystals of thymol, which slows the evaporation rate. In my experience, its efficacy is dependent upon the bees physically carrying away the particles, rather than simple evaporation.

I’m also aware that a number of beekeepers mix up their own “homebrew” thymol formulations, sometimes with the addition of other essential oils. I’ve tested some of these homebrews (which can be efficacious), but am most interested in straight thymol.

Understanding Thymol

Thymol appears to affect the mites by exposure to its vapor. It not only causes direct toxicity, but may also irritate them, or perhaps affect their critical sense of smell. In addition, thymol vapors can cause bees to engage in more intense cleaning behavior, or even brood removal. But it’s clear that thymol has a far greater adverse effect upon varroa than it does upon the bees (especially adult workers, who “get used to it”).

Contrary to popular belief, thymol is not highly volatile — if you put a few thymol crystals out in a tray to evaporate, it takes many days for them to do so, even at broodnest temperature. It’s really difficult to find data on thymol’s vapor pressure, but it appears to be far less volatile than water, kerosene, or perhaps even vegetable oil. If I stick my nose into the mouth of a jar of thymol crystals I can smell a faint odor; if I do the same with a jar of menthol crystals, the odor is intense, and my eyes and lungs burn.

The slow evaporation of thymol is affected by temperature, as well as the crystals’ exposure to air flow (which can be notably increased by the fanning of the bees). With Apiguard, the thymol is ground into fine crystals, which are suspended in a mixture of water and absorbent globules of sodium polyacrylate — very little thymol actually dissolves into water. Thus, the bees must actually remove the globules of gel to expose the surfaces of the crystals to evaporation.

With Thymovar, the crystals are more exposed, and can evaporate more quickly (sometimes causing excessive brood issues in hot weather).

Practical application: I wondered whether there was another way to apply thymol, that extended its release rate to avoid the need to reapply the treatment, that did not encourage propolization, that did not require the addition of a rim, and possibly be a matrix that the bees would remove themselves after treatment (such as happened when I applied a low dose of Apiguard between the brood chambers on an index card [[4]]). Ideally, a perfect treatment would not be excessively irritating to the bees, and would not shut down broodrearing for an extended period.

Reaction to the delivery matrix by the bees

Bees are repelled by a strong odor of thymol, and will attempt to enclose it in a curtain of propolis. But once its outgassing slows, the workers acting as “housecleaner/janitors” will either encase it in propolis (Figure 4), or pick off pieces of the matrix and remove it from the hive (Figure 6).

Fig. 4. If the cleaner bees are unable to chew away a matrix that smells of thymol, they will either build a wall of propolis around it, or encase it in a layer of propolis as above.

Thymol in Shop Towels

One of the delivery matrices for thymol that I tried was the commercial beekeepers’ standby — the blue shop towel. I found that a saturated solution of thymol could be easily made by dissolving it in denatured alcohol (especially if the alcohol was warmed) (Figure 5).

Fig. 5. A gram of thymol readily dissolves into a milliliter of denatured alcohol, resulting in ~2 mL of a saturated solution. The solution wicks readily into a shop towel. I then just laid the towels out to dry for an hour, by which time (at least in our California dry air) the alcohol had all evaporated. Due to the low vapor pressure of the thymol, there is nearly no weight loss of thymol. I then placed the dried towels into an airtight container for storage.

I applied the towels to test for removal and adverse effects. I placed half towels (containing a typical dose of ~12 g of thymol), in hot weather, either between the brood chambers, or in rims below the hive cover (Figure 6). It was quickly apparent that 12 grams on a towel in the middle of the cluster was too much, as the colonies roared and bearded out the entrances (I was afraid that they might abscond). That intense a dose also resulted in some removal of sealed brood. A quarter towel (containing ~6 g of thymol) was better tolerated.

Fig. 6. Half a thymol-impregnated shop towel in a rim. At first the bees are repelled by the fumes and fan them away. But as soon as the intensity of vapors drops, they rapidly remove every trace of the towel, rarely propolizing it.

What was interesting to me was the surprisingly rapid rate that the bees evaporated the thymol from the towels placed in the middle of the cluster. Unlike with Apiguard or Thymovar, it was like a “flash” treatment, and after a couple of days I could barely smell thymol in the remaining towels. Apparently, the temperature of the broodnest, coupled with the strong fanning, results in rapid thymol evaporation from this thin matrix, and janitor bees then quickly remove the remaining mildly scented towels.

A friend tried the towels in a few colonies, taking before and after mite counts. The results were that a half towel placed in the middle of the cluster resulted in substantial mite reduction, but was very stressful to the colony. A quarter towel was much better tolerated, but required a repeat application for efficacy. The towels placed in a rim above the hive gave variable results.

Practical application: The take home is that a shop towel, due to its high ratio of surface area relative to volume, allows for too-rapid release of the thymol for most purposes (but might be used in broodless colonies [[5]]). This thus raises the question of whether a cellulose matrix thicker than the Thymovar sponge would allow for a more controlled and extended release of thymol vapors as the thymol vapor from the interior of the matrix slowly diffused to the surface.

Removal and dispersion of thymol matrices by the Bees

Practical application: I’ve found that when Apiguard is placed above the colony, unless the bees physically remove Apiguard from the card it exhibits low efficacy against varroa. I suspect that the process of physical transport through the cluster is important, since it would expose the surfaces of the chewed particles of the matrix to allow for evaporation of the thymol. So an important question is, do the cleaner bees drag the particles down through the brood area (thus exposing the mites there to the fumes), or do they avoid that area?

We made an interesting observation some years ago when we had stickyboards under colonies treated with shop towels infused with oxalic acid and glycerin (Figure 7).

Fig. 7. When the housecleaner bees chewed away shop towels infused with oxalic acid and glycerin, the particles of debris nearly all wound up piled on the stickyboards, heaped just inside the entrance, suggesting that the janitor bees took routes that avoided the broodnest.

Curious, we recently set up a hive to determine whether the cleaner bees do the same with debris containing thymol. We put a stickyboard under a colony, and applied two different thymol treatments on the top bars between the brood chambers — one a half shop towel impregnated with thymol, the other scoops of Apiguard (mixed with a fluorescent tracer). We placed each treatment separately on different sides of the hive. I wanted to see whether the bees would again carry the debris to the front of the hive, or whether it would be taken (or fall) down through the broodnest (Figure 8).

Fig. 8. We placed fluorescent-tagged Apiguard on the top bars of one side of the broodnest (the far side in this photo), and a half thymol-infused shop towel on the near side. This image (under UV + natural lighting) was taken four days later, confirming that plenty of particles of each matrix somehow sifted their way directly down through the broodnest, rather than being carried out away from the brood (the gray-bluish debris in the foreground is from the shop towel). This dispersion of the matrix through the broodnest likely increases the exposure to the mites to the thymol vapors, since the thymol would evaporate rapidly from the surfaces of the small particles of debris as they were carried (or sifted their way) down.

Extended-Release Thymol?

Note that all the registered thymol products call for repeated applications to achieve good efficacy. If we could come up with a treatment that extended the release of thymol even longer than does Apiguard, we might be able to get by with only a single application.

This got me wondering whether we could use a thicker cellulose matrix than used by Thymovar, to throttle the evaporation rate over a longer period of time, allowing us to get by with a single application. And would a matrix that encouraged physical transport by the janitor bees increase efficacy?

A Thicker Matrix

I was intrigued by the Homasote insulation board (made from recycled newspaper) used by NOD to make its MiteAway II pads. So back in 2017, assisted by Tara McKinnon, we experimented with it. Other beekeepers were dissolving thymol in vegetable oil or Crisco for their homebrews, so I wondered whether the thymol would work better if canola oil was added to slow evaporation, or possibly help it to stick to the bees’ bodies. So we made up a 4-way grid of sixteen different thymol and canola oil concentrations (Figure 9), dissolved them in ethanol, and saturated 1” square blocks of Homasote with them (Figure 10).

Fig. 9. We tried sixteen different ratios of thymol and canola oil. The amount of each substance that would remain in each 1” square block of Homasote is shown above. Those amounts ranged from 0.6-2.4 g of thymol, and zero to 1.9 g of oil.

Fig. 10. Preparing the test blocks. These have just absorbed the solutions.

We then placed sets of 16 test blocks into four hives between the brood chambers (Figure 11). This allowed us to test for how the bees responded to the intensity of thymol, as well as to the addition of oil.

Fig. 11. Note the range of oil saturation, as indicated by the darker color of the blocks containing more oil. There were thymol crystals on the surfaces of all the blocks other than the most oil-saturated.

Tara then opened the hives daily, and recorded how the bees were responding to the blocks, as far as avoidance, chewing, or propolization (Figure 12).

Fig. 12. There was of course colony-to-colony variation, but we learned that the bees tended to propolize blocks containing more oil, whereas after a few days they started chewing away blocks with less oil.

This finding suggested that blocks of Homasote containing thymol alone (no oil), might work well as an extended-release cousin of the Thymovar strips.

moving ahead to 2022

I shelved the above thought until this year. Since I’ve recently been experimenting with extended-release formic and oxalic acids, I figured that I should pick back up on my experimentation with thymol and Homasote. But I’m out of space, so you’re going to have to wait until next month to see what we learned.

Citations and notes

[1] Understanding Unapproved Use of Approved Drugs “Off Label” | FDA

[2] Varroacide Registration : USDA ARS

[3] IPM 7 Fighting Varroa The Arsenal: “Natural” Treatments – Part 2 – Scientific Beekeeping The application instructions on the label are surprisingly confusing, but Veto Pharma has some useful videos on its website.

[4] The Learning Curve – Part 3: The Natural Miticides – Scientific Beekeeping

[5] Chiesa, F & M D’agaro (1991) Effective control of varroatosis using powdered thymol. Apidologie 22(2): 135-145.

Formic Pro in Hot Weather — Slowing the rate of vapor release

Contents

Hard “Flash Treatment” vs Slow-Release. 1

Flash Treatments 1

Extended-release application. 2

our Follow-up experiments. 3

But did the wrapped strips kill mites?. 8

Discussion. 9

Citations and notes 9

Formic Pro in Hot Weather —

Slowing the rate of vapor release

First published in ABJ in October 2022

Randy Oliver
ScientificBeekeeping.com

Last month I wrote about some “quick and dirty” preliminary experiments to investigate methods to possibly reduce queen turnover when applying Formic Pro during hot weather.  We followed up with some more experimentation with the strips.

Hard “Flash Treatment” vs Slow-Release

The challenge when using formic acid for varroa control, especially in hot weather, is to find the “sweet spot” of enough vapor intensity and duration to kill the mites, without knocking out the queen.

Flash Treatments

A flash treatment is of short duration, measured in hours, as opposed to days.  An overnight hard flash application of formic acid can indeed penetrate the cappings and kill the mites thereunder, but at the potential cost of causing the loss of the queen.

Dr. Jim Amrine wrote about using a hard “flash” treatment to control varroa [[1]], and my sons and I played with it a bit some years ago [[2]].  Amrine used 80-110 mL of a “weak” 50% formic acid solution, and obtained good kill of mites under the cappings.

Another flash treatment widely used by Canadian beekeepers, typically involves pouring 30-40 mL (sometimes more) of  a more concentrated (60-65%) formic acid solution into meat pads (“mite wipes”), applying them on the top bars, and repeating three or more times, 7-10 days apart.

Practical application:  The necessity of reapplying the lower dose of formic acid indicates a lack of efficacy at the killing the mites in the brood.  There is a clear tradeoff between applying formic vapors strong enough for penetration and kill off the mites, but being “gentle” enough to not kill the brood and queen.  The labor involved in the necessity of repeated applications led others to develop extended-release methods.

Extended-release application

Years ago, the Europeans, as well as others, developed extended-release methods to apply formic acid, involving placing an absorbent matrix saturated with formic into a perforated zipper-top bag or some sort of fancier plastic application device to regulate the rate of release.

Practical application:  The evaporation rate of formic acid is dependent upon the exposed surface area of the delivery matrix, the temperature, and the humidity.  Within the cluster, temperature and humidity are largely held fairly constant by the bees, but when an irritant such as formic acid is introduced, the bees may fan vigorously, causing more rapid ventilation, resulting in inadequate penetration of the vapors into the capped brood cells. 

On the other hand, at lower concentrations of formic vapors, the bees quickly acclimate and ignore the fumes, and I suspect that they maintain near-normal temperature and humidity around the delivery matrix or device (if anyone has recorded data on this, I’d love to see it).

There are a number of adjustable formic application devices sold in Europe.  The Nassenheider evaporator, invented by the German beekeeper Bruno Becker in the 1990s, is also widely used.  It allows the user to precisely adjust the rate of formic vapor release.  One suggested daily evaporation rate when using 60% formic acid is 15 – 20 mL in warm weather, or 6 – 10 mL when cooler.  If the evaporation rate is below 6 ml per day, the treatment may not be successful [[3]].

In North America, lately-deceased beekeeper Bill Ruzicka developed the slow-release MiteGone pads [[4]].  At the normal broodnest relative humidity of  45%-65%, the evaporation rate of 65% formic from a MiteGone pad  hit a target minimal release rate of around 6 grams, but at 85% humidity, the evaporation rate dropped to 3 grams a day, and additional pads needed to be added [[5]].

Practical application: Note that for the products above, the recommended daily rate of release of 65% formic was in the 10-20 g range, as compared to the recommended 60-80 g (or more) for an overnight flash treatment.

Years ago, Medhat Nasr and David VanderDussen developed a formic-saturated pad in a plastic bag punched with ventilation holes.  NOD Apiary Products later marketed them as Mite-Away, and later Mite-Away II pads, designed to be applied in a spacer rim above the cluster.  NOD later replaced that product with slow-release strips to be placed directly within the cluster (to avoid the need for a rim),  first releasing MiteAway Quick Strips, and more recently Formic Pro (which has a longer shelf life).

Over the years, I’ve tested the above products, and collected data on their rates of release of the formic acid vapors (Figure 1).

Fig. 1 Mixed data on mean daily weight loss from various NOD Apiary products.  High-temperature, MAII, and MAQS data are from 2016; Formic Pro data from 2021 (at highs of ~95°F).  Note how the early MAII pads, designed to be applied in a rim above the cluster, gave a fairly steady daily release of 30-40 grams, compared to the later strips, which give a more-intense formic “flash” the first day, then a rapidly-dropping rate of release on the ensuing days.

Practical application:  The initial flash of vapors from MAQS or Formic Pro are associated with queen turnover or loss when applied at above the recommended maximum temperature for application (85°F) (From the label: ”Hot temperatures (>92°F) during the first three days may cause excessive brood mortality and queen loss”).  But note that by covering the Formic Pro strips with their wrapper, that first-day flash can be eliminated (solid black plot).  Our experiments last month suggested that partial covering of the strips might be a way to avoid queen issues, while still obtaining reasonable efficacy against varroa.

Add:  The old MiteAway II pads gave a more consistent and extended offgassing of formic acid.  I’m not sure that the new products are actually an improvement!

Since we are stuck with increasingly hot summers, we may need to get creative to be able to utilize formic acid during summer weather.  I’m unable to test under conditions of high humidity, but followed up on the exploratory research detailed in my previous article — specifically, what would happen if we further reduced and extended the release of the vapors?

our Follow-up experiments

My helpers and I, encouraged by the minimal amount of queen turnover when we covered single Formic Pro strips with their wrapper, wondered about applying two strips at once, but greatly restricting their evaporation rate by leaving them in their original wrapper with only the ends cut off.  This would greatly reduce the amount of evaporative surface area, and perhaps provide a low-intensity extended release similar to the Nassenheider or MiteGone delivery methods.

We tried two different application methods (Figures 2 & 3), comparing them to the covered strip method detailed last month.

Note and Objectives:  These experiments were not according to label directions.  This was exploratory research to (1) observe colony response to a slow-release application of formic acid, and (2) determine whether it was able to substantially reduce the mite infestation rate.  We had a limited number of high-mite colonies to work with, so were not able to run tests on as many hives as we would have liked.

Fig. 2 In three colonies, we left the two strips within their original wrapper, with only the two ends cut off, placing them directly over the brood combs in the lower brood chamber.

Fig. 3 After observing minimal colony disruption due to the previous treatment, the next day we doubled the amount of evaporation area in seven additional colonies, by cutting the packages of strips crosswise, which also better spanned the brood area.

As positive controls, we also treated five colonies with a single unwrapped strip (outer membrane intact), loosely covered on top with its wrapper, and treated a single colony with two separated unwrapped Formic Pro strips, also covered with wrappers.

For all the colonies, we restricted the entrances to see how they would handle the various application methods in hot weather (Figure 4).  I inspected the hives daily for the first several days after application (Figures 5 & 6).  The experiments were performed in late July through mid-August, with the daily temperature highs in the mid-90s.  The colonies were in double or triple deeps, and varied in strength.  There was a moderate nectar flow on, which may have raised cluster humidity.  We performed follow-up mite washes after 21 days.

“

Fig. 4 We inserted wedge-type entrance reducers with a single 3/8” x 3” opening.  For the unwrapped but covered strips, there was noticeable bearding the first couple of days.  But for the wrapped strips, the photo above was typical of the maximum amount of bearding on the first day, with no noticeable bearding or fanning afterwards.

Fig. 5 Formic vapors often kill any vegetation immediately in front of the entrance of a treated hive, but during our experimentation, the grass was already dried up.  For the first several days, I checked for the odor of formic vapors in the exhaust from the reduced entrances — formic fumes were strong only from the colony treated with two covered strips, and noticeable for the single covered strips.  I was surprised that there was no obvious formic odor exhausting from the entrances of any of hives containing still-wrapped strips, even on the day after application.

Fig. 6 The colonies with the wrapped strips initially reduced broodrearing a bit, but continued to store honey, and quickly reestablished healthy broodnests.

Of interest, when we removed the wrapped strips after 21 days, was that they were still quite moist within the wrappers, and still smelled of formic acid, but not very strongly.  It appeared that the acid had evaporated more than had the water.  Due to the remaining water in the strips, I was unable to calculate the amount of formic acid released during the 21-day treatment intervals.

But did the wrapped strips kill mites?

OK, the colonies tolerated the wrapped strips just fine.  Unfortunately, the slow-release treatment didn’t appear to bother the mites very much either.  I’ve plotted the results in Figure 7.

Fig. 7 Treatment of one colony with two strips, separated and covered with their wrapper material, was quite efficacious (resulting in an ending mite count of zero).  A single application of only one covered strip to five colonies, resulted in a “knockback” of the mite infestation rates in the ballpark of 45% (indicating that a second application would be required to obtain full efficacy).  But there’s too much red showing for either of the slow-release treatments involving strips still wrapped in their packages, with an overall mite reduction for the ten colonies of only 12%.

Not unexpectedly, only one colony in the wrapped strips groups was queenless at 21 days.  Perhaps of interest is that the 2-strips covered colony, although still queenright, had supersedure cells (not unusual for a 2^nd-year queen).

Discussion

Formic Pro works fine when applied according to its label instructions at recommended temperatures — these experiments were to see whether we could reduce queen turnover when formic treatment is applied in hot weather, while still attaining desired mite reduction by slowing the rate of vapor release.

There were too few colonies involved in these quick and dirty experiments to draw firm conclusions, but the rate of formic release from Formic Pro strip packages with only the ends cut off appeared to be insufficient for varroa control.  However, partial covering of Formic Pro strips to reduce the first-day flash off, may in hot weather may be a means of minimizing queen turnover or loss.  If you try this yourself, I’d be interested in hearing your results!

Citations and notes

[1] Amrine Jr, J, & R Noel (2006). Formic acid fumigator for controlling varroa mites in honey bee hives. International Journal of Acarology 32(2): 115-124.

[2] Messin’ With Varroa 2014 – Scientific Beekeeping

[3] The Nassenheider Evaporator (dave-cushman.net)

[4] MiteGone for effective control of the varroa & tracheal mites

[5] Treatment Modification March 21 Y doc (mitegone.com)

Formic Pro and Queens in Hot Weather

Contents

Formic for mite management 1

A trick for using formic pro on weak colonies 2

Queen loss in hot weather 5

Why would queens be more susceptible to formic than workers?. 6

A test of queen loss in tiny colonies. 7

Taking advantage of opportunity. 10

Trial #1 — Would foil covers reduce queen loss or turnover?. 11

Trial #2 — Does covering the strips affect efficacy?. 12

Results. 13

Trial #1. 13

Trial #2. 14

Wrap up. 15

Citations and notes 15

Formic Pro and Queens in Hot Weather

First published in ABJ in September 2022

Randy Oliver
ScientificBeekeeping.com

Formic acid has a lot going for it when used for varroa management.  A recurring question is how to avoid the common complaint about queen loss, especially when applied during hot weather.  I recently had the chance to run three impromptu “quick and dirty” experiments to investigate.

Formic for mite management

Beekeepers worldwide have long used formic acid — applied by various methods — for control of varroa.  Formic is able to achieve rapid mite reduction — and notably, kill mites beneath the cappings — while not contaminating the combs or honey.  My sons and I use it extensively in our operation (in rotation with oxalic acid and thymol), and have experimented with a number of application methods, including Formic Pro strips.

Since the recommended temperature range for application of Formic Pro lies between 50 and 85°F (10-30°C), using it during summer may be problematic.  There is a common concern about formic causing queen loss (or inducing queen turnover) during hot weather.

Practical notes: 

• In my 2020 hot-weather trial [[1]], although a number of colonies replaced their second-year queens after treatment with Formic Pro, 28 out of 29 had a laying queen a month after the second application of a strip.
• Formic acid is great for springtime treatment, since its induced brood break may help to avert swarming, and colonies can easily replace a lost queen at that time of year.
• The release rate of formic acid from Formic Pro is essentially the same as that from Mite Away Quick Strips [[2]], so the results of the experiments below would likely apply to either type of strip.

A trick for using formic pro on weak colonies

I routinely treat nucleus colonies with Formic Pro strips, even in hot weather.  A trick for preventing queen loss is to press the hive cover down tight onto the top of a half strip (Figure 1).

Fig. 1 A nuc can be treated with a half strip of Formic Pro, if the top surface is tightly pressed on by the hive cover.

Take a look at the photo above.  Note that evaporation from the bottom side of the strip is largely blocked by the top bars.  If evaporation on the upper surface is blocked by the hive cover, that leaves only the outer edges and two 3/8” strips from which the fumes can escape.  This greatly slows and extends the release rate of the formic acid.  Unlike strips placed between two brood chambers, which rapidly gas off and dry to stiffness, strips placed directly under the cover as above, will even after 10 days of hot weather still be soft and outgassing formic acid (Figure 2).  It’s important to press the hive cover down firmly over the strip (Figure 3).

Fig 2 Tammy giving Rose her first introduction to formic vapors.  These strips had been in hives for 10 days, with their foil cover placed on top.  As you can see from the grimace on Rose’s face, there were still fumes being emitted.  The ladies are standing at our mobile mite wash station.

Fig. 3 Helper Rose Pasetes performing a balancing act while demonstrating the pressing method that we use for the nuc cover.  Caution: this dangerous technique should only be used with appropriate safety equipment to prevent falls [[3]].

Practical application:  The label directions for treatments don’t necessarily cover all the “tweaks” necessary for optimal application under various conditions and situations.  Small adjustments to an application method can make a big difference!

Queen loss in hot weather

Despite the temperature recommendations, during our increasingly-hot summers, we still sometimes need to get the mite count down quickly in a colony, and formic fits the bill.  We often apply a single Formic Pro strip during hot weather for a “knockback” treatment while honey is still on the hive, and don’t observe queen problems.   And once we’ve pulled honey, we routinely break high-mite colonies with second-year queens down into singles, and then blast them hard with a strong formic treatment when the temperature is in the high 90s.  A handful of (presumably weak, old, or newly-emerged) dead workers in front of the hive the next day indicates that an adequate dose was applied, but surprisingly, even with these extreme formic blasts, the (unwanted) queens still often survive!

Why would queens be more susceptible to formic than workers?

So why, of all the bees in the hive, would the well-fed and long-lived queen be the most susceptible individual to formic acid?  It’s easy to observe that queens simply move away from irritating fumes.  I’ve long been curious about an observation by Amrine & Noel [[4]], during their development of the 50% formic “flash” treatment:

Essential oil components in Honey-B-Healthy [consisting of spearmint and lemongrass oils] modify the effect of Formic Acid (FA) treatment on bee hives, such that queens are not lost. This aspect is extremely important for any beekeepers using formic acid to treat varroa mites.

The idea for using lemongrass oil may have come from a prior study by one of Amrine’s students.  Vargas-Sarmiento [[5]] found that although application of lemongrass oil could cause queen supersedure on its own, it appeared to suppress the removal of foreign material from the hive.

To my great surprise, I can’t find follow up research on using essential oils to mitigate queen loss when using formic.  This is especially surprising because both oils exhibit miticidal properties in their own right [[6]^,[7],[8]], so adding them might also improve the efficacy of formic acid treatment.

Practical application: Could it be that the queen loss associated with formic treatment is due to the workers, in response to the stress from formic vapors, killing the queen themselves?  Or perhaps, since formic fumes kill all the eggs and very young larvae, is the resulting sudden reduction in young brood pheromone a cue that triggers the bees to supersede an apparently failing queen?

And could lemongrass oil, which contains some of the same components as orientation pheromone, suppress such behavior?  I’ve been  wanting to test the effect of adding these essential oils to formic treatment for some time, but first need to determine an application method that consistently kills queens.

To that end I’ve placed queens into push-in cages directly below formic applications, and found that even if they were constrained from moving away from the fumes, some queens survived even when workers in the same cage died (Figure 4).

Fig. 4 You can see the dead workers in this cage, but I was unable to get the still-living queen to pose for the photo.  It surprised me to find that queens in push-in cages could sometimes survive being placed directly below strong formic applications, with or without attendants in the cage.

And then in June I had an unplanned opportunity to run a spontaneous test…

A test of queen loss in tiny colonies

Assisted by Tammy Hayden and Rose, I was setting up colonies in an outyard for an experiment, when my son Eric called to say that he was heading over to formic blast some “dinks” in the same yard.  The colonies (made from splits with queens in their second year) had never built up, and he hoped to both kill the mites as well as the poor queens with the treatment, before adding the boxes to other hives .  Realizing an opportunity, I asked him to grab some push-in cages to bring along, but he couldn’t find them, so brought some JZs BZs introduction cages instead.

The colonies were all weak and in single deeps.  We quickly found and caged the queens, pressing them into a center comb to be replaced directly below a single Formic Pro strip (Figure 5).

Fig. 5 These “dink” colonies all had second-year queens, and most had poor brood patterns.  The weather was hot and dry.  We placed the queens in cages without candy or attendants (which if we’d had the push-in cages, we’d have done differently).

Practical application: Many of us have noticed that formic tends to “knock out” poor queens more so than young high-quality queens.  So we’re not concerned about the loss of poorly-performing queens.

Not wanting to kill the brood in the weaker colonies, and knowing how the pressing of the lid on top of a strip reduces queen loss, the boys placed 1.5” rims only on the stronger colonies (Figure 6) (to allow for more evaporation from the strips.  This proved to make a big difference.

Fig. 6 Rose placing a strip in a 1.5” spacer rim.    For the weaker colonies, we pressed the migratory cover directly on top of the strips.

Important note:  All hives used in the trials in this article had unrestricted 3/4” high entrances.

We checked two days later for queen survival (Table 1).

Table 1. Note the higher survival rate of the queens in the weaker colonies.  I can’t say for sure, but the variable of restricting evaporation from the top of the strips appeared to make a difference.

Practical application:  Had Eric been able to find push-in cages, I would have supplied the queens with food and attendants.  So I can’t say whether the queens died from the formic fumes, or due to not being fed through the cage openings, but there was clearly a direct or indirect effect of formic upon some queens.  I still need to test for the effect of adding essential oils.

The above was not the only opportunity to arise for an unplanned experiment…

Taking advantage of opportunity

Shortly after the above experiment, my helpers and I had taken final mite wash counts from colonies in two different trials (one yard having been treated with different kinds of oxalic dribbles, the other with an experimental thymol gel).  Since some of the mite counts were still high at the end of the experiments, we planned to hit those colonies immediately with formic acid.

It occurred to me that those ending mite counts could serve as starting counts for new field experiments with formic, if we started them immediately.  So I made the spur of the moment decision to take advantage of this opportunity to follow up on my observation of the substantial amount of queen replacement that occurred in my testing of Formic Pro in hot weather last year.

Practical application:  Bees initially move away from formic fumes and fan, but very quickly get used to the odor (and presumed irritation), and even walk right over active strips.  Queen “turnover” appears to be due to the initial high-intensity “flash” of vapors that occurs with most application methods.  Perhaps by controlling the initial “flash” (similar as to how we treat nucs) we may be able to reduce queen issues.

Experimenting with ways to reduce the initial flash, I thought of using the foil wrapper that Formic Pro strips come packaged in to simply cover the upper surface of the strip when applied between two brood chambers.  So last year I measured the rate of weight loss of a couple of pairs of Formic Pro strips placed in a double deep of drawn combs (without bees, to avoid both the variable of fanning, as well as weight gain from propolis deposition) during hot weather, covering one of each pair with its foil wrapper.  The results are shown in Figure 7.

Fig.  7 Covering the upper surface of a strip with its wrapper reduced its daily weight loss (from the release of the formic vapors).  This greatly reduced the first-day “flash,” and extended the lower-level release afterwards.  Could this method of application improve the treatment?

Practical application: Over the years I’ve measured the daily release rate of formic acid from various application devices and methods.  After extensively reviewing published and unpublished studies, it appears that the optimal continuous-release application rate for 65% formic acid is somewhere in the range of 8-22 g per day (as indicated in Figure 6), depending upon ambient temperature, colony strength, and entrance restriction.  That said, we’ve treated a lot of hives with the old Mite Away II strips, which released 30-50 g per day, and overnight flash treatments of up to 90 g of 50% formic [[9]].

Back to us having two yards with starting mite counts, it occurred to me that this was an opportunity to test covering the strips in colonies with bees to see whether it reduced the amount of queen turnover.  So we started two ad hoc, hastily-planned experiments — one focusing upon queen loss, the other focusing upon efficacy.

Trial #1 — Would foil covers reduce queen loss or turnover?

We ran both trials starting on June 24, during our honey flow.  All colonies had been started from nucs with second-year queens.  Due to awful weather during their buildup period, the colonies were still relatively weak, all in double deeps with the brood in the lower box, and the bees just starting to work into an upper box containing frames of foundation (we gave a few of the stronger colonies a honey super at the second formic application).  Helped additionally by Corrine Jones, we applied formic strips, centered over the clusters, so the combs with brood got direct exposure to the fumes (Figure 8).

Fig. 8 We ran this trial during hot weather, with daily highs often in the upper 90s (35C+).  The colonies in this yard were the stronger of the two, and had higher mite counts, so we wanted to give them a double application of Formic Pro, with a second strip applied after 10 days (replicating the 2020 field trial).  In this yard, we covered the tops of the Formic Pro strips with their wrapper in all the colonies in order to reduce evaporation from the upper surface (I opened the wrappers carefully and cut each in half).

Trial #2 — Does covering the strips affect efficacy?

I hadn’t planned either of these spontaneous trials, and made up the protocols as we were driving to the yards.  The colonies in the second yard had lower mite counts overall, so in this yard we ran Test and Control groups — with the strips either covered or uncovered.  I haphazardly assigned treatments as we applied them, using the full wrapper, pressed flat, to cover one of each pair of strips.  Once home, I noticed that based upon the May counts that were still on the hives from the previous experiment, that I’d wound up over-favoring the lower-mite colonies with the uncovered strips (so they could not be used as proper Controls in order to calculate efficacy).

Sampling timing explanation:  Since what I was interested in with this experiment was the effect of covering the strips upon efficacy at mite reduction, we only treated once, taking final mite counts 21 days after application of a single strip, which allowed for mites that survived treatment  while they were in the brood to emerge, and reestablish the equilibrium of the proportion of mites in the brood relative to those on adult bees, so that a mite wash of adult bees would again reflect the overall mite population in a hive.  Details, details, details…

Results

Trial #1

Unlike what occurred in my hot-weather trial of Formic Pro in 2020 [[10]], in which there was a goodly amount of queen “turnover,” in this experiment, every single queen was still alive and had plenty of young brood 10 days after the application of the second strip. Go figure!

As far as reduction in the mite infestation rate of the adult bees, the mean (as well as the median) reductions in mite counts were around 54%.  This figure is roughly the same as we obtained in 2020 (85% median reduction, 54% mean).  Since the colonies varied considerably in strength, I sorted the results by ending colony strength (Figure 9), to see whether cluster size affected mite reduction.

Fig. 9 I didn’t run a control group, so can’t compute efficacy, but can show the degree of mite count reduction at 21 days (10 days after application of the second strip), by which time most remaining live mites in the brood would have emerged.  On average, mite counts dropped by slightly more than half, and were reduced in nearly all the colonies, but there was considerable variation.  Note that you see less red relative to blue on the left, indicating that the treatment tended to be somewhat more efficacious in the weaker colonies, in which most of the cluster was located below the strip.

Practical applications:  Once again there is great hive-to-hive variation in the amount of mite reduction due to this treatment method.  But unlike as in 2020, not a single queen got replaced.  But was that due to covering the strips?

Trial #2

In this trial, with only a single strip applied to relatively weak colonies, at 21 days after application, we lost no queens in the 11 covered-strip colonies.  But surprisingly, we lost only 1 queen out of 12 in the uncovered strip group (in one of the stronger colonies).  So it’s hard to attribute the lack of queen loss in Trial #1 to covering the strips.

Practical application:  I have no idea as to why we didn’t see as much queen turnover this year, but covering the strips didn’t appear to make much difference in relatively weak colonies with plenty of room in the second brood chamber. 

So how about the effect of covering the strips upon efficacy in mite reduction?  Three weeks after applying a single strip, both groups in this yard averaged roughly the same mite counts that they started with, but again with wild variation (Figure 10).

Fig. 10 As far as mite reduction, we didn’t get much from a one-strip “knockback” treatment.  There was again considerable hive-to-hive variation, and again I have no idea as to why. I hesitate to draw any conclusions as far as efficacy against varroa, since these were relatively weak (and well-ventilated) colonies.

Wrap up

Allow me to first make abundantly clear that in the above experiments we were applying Formic Pro at well above the recommended temperatures on the label, so its relatively moderate efficacy would not necessarily reflect its performance when applied at lower temperatures.

That said, we observed only a single queen being lost out of 47 treated colonies.  I have no explanation as to why our colonies, treated with the same application of Formic Pro, in similar yards at similar temperatures, had such different rates of queen turnover when replicated in two different years.

Nor do I understand the inconsistency in mite reduction with Formic Pro, but suspect that it has something to do with colony ventilation.  Once ambient temperature exceeds broodnest temperature (95°F; 35°C), colonies are heat stressed and must increase fanning and evaporation within the hive (but varroa mites are also stressed above this temperature [[11]]).  Thus the hive-to-hive variation in mite reduction may have to do with differences in fanning behavior.

There appears to be a fine line, especially during hot weather, between the intensity of exposure to formic acid that it takes to kill the mites under the cappings, and that which causes queen issues.   Underwood’s [[12]] research suggested that “queens are affected by acute rather than chronic exposure to formic acid,” and this observation has been confirmed by many others.  The trick for formic application appears to be to slow down the initial flash of vapors, while providing an extended lower level of fumigation to chip away at the mites.  Along this line, we did so by covering the strips with their wrappers.  As I type these words, I’m thinking that perhaps we should also have reduced the entrances (and may return to those yards today to run another experiment).

Since Formic Pro and Mite Away Quick Strips are the only registered formic acid products in the U.S., and since our summers don’t appear to be getting any cooler, it’s likely worthwhile  for us to keep experimenting to figure out how best to use them if we need to attain quick knockdown of mites during hot weather.

Practical note:  We’re still experimenting with extended-release oxalic acid.  This application method takes about two months to attain full efficacy, and in our informal tests, appears to work well in combination with a simultaneous application of formic acid.  We need to collect more data on this…

Citations and notes

[1] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-3/

[2] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-3/

[3] Hey, I gotta cover my butt against lawsuits.

[4] Amrine Jr, J, & R Noel (2006). Formic acid fumigator for controlling varroa mites in honey bee hives. International Journal of Acarology 32(2): 115-124.

[5] Vargas-Sarmiento, M (2000). Essential oil treatments to control Varroa destructor Anderson and Trueman 2000 (formerly Varroa jacobsoni Oudemans 1904)(Mesostigmata: Varroidae). West Virginia University.

[6] Islam, N, et al (2016) Management of Varroa destructor by essential oils and formic acid in Apis Mellifera Linn. colonies.  Journal of Entomology and Zoology Studies 4(6): 97-104.

[7] Ariana, A, et al (2002). Laboratory evaluation of some plant essences to control Varroa destructor (Acari: Varroidae). Experimental & Applied Acarology 27(4): 319-327.

[8] Refaei, G (2018). Comparing effect of plant-derived oils on Varroa destructor Infesting honeybee, Apis mellifera. Acarines: Journal of the Egyptian Society of Acarology 12(1): 61-64.

[9] https://scientificbeekeeping.com/messin-with-varroa-2014/

[10] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-2/

[11] https://scientificbeekeeping.com/a-test-of-thermal-treatment-for-varroa-part-1/

[12] Underwood, R & R CURRIE (2007) Effects of release pattern and room ventilation on survival of varroa mites and queens during indoor winter fumigation of honey bee colonies with formic acid. Canadian Entomologist 139: 881–893.

Selective Breeding for Mite Resistance, Part 3; Shifting the Genetics of a Breeding Population

Selective Breeding for Mite Resistance, Part 3

Shifting the Genetics of a Breeding Population

Randy Oliver
ScientificBeekeeping.com

The alleles necessary for varroa resistance already existed in my stock of bees, so I didn’t need to “create” anything new. What I’m attempting to do via strong selective pressure is to (1) eliminate from our breeding stock the alleles that favor varroa, (2) increase the prevalence of allelic combinations that confer mite resistance, and (3) “fix” those alleles in the genome of our breeding population.  During this process, I want to (4) minimize any deleterious “bottlenecking” of our stock’s genetics.  So let’s talk about those genetics.

Several important terms

Breeding population: a population within which free interbreeding takes place and evolutionary change may appear and be preserved.

Genome: The entire genetic structure of an individual or breeding population.  All the individuals have the same genes, but the breeding population may contain a number of different forms (alleles) of any gene.  In this case, we’re selecting for combinations of alleles that work together to confer mite resistance.

Genotype vs phenotype: An organism’s genotype is the set of alleles that it carries. An organism’s phenotype is all of its observable characteristics — which are influenced both by its genotype and by the environment.  We’re selectively breeding for the phenotype of exhibiting very low mite counts; we can only guess at the genetics involved.

Fixation: In population genetics, fixation is the change in a breeding population so that only one allele of a specific gene remains in that population (such as the recessive allele that results in the cordovan phenotype).

Genetic bottlenecking:  A reduction of allelic diversity in a breeding population.  In the case of our selective breeding, we are intentionally trying to capture and bottleneck the diversity of the alleles associated with the trait of mite resistance (favoring the alleles that confer resistance), but want to conserve the diversity of the rest of genome.

Recurrent selection:  A cyclic selection process that is used to increase the frequency of desirable alleles for a character or trait that already exists within that breeding population at low levels.  The method involves repeated selection of, and breeding from, generation after generation of individuals exhibiting the desired trait.  The interbreeding of their offspring then allows for novel genetic recombinations to occur, and ideally to eventually become “fixed” in that breeding population, without excessive reduction of overall genetic diversity within that population.

Practical application:  We’re using the tried and true method of recurrent selection — breeding only from the queens of colonies that exhibit resistance to varroa — in the hope that we can fix the genetic recombinations that confer mite resistance into the genome of our entire breeding population.

The drone pool and avoidance of excessive inbreeding

Selective breeding of honey bees requires the genetic management of an entire breeding population, in this case, the thousand-plus colonies of our own stock dedicated to our program (since we replace every queen in our operation each year with one from our selected breeders).  We bring our colonies back from almond pollination chock-full of drones, and flood our mating yards with the sons of last-season’s queens.  We also provide free queen cells of our stock to nearby hobby beekeepers.  Based upon research by Hellmich [[1]], I have reason to believe that we control most of the genetics of our drone pool.

Practical application:  The 50+ bee yards in our operation are largely in wooded areas (which provide suitable cavities for feral honey bees).  And although we largely manage swarming, in some years swarms do fly off and establish feral colonies.  I do not know how much genetic difference there is between our local ferals and our managed stock, but the influence of our drones upon feral genetics would likely be considerable. 

Any of our swarms that successfully establish, but don’t carry the alleles for strong mite resistance, will become feral “mite factories” in late summer.  My own yet-unpublished data suggest that they account for much of the mite immigration coming into our managed hives.  The good news is that non-resistant feral colonies will likely perish during the winter, and won’t be sending out drones the next spring, meaning that any drones coming from the feral population will likely carry alleles for resistance.

Another poorly-understood variable observed by Couvillon [[2]], is that drones from some mothers may be disproportionally more successful at actually inseminating a queen.  Whether that has any linkage to the alleles for mite resistance is unknown.

Selection vs maintaining Diversity

It’s also important to maintain a large enough diversity of queen mothers each generation.  Otherwise, excessive bottlenecking of the genetic diversity of your breeding population will catch up with you, resulting in an inadequate number of sex alleles in the population, and queens laying diploid drones (as well as lack of general genetic diversity to allow for environmental adaptation, and to mask the effects of deleterious recessive alleles).  As explained by King [[3]]:

Non-random, imbalanced mating designs in which better parents are crossed more often does increase the per generational gains by nearly 10%.  However, there is a cost associated in terms of reduced effective population size.

Practical application: In our breeding program, we graft off a minimum of 30 queens (all having mothered a mite-resistant colony) every year in order to maintain an adequate amount of genetic diversity (remember that every queen carries the genetics of dozens of drones) (Figure 1).  I keep an eye on both brood patterns, as well as the wide variation in coloration in our queens as indicators of diversity.

Fig. 1 We maintain any colonies marked as “potential breeders” (due to having very low mite counts) in our outyards along with the rest of our colonies, subject to plenty of mite drift.  Only after almond pollination do we move the 30-40 “best of the best” to our home yard (shown above) to provide larvae for grafting.  To make “breeder grade,” a mite-resistant colony must come back from almonds chock-full of bees and almond honey.  To prevent them from swarming, we reduce them to small singles, and only after grafting add back a second brood chamber back to allow them to regrow.

Trying to figure out the genetics involved

It’s important to understand that the queen is not directly involved in varroa resistance — it is the single or combined responses of the workers of the colony that affect the mites.  Keep in mind that a colony consists of as many “patrilines” of full-sisters (also called “supersisters”) as the number of drones that the queen mated with — each patriline potentially exhibiting differences in behavior.

It could be that it takes only a single patriline of daughters to initiate varroa-sensitive uncapping behavior.  And that behavior could be due to that patriline of workers inheriting a single dominant allele from their father.

Practical application:  I’ve seen over and again that there can indeed be strong, gentle, and productive colonies that are absolutely bulletproof to varroa.  But the queen’s genetics may have little to do with the resistance exhibited by the colony, due to the genetic influences of the different drones with which she mated. Thus, unlike the genetic heritability of a trait carried by the mating of a single mother with a single father, a trait exhibited at the colony level may be the result of a large number of different paternal bloodlines of workers all interacting with each other.  This means that duplication of the genetics of a resistant colony is nearly impossible with open-mated queens, since there were so many fathers involved — any one of which could have been a rare “wild card.”

Allow me show you a couple of examples…

“Old School” Mendelian genetics

We’ve all heard of Mendelian genetics, and how to figure out the expected results from crosses of the genetics of two parents by using a grid of Punnett squares.  Let me give the classic example for pod coloration of pea plants, involving a dominant allele (G) for green color, and a recessive allele (g) for yellow pods (Figure 2).

Fig. 2 One of Mendel’s Punnett squares, showing the results a test cross between two pea plants heterozygous for pod coloration, with coloration being determined by the alleles of a single gene.  Note that the effect of the dominant allele (G), outweighs that of the recessive allele (g), so that the phenotypes of the parents (the green pods that we see) do not reflect their heterozygous genotypes.    Caveat: in light of current knowledge, I’m not sure that pod coloration is actually this simple.

It gets far more complicated with honey bees, because the phenotype of the colony (such as resistance to varroa) may involve the genetics of a large number of parents.  A diploid honey bee queen mates with dozens of haploid drones.  Each drone thus fathers two patrilines of workers — one carrying the alleles of the drone combined with the alleles of the queen’s father, the other a combination with those of the queen’s mother [[4]].  I’ve illustrated this in the Punnett squares of Figure 3.

Fig. 3 Let’s hypothetically assume that there is a gene for uncapping behavior with several alleles — a, b, and c being common, but recessive and not triggering uncapping.  But there’s also a rare allele R (for resistance) which is dominant, and does induce uncapping behavior.  If a non-resistant queen mates with 25 drones, with only one carrying the R allele, this will create a colony consisting of 50 daughter patrilines (the yellow cells), of which only two (the green cells) will exhibit the behavior to uncap cells containing a mite — which may be able to confer resistance to the entire colony as a whole.

Practical application: The colony containing the 50 different genetic combinations of workers shown above might exhibit mite resistance, due to the performance of only the two patrilines of daughters from a single drone.  But the queen herself would not carry any alleles for resistance.

If I were to graft daughters from the queen of the resistant colony above, only two out of 50 daughters would carry the R allele for resistance in their maternal bloodline; although some of the rest might be able to exhibit resistance at the colony level by the lucky chance of mating with an unrelated drone that did carry the allele.

Practical application:  A great deal of luck is thus involved in picking the right daughters, since we don’t know whether the colony’s alleles for resistance came from the queen, or from one or more drones.  It would be easy if the critical alleles were recessive, since the queen of any resistant colony would have needed to have carried that allele, and thus conferred it to half her daughters.  But it’s much more difficult to pick a queen actually carrying a dominant allele, since any of the other 48 daughters might head a resistant colony if she happens to mate with a drone carrying that dominant allele.

In the case of selecting for a dominant allele, one must perform “progeny testing” — tracking the performance of the colonies of daughter queens, looking for queen mothers for which half of their daughter colonies exhibit resistance (Figure x), and then going back and breeding off them or the original mother.  Last year was the first time that we saw this happen.  We’ve got those resistant daughter colonies identified, and plan to move them to isolated mating yards this summer to attempt to “fix” the winning genetics into maternal bloodlines.  Keep your fingers crossed!

Figure 4 shows the expected results of what we’d expect to occur if we got lucky (2 chances out of 50) and happened to graft from either of the two daughters above that carried the R allele in their maternal bloodline.  In this case, not only would half the workers in her colony carry the R allele (and the colony likely exhibit strong resistance), but half of her daughter colonies would also be expected to exhibit resistance.

Fig. 4 An example of progeny testing. Same simulation as above, but this time with a queen carrying the R allele for resistance.  In this case, fully half of that queen’s daughter colonies would now carry the critical R allele in their maternal bloodline.

Practical application:  In one of our yards last season, 24 out of 48 colonies exhibited resistance.  We’re crossing our fingers that we may have hit the jackpot!

I’ve spent hundreds of hours these past months running huge Punnett square series (up to 50 x 50 grids) to figure out the genetics involved in order to match our actual progress (shown in my previous article).  I’ve run simulations with single alleles, double alleles, dominant or recessive alleles, epistatic dominant or recessive effects, etc.  But every simulation suggests that even if the trait came originally from the rare drone, that by breeding only from mothers whose colonies demonstrate full resistance, we “should” be seeing more rapid progress.

Practical application:  It’s relatively straightforward to select against a dominant allele with an observable phenotype, by culling individuals expressing that trait (e.g., cull the queens of any “hot” colonies).  And it’s pretty straightforward to select for a recessive allele, by breeding only from individuals expressing that trait (e.g., breed only from cordovan-colored queens).

On the other hand, it’s difficult to select for a dominant allele (meaning against other recessive alleles), since you can’t tell whether a queen is carrying a recessive allele unless she’s homozygous for the recessive allele (refer to the pea illustration).  See [[5]] for some good visuals of what I’m talking about. 

And in the case of the “trait” of varroa resistance, although we might phenotypically observe uncapping behavior, the only proof that it actually conferred resistance to the colony would be to take mite wash counts.

Another reason that the genetics involved are so hard to understand is that the expression of particular traits is far more complicated than simple Mendelian genetics and Punnett square combinations.  As explained by Miko [[6]]:

The relationship of genotype to phenotype is rarely as simple as the dominant and recessive patterns described by Mendel… Mendel’s early work with pea plants provided the foundational knowledge for genetics, but Mendel’s simple example of two alleles, one dominant and one recessive, for a given gene is a rarity. In fact, dominance and recessiveness are not actually allelic properties. Rather, they are effects that can only be measured in relation to the effects of other alleles at the same locus. Furthermore, dominance may change according to the level of organization of the phenotype. Variations of dominance highlight the complexity of understanding genetic influences on phenotypes.

Miko goes on to explain the concepts of partial dominance, co-dominance, overdominance, multiple alleles and dominance series.  That said, in the simplest cases, one may be able to select for or against some easily-identifiable dominant or recessive traits, such as color, “gentleness,” or hygienic behavior.  But mite resistance doesn’t appear to be so simple.

Practical application:  The bottom line is that selective breeding for mite resistance is a challenge, especially since there are dozens of fathers involved in any resistant colony.  I’ve given up on trying to understand all the complex genetics (and epigenetics) involved in mite resistance.  So we just keep plugging away with our “recurrent selection” approach — which has a long history of success.  In short, we simply requeen our entire operation each year solely with daughters of queens whose colonies exhibited control of varroa for the entire previous year. 

What can small-scale beekeepers Realistically hope to do?

I often get asked by small-scale beekeepers how they could engage in a breeding program of their own.  Unless they plan to instrumentally inseminate each of their queens, the question then is to what extent can they realistically expect to control the genetics of their drone pool?

Practical application:  Honey bees may be the most difficult animal on Earth to selectively breed, due to the uncontrolled polyandry of the queens with the drones of the surrounding breeding population.  The selective breeding experiment that my sons and I are engaged in is targeted for large-scale queen producers who run enough colonies to manage the genetics of an entire breeding population. 

As you can see from our results, in which we essentially control the genetics of the drone pool, but also apply extreme selective pressure (which requires a lot of colonies to pick from), that it takes time and dedication.

The reality is that it’s difficult to imagine that any hobby beekeeper (unless they live on an isolated island) could be expected to significantly affect the breeding population surrounding their apiaries.

That said, if they are surrounded by an unmanaged population of native or feral honey bees, upon which Mother Nature herself has impartially applied enough selective pressure (“live and let die”) upon the free-living breeding population to gain resistance to varroa, the beekeeper could work in collaboration with the “natural” evolutionary process (nicely discussed by van Alphen [[7]]).

They could do so by eschewing bringing in non-resistant commercial stock, and instead populating their hives with swarms from the surrounding “wild” population.  The beekeeper could then retain only colonies that were amenable for beekeeping (such as being gentle and productive), cull the queens of those that were not, and rear replacement queens from the “good ones.”  I get reports from beekeepers in areas with viable unmanaged honey bee populations of doing just that, and being happy with the results.

Yes, such a beekeeper could consider themselves to be a “breeder,” but most of the credit would actually go to Mother Nature, with the beekeeper simply culling out bloodlines from their few managed colonies that were too spicy or swarmy.  Nothing wrong with that!

Acknowledgements

I want to express my appreciation to my intellectually-gifted beekeeping friend Richard Cryberg, who has for years been an invaluable resource to answer my deep questions on chemistry, pesticides, biology, disease, and especially animal breeding and genetics.  His deep knowledge and experience in the breeding of racing pigeons has helped me immensely in my experiment to selectively breed for varroa-resistant honey bees.

References

[1] Hellmich, R & G Weller (1990) Preparing for Africanized honey bees: Evaluating control in mating apiaries.  ABJ 130(8): 537-542.

Hellmich, R, et al (1993) Evaluating mating control of honey bee queens in an Africanized area of Guatemala.  ABJ March 207-211.

[2] Couvillon, MJ, et al (2010) Sexual selection in honey bees: colony variation and the importance of size in male mating success. Behavioral Ecology, 21(3): 520-525.

[3] King, J & G Johnson (1993) Monte Carlo simulation models of breeding-population advancement. Silvae Genetica 42(2-3): 68-78.

[4] Recombination and crossover of chromosomes during the process of meiosis complicates this even further — in reality every single worker bee (or potential queen) could theoretically be genetically unique.

[5] Selection for qualitative phenotypes.  https://www.fao.org/3/v8720e/V8720E03.htm

[6] Miko, I (2008) Genetic dominance: genotype-phenotype relationships. Nature Education 1(1):140.

[7] Van Alphen, J & B Fernhout (2020) Natural selection, selective breeding, and the evolution of resistance of honeybees (Apis mellifera) against Varroa. Zoological letters 6(1): 1-20.

Selective Breeding for Mite Resistance, Part 2; Mite Resistance and Genetic Expression

Contents

The Achilles’ heel of varroa. 2

Genotype vs. Individual- or colony-level phenotype. 2

gene regulation. 3

An example of the differential expression of genes in the honey bee. 4

What Is our own bees’ mechanism(s) for Resistance?. 6

Testing for Uncapping behavior 9

Is there a Cost to the colony for resistance?. 10

Our Traditional Breeding Experiment 11

References. 11

Selective Breeding for Mite Resistance, Part 2

Mite Resistance and Genetic Expression

First published in ABJ in July 2022

Randy Oliver
ScientificBeekeeping.com

A selective breeding program is all about genetics. But humans bred plants and animals for thousands of years before we knew anything about DNA or genetics. What breeders selected for were traits. So how are my own varroa-resistant colonies actually preventing varroa mites from building up in their hives? And why does the trait appear to exhibit low heritability?

I’ve already mentioned that I am selecting for varroa-resistant bees, as opposed to them being merely varroa-tolerant. Resistance to varroa takes place at the colony level. But that colony-level resistance depends upon the interactions of individual bees with individual mites.

It’s easy to visualize individual bees recognizing and attacking mites (so called “grooming” and/or “biting” behaviors), but this agile parasite has evolved to avoid harm. Varroa mites are able to survive for long periods in colonies of their original host (Apis cerana), despite the fact that cerana workers vigorously attack and groom mites not only off their own bodies, but also off their nestmates. So although grooming is certainly a useful behavior to select for, it’s unlikely that it alone would be enough to confer a colony with adequate resistance [[1]^{, [2]}].

The Achilles’ heel of varroa

All mites eventually die of old age. For mites to become a problem in the hive, they must reproduce more quickly than they die. Thus, as far as a honey bee colony is concerned, varroa’s Achilles’ heel is its ability to reproduce, especially in worker brood. Thwarting this may require a “team effort” by the bees. For example, a mite-infested larva or pupa might emit an olfactory signal, or even self-sacrifice (“social apoptosis”), a nurse bee would then need to recognize and respond to that signal and initiate uncapping of the cell, and then a different worker might either remove or cannibalize that infested pupa.

My point is that all of the above responsive behaviors are controlled by genetics, and may require the combined efforts of two or more workers (as well as the infested larva or pupa), each carrying different critical alleles (different “flavors” of any gene) that somehow code for the initiation of each specific behavior.

And then it gets even more complicated; it is not enough that a worker carry the right alleles (the worker’s genotype, or “library” of genetic instructions), those genes must then be read and expressed in the worker’s phenotype — its morphology, physiology, and behaviors. Molecular biologists now talk about the “transcriptome,” which reflects the genes that are being actively expressed from the DNA of the genome into different forms of RNA (the transcriptome) at any given time.

Practical application: The RNA in the transcriptome can code for the manufacture of proteins, or have regulatory functions. I suspect that resistance to varroa is gained mainly from shifts in the regulatory aspect of the transcriptome. Some genes being actively expressed in the transcriptome (at the moment) might be triggered by a nurse detecting a specific odor, which may then unleash “regulatory cascades” that result in the initiation of behaviors involved in mite resistance.

The coding and non-coding genes work together. For example, for a nurse bee to detect that a mite is reproducing under the capping of a cell may require an allele that specifically codes for the formation of an olfactory receptor protein which might bind to a specific odor emitted by immature mites (or a cuticular hydrocarbon from an infested pupa). Binding of that specific odor molecule at the tip of the nurse’s sensory papillae could then initiate a behavioral response cascade, resulting in that nurse bee initiating the chewing away of the capping.

Practical application: If you can’t smell it, you can’t respond to it! For a nurse to respond to a particular scent of a mite or an infested pupa, she must possess a specific olfactory receptor protein that binds to that scent molecule. The coding alleles for such critical receptor proteins may be rare in commercial bee stocks. But once detected, the alleles for the behavior of uncapping targeted pupa are already common in any breeding population, so it may take only the slightest genetic tweak to shift non-resistant bees toward resistance.

Genotype vs. Individual- or colony-level phenotype

Mechanisms for varroa resistance take place at both the individual worker (or larva) level, as well as at the colony-as-a-whole level. The phenotype is where the rubber of the genotype hits the road, and is where the end results of any particular assortment of alleles become evident.

Resistance for varroa may involve the individual larva or pupa (by olfactory signals, social apoptosis, behavior, or gene expression [[3]]), or the behavioral actions of individual adults (grooming, uncapping, or hygienic behavior)[[4]], or take place at the colony level (temperature regulation, absconding, or brood management). All the above phenotypes result from the “expression” of the genotype. As summarized by a recent study that investigated SMR and VSH (Suppression of Mite Reproduction and Varroa-Sensitive Hygiene) [[5]]:

All of the above demonstrate that [mite non-reproduction] is a complex mechanism combining multi-factorial effects, such as adult bee behavior, brood and mite physiology, and bee and mite genetics.

Practical application: The above-cited study is open access, and is must reading for anyone engaged in a selective breeding program. The complexity of mite resistance may help to explain why it’s more difficult to breed for than some other traits.

Gene regulation

Left to themselves, honey bees have shown that they can fairly rapidly adapt to varroa. The process of selected breeding is simply human-directed evolution (forcing adaptation by the breeding population to our selective pressure). The fastest and most efficient way for a species to adapt to environmental changes (such as the gaining of a new parasite) is by gene regulation.

The above concept is explained by Boyle [[6]]:

For typical traits, even the most important loci in the genome have small effect sizes and that, together, … only explain a modest fraction of the predicted genetic variance … in contrast to Mendelian diseases — which are largely caused by protein-coding changes — complex traits are mainly driven by noncoding variants that presumably affect gene regulation. [Highlighting mine]

Albert [[7]] found that almost every gene is influenced by a complex set of regulatory regions all over the genome. Some hotspot regions (“expression quantitative trait loci”) were found to influence the expression of thousands of other genes. I suspect that the genetics of breeding for varroa resistance has mostly to do with selecting for the genetic regulation of alleles already existing in our breeding population (since we clearly see some existing colonies that are able to keep varroa in check) [[8]]. As stated eloquently by Alyson Ashe [[9]]:

The possibility of extreme phenotypes must lurk within the normal genome. These extreme phenotypes are only expressed when an environmental or genetic challenge is sufficient to reveal them. Therefore, the selection on the regulation of a gene network alone should be sufficient to produce the same extreme phenotype without change in the average genotype of the genes that directly contribute to a trait.

The Takeaway: The alleles of a few critical genes act as “switches” to influence or initiate regulatory cascades that control the expression of many other genes involved in physiology or behavior. Evolution may not depend upon novel mutations (adding new tools to the toolbox), but rather upon using the existing tools in the genome in new ways. In our selective breeding experiment we’re not hoping for “magic,” but simply selecting for colonies from our own stock that are using their existing toolboxes to effectively control varroa. In other words, simple “old school” selective breeding.

An example of the differential expression of genes in the honey bee

Allow me to offer a graphic example of how the genotype of a honey bee egg can be very differently expressed in the phenotype of the resulting adult. The one-celled zygote formed upon fertilization contains two sets of chromosomes — one from the queen, and one from a drone with which she mated. Let’s focus upon one matching pair of chromosomes in that zygote, which may have received from each parent a slightly different “spelling” (or not) of a single allele at one critical gene. That minuscule difference, coupled with the “environmental effect” of what the larva gets fed, can result in the formation of any of three strikingly different sexual phenotypes during the development of that egg into an adult.

Surprisingly, the default for any fertilized honey bee egg is to become a male (a viable diploid drone) [[10]]. Only if the matching pair of the Complementary Sex Determiner (csd) gene happens to carry two different alleles (out of over a hundred known), will the csd gene then produce messenger RNA to activate the Feminize gene (fem), which will then kick in to initiate a genetic “regulatory cascade” that results in the development of a female bee.

And once differentiated into a female, depending upon the “environmental influence” of how that now-female larva is fed, “she” can develop into either a worker or a queen (Figure x) [[11]]. Also surprising is that the young larval transcriptome is more influenced by diet than by sexual differentiation [[12]].

Fig. 1 A diagram of how a triggered genetic regulatory cascade, or the environmental effect of feeding, can differentiate the expression of the genes in a fertilized egg into developing into a drone, or either caste of female. Credits [[13]].

My point is that any genome has the potential to develop into substantially different morphological or behavioral phenotypes, depending upon which genetic “switches” are flipped. A honey bee embryo exhibiting virtually identical genetics (with only the slightest difference of a single allele at one regulatory gene) has the potential to develop into either a drone, a queen, or a worker. Further regulation of the transcriptome can then shift that worker from acting as a jelly-producing in-hive nurse, to a wax-producing “mid-age” bee, a guard at the entrance, or eventually into a specialized free-flying pollen, nectar, water, or propolis forager.

Practical application: The above is an example of how differential expression of a fixed set of genes can result in vastly different phenotypes (morphologically, physiologically, or behaviorally). Breeding for mite resistance may not be so much about finding novel genes, but rather about selecting for bloodlines that differentially express existing regulatory genes in response to certain cues (such as the odor of varroa mating pheromone, or mite-stressed pupae). Slight differences in how even a single patriline of workers in a colony flip their genetic “regulatory switches” may allow the colony as a whole to exhibit resistance to varroa.

An addition following a Letter to the Editor:

I was in no way trying to suggest that diploid drones were “normal.”   Drones are of course normally haploid, coming from unfertilized eggs, and diploid drone larvae are normally quickly consumed by the nurse bees.  Since, as you say yourself that “It is fairly common knowledge that the common male drone bee is from a HAPLOID zygote,” I mistakenly assumed that my readers would know that, and it didn’t occur to either me or the Editor that further explanation was necessary.

The point of my example was that any fertilized egg has the “choice” of either of two (as termed by Herrmann¹) “sex options” —  taking either of two different developmental pathways resulting in the development of either male or female organs and body morphologies.  Which pathway it takes is determined solely by whether it carries the same, or a different, pair of alleles at a single gene.

Thank you for bringing this possible confusion to my attention; I will add this correction to the article when I post it to my website.

Regarding your claim that I failed to attribute credit, I make a point of always acknowledging the source of images in my illustrations, as I did for this one in the legend.  One cannot cite USDA as an author or illustrator, one must cite the human being who actually created the work, which I did.

¹Herrmann, M, et al (2005) Characters that differ between diploid and haploid honey bee (Apis mellifera) drones. Genet. Mol. Res. 4(4): 624-641.

What Is our own bees’ mechanism(s) for Resistance?

I don’t limit the “genetic creativity” of my bees by telling them how to do their job; I just fire all the ones that don’t get the job done. After a few years of strong selective pressure, I observe quite a bit of ”bald brood” (Figure 2).

Fig. 2 “Bald brood,” likely indicating uncapping/recapping behavior, involves some workers (perhaps only a patriline of full sisters fathered by a single drone) chewing the cappings off apparently-healthy sealed pupae, but not necessarily removing those pupae (removal would be termed “Varroa Sensitive Hygiene” (VSH) [[14]]). The pupae may then be capped back over, perhaps by a different patriline of workers (recapping is a common trait).

Uncapping/recapping behavior was described in 1998 in Brazilian Africanized honey bees by Corrêa-Marques and de Jong [[15]], elaborated on by Harris, Danka, and Villa in 2010 [[16]], recently identified as a common trait for resistance in bee populations around the world by Stephen Martin in 2020 [[17]], and elaborated upon by Melissa Oddie in 2021 [[18]].

Since I keep an eye on the first brood patterns of our new queens (in order to look for signs of excessive inbreeding resulting in diploid drones), I note that the first patterns are generally quite solid (Figure 3).

Fig. 3 I monitor the brood patterns of our new queens each year to make sure that we’re not inbreeding excessively, which would result in spotty brood patterns due to diploid drones.

Compare the above to a typical pattern of one of our mite-resistant colonies later in the season (Figure 4).

Fig. 4 Although strong and healthy, with a mite wash count of zero, note the typical presence of open cells in this resistant colony later in the season. I suspect that the open cells have to do with some aspect of mite resistance, such as VSH, perhaps following uncapping behavior.

Practical application: I rarely observed bald brood before varroa, but now see it regularly in our own resistant colonies. But it’s not always easy to observe, because if a colony holds its infestation low, there won’t be many infested cells at any time. But what I do see is that there appears to be some threshold of mite infestation at which uncapping and removal of infested pupae really kicks in, especially in my resistant colonies.

I have noticed that bald brood appears to occur in patches, which is explained by an illuminative paper by Grindrod and Martin [[19]]:

These findings are important as they suggest first that all colonies have the ability to detect and thus potentially to remove mite infested brood, and secondly that whether a cell is checked for Varroa is influenced by the infestation status of its surrounding cells.

Grindrod’s findings suggest that the olfactory cue from mite-infested cells may trigger, at least in certain workers, upregulation of their inspection and uncapping behaviors, similar to how other environmental cues alter bee behavior.

Practical application: It’s not clear whether uncapping behavior or even the removal of infested pupae results in the death of any adult mites in the cells, but it likely decreases their overall reproductive success (varroa’s Achilles’ heel).

Testing for Uncapping behavior

During visual inspection, such as in the above photos, one may see uncapped pupae and/or scattered open cells, but you can’t tell the degree to which cells have been uncapped and then recapped without the removal of the pupae. Finding that out requires removing the cappings over sealed brood, and inspecting for signs of being recapped. When recapped in a dark comb, the underside of the capping of a recapped cell will look darker, due to the light-colored silk cocoon having been chewed away.

This process is tedious to do with forceps. In discussion with other researchers, I’ve tried using molten beeswax and gauze or sticky tape to remove a large number of cappings at once. But neither worked well, for various reasons. Luckily, I got a tip from Dr. Ralph Büchler to try using depiliatory strips [[20]], which work as well to remove cappings as they do for bikini waxing (Figure 5).

Fig. 5 This assay must be performed on capped pupae (capped larvae may not have yet spun their cocoons). Look for a patch of pupae at pink-eyed stage, so that the nurses have had plenty of time to detect any cue for uncapping. It’s easy to pick out the dark recaps, compared to the intact undersides still showing light-colored silk cocoons.

Practical application: I’ve only just begun to experiment with this assay, and may write more about it as I learn more. It could be a quick assessment method for mite resistance. Let me be clear that this is only one mechanism for resistance that happens to stand out in our colonies — I have no idea how many different mechanisms that they are actually using (or whether all bloodlines are using the same mechanisms).

Of course I’m curious as to how our resistant colonies keep varroa under control, but as concluded by Eynard [[21]]:

[Mite Non-Reproduction] measurement remains one of the few measurements for varroa resistance in honey bee populations, which can be achieved in the field on a relatively large scale. Although time consuming and tedious to implement, it also gives a lot of different information which can help us to better understand the control mechanisms that bees use to counteract the varroa mite. [Boldface mine]

In our selective breeding program, we do observe a great deal of uncapping behavior and some VSH. But because a resistant colony contains very few mites, it would indeed be tedious to confirm. I’ll leave it up to someone else to figure out how our zero-count colonies manage to control the mite.

Is there a Cost to the colony for resistance?

One advantage of uncapping/recapping behavior for mite resistance is that there is not as much cost to the colony as from brood sacrifice, such as with “social apoptosis” or Varroa Sensitive Hygiene [[22]]. I don’t see any negative tradeoff in productivity or gentleness in our most-resistant colonies — they’re often the most productive hives in a yard. This could be due to their not having to deal with having their all-important fat bodies getting destroyed by the mites, as well as benefitting from not having mites injecting their weakened bodies with viruses.

Our Traditional Breeding Experiment

It’s clear that mite resistance is attainable, but it may take a while to “fix” the trait in our breeding population [[23]]. We’re gonna stick with Harbo and Harris’s suggestion to simply identify those colonies that by some means are able to prevent varroa from building up over the course of the season [[24]]:

We define mite resistance as the ability of a colony of honey bees to impede the growth of a population of V. jacobsoni. With this definition, a highly resistant colony of bees would cause a mite population to decline and then to either disappear or be maintained at a very low level. This is the breeding objective.

Exactly! Funded researchers can perform the tedious work. I just define the job description for our bees, and breed only from those that do the job, as evidenced by, however they do it, of maintaining mite counts of zero!

References

[1] Vandame, R, et al (2002). Parasitism in the social bee Apis mellifera: quantifying costs and benefits of behavioral resistance to Varroa destructor mites. Apidologie 33(5): 433-445.

[2] Borba R, et al. (2022) Phenomic analysis of the honey bee pathogen-web and its dynamics on colony productivity, health and social immunity behaviors. PLoS ONE 17(1): e0263273. “… our data failed to show any significant relationship between damaged mites and the mite infestation indices measured.”

[3] Conlon, B, et al (2019). A gene for resistance to the Varroa mite (Acari) in honey bee (Apis mellifera ) pupae. Molecular Ecology. 28. 10.1111/mec.15080.

[4] Scannapieco, A, et al (2016). Individual precocity, temporal persistence, and task-specialization of hygienic bees from selected colonies of Apis mellifera. Journal of Apicultural Science 60(1): 63–74.

[5] Eynard, S, et al (2020) Descriptive analysis of the Varroa non-reproduction trait in honey bee colonies and association with other traits related to Varroa resistance. Insects 11(8): 492.

[6] Boyle, E, et al (2017) An expanded view of complex traits: from polygenic to omnigenic. Perspective 169 (7): 1177-1186.

[7] Albert, FW, et al (2018) Genetics of trans-regulatory variation in gene expression. Elife 7:e35471. doi: 10.7554/eLife.35471.

[8] Albert, F & L Kruglyak (2015) The role of regulatory variation in complex traits and disease. Nature Reviews/Genetics 16:197.

[9] Ashe, A, et al (2021) How does epigenetics influence the course of evolution? Phil. Trans. R. Soc. B3762020011120200111.

[10] Gempe, T, et al (2009) Sex determination in honeybees: Two separate mechanisms induce and maintain the female pathway. PLoS Biol 7(10): e1000222. doi:10.1371/journal.pbio.1000222. “in the absence of the female-specifying signal, the male variant is produced that is the default regulatory state.”

[11] The developing worker larva is also affected by the presence of queen pheromone. Woyciechowski, M, et al (2017) Honeybee worker larvae perceive queen pheromones in their food. Apidologie 48: 144–149.

[12] He, X-J, et al (2019) A comparison of honeybee (Apis mellifera) queen, worker and drone larvae by RNA-Seq. Insect Science 26: 499–509. “For young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination.”

[13] I arbitrarily placed the csd gene on a fanciful chromosome. Bee drawings from: Kauffeld, M (1980) Seasonal cycle of activities in honey bee colonies. In, Beekeeping in the United States, Agriculture Handbook 335: 30-32.

[14] Harris J, R Danka, J Villa (2021) Honey bees (Hymenoptera: Apidae) with the trait of Varroa sensitive hygiene remove brood with all reproductive stages of Varroa mites (Mesostigmata: Varroidae). Ann Entomol Soc Am. 2010; 103(2):146–52.

[15] Corrêa-Marques, M, D de Jong (1998) Uncapping of worker bee brood, a component of the hygienic behavior of Africanized honey bees against the mite Varroa jacobsoni Oudemans. Apidologie 29 (3): 283-289.

[16] J Harris, R Danka, J Villa (2010) Honey bees (Hymenoptera: Apidae) with the trait of varroa sensitive hygiene remove brood with all reproductive stages of varroa mites (Mesostigmata: Varroidae). Annals of the Entomological Society of America 103(2): 146–152.

[17] Martin, S, et al (2020) Varroa destructor reproduction and cell re-capping in mite-resistant Apis mellifera populations. Apidologie 51: 369–381.

[18] Oddie, M, et al. (2021) Reproductive success of the parasitic mite (Varroa destructor) is lower in honeybee colonies that target infested cells with recapping. Sci Rep 11: 9133.

[19] Grindrod, I & S Martin (2021). Spatial distribution of recapping behaviour indicates clustering around Varroa infested cells. Journal of Apicultural Research 60(5); 707-716.

[20] I used Veet brand “Ready-to-use wax strip kit” (a gel adhesive used without heating).

[21] Eynard, S, et al (2020) op cit.

[22] Vandame, R, et al (2002) op cit.

[23] By us continually removing from the breeding population any queens who apparently didn’t carry alleles that conferred resistance to her colony.

[24] Harbo, JR & JW Harris (1999) Selecting honey bees for resistance to Varroa jacobsoni. Apidologie 30: 183-196.

Walking the Walk Selective Breeding for Mite Resistance; 2022 Update, Part 1

Contents

Resistance vs. Tolerance or “Survival” 1

Managed apiaries vs. natural evolution. 2

Background. 3

The necessity of Mechanical Agitators 3

The resistant colonies 6

So what’s our progress so far?. 10

A built-in lag inherent in open mating programs 13

Exhibit A: Mite-count tracking for our 2022 breeders 14

Coming. 16

References. 16

Walking the Walk

Selective Breeding for Mite Resistance; 2022 Update, Part 1

First published in ABJ June 2022

Randy Oliver
ScientificBeekeeping.com

I’ve been selectively breeding queens for many years. Breeding for color, gentleness, or AFB resistance was easy. Ditto for tracheal mite resistance, as well as for the ability to deal with Nosema ceranae. But breeding for varroa resistance has proven to be more difficult.

The obvious long-term solution to The Varroa Problem is to stock our hives with varroa-resistant bees. Varroa’s original host, Apis cerana, fits that bill. And left to their own, the hard hand of natural selective pressure applied to unmanaged bee populations has resulted in the evolution of resistant bees in various areas. But progress has been slower for those engaged in the selective breeding of managed European honey bees; their worldwide efforts are well reviewed by Le Conte [[i]]. So the question is, why is it taking so long for reliably mite-resistant bees to hit the market?

Resistance vs. Tolerance or “Survival”

Before I get going, please first allow me to clear up some terminology. I often hear people use the terms “resistance” and “tolerance” interchangeably. They actually have two very different biological definitions, as explained by Ayres and Schneider [[ii]]:

A host can evolve two types of defence mechanisms to increase its fitness when challenged with a pathogen — resistance and tolerance … Resistance is defined as the ability to limit pathogen burden while tolerance is defined as the ability to limit the health impact caused by a given pathogen burden. The sum of these two mechanisms defines a host’s defensive capacity.

There are “varroa tolerant” or “survivor” colonies, that may thwart the mite by maintaining tiny colony sizes, or by swarming or absconding frequently, or by fiercely attacking any invaders, but beekeepers are generally not interested in bees with those traits. Or “tolerant” bees may exhibit resistance to the mite-vectored viruses, even at high mite infestation rates.

But what I want are bees that take me back to the (pre-varroa) ”Good Old Days.” So I’m interested in bloodlines that “just say no” to varroa, and are by some means able to prevent the mites from successfully reproducing in their colonies.

Practical application: I’m breeding for resistance, not tolerance. A varroa-tolerant colony, due to its inability to control its mite population, will eventually suffer from the burden of its parasites and pathogens.

Managed apiaries vs. natural evolution

We might expect the hand of evolutionary pressure (natural selection) to come up with a “benign” mite, or a “stable” host-parasite relationship, but that’s not gonna happen so long as honey bees exist as managed livestock. This is because there’s no downside to varroa (or its associated viruses) if they inadvertently kill their host colony — so long as we keep providing fresh colonies to replace them.

In addition, our keeping of hives of bees closer than a mile apart means that mites can use the bees themselves as vectors to transmit those parasites to other hives in the vicinity. As pointed out by Ewald [[iii]], parasites that spread via flying vectors can be transmitted effectively from sick or dying hosts to new hosts, and therefore are not constrained from evolving to become even more virulent. (This would be the case whether we’re talking about the vector being mosquitoes, or drifting or robbing bees.) On the other hand, parasites that require their hosts to remain alive and healthy enough to transmit the parasite vertically to their offspring (in the case of bees, to their swarms), may develop a stable host-parasite relationship (as in the case of humans and our body lice or follicle mites).

Practical application: The above means that there’s no evolutionary pressure upon either varroa or Deformed Wing Virus to become less virulent — The Varroa Problem is not going to get better until we start keeping resistant bees. Thus our only long-term solution is to select for honey bee stocks that are able to reduce varroa’s reproductive success to the extent that it becomes only a minor pest in the colony. Selective breeding is simply human-directed evolution; in this case working with Nature for the benefit of our beloved (and beleaguered) honey bees.

Background

For those who have not been following my “Walking the Walk” series on breeding for varroa resistant bees, you can catch up here [[iv]].

Practical application: My sons and I are running a long-term proof of concept experiment, for the benefit of the commercial queen breeders/producers. My goal is to determine the plausibility/feasibility, labor cost, and rate of progress from a simple “traditional” breeding program, starting with one’s own mongrel stock.

Our experimental program involves managing a dedicated breeding population, by the open-mating (within that breeding population) of a yearly cohort of at least 1000 new queens, all bred from roughly 30 selected breeder queens from the previous year. The selected breeders each spring are chosen from colonies that exhibited the ability to hold their mite infestation rate to near zero over the course of an entire year (without any treatments), but just as importantly, continue to have the desirable properties of being gentle and productive.

I’m intentionally avoiding instrumental insemination, genetic analysis, brood dissection, diligent maternal line tracking, or other costly or time-consuming methods. Our only assessment method is taking mite washes (roughly 2000 per year), coupled with visual inspection for general colony qualities. Any colonies rejected as potential breeders are treated; no colonies need be lost to varroa.

The necessity of Mechanical Agitators

The key to this method is to be able to perform rapid-fire mite washes. This requires portable mechanical agitators, and a system that allows a crew to work together without confusion, described at [[v]]. Since that article, I’ve spent many hundreds (my wife says thousands) of hours in the shop, perfecting new generations of portable mite wash agitators (Figure 1).

Fig. 1 I built several of these Generation 2 portable mite wash agitators. My assistant Brooke Molina has her finger over the start button. The agitator then performs ~300 revolutions and shuts itself off after 60 seconds. We’ve validated that this agitation recovers of at least 95% of the mites.

The agitators above have served us very well, but when we shifted from using alcohol to Dawn detergent instead, I realized that I could improve them further. After building, testing, and fine-tuning many designs and prototypes, I’ve now developed Generation 4, with better cups, and a simpler design (Figure 2).

Fig. 2 The Gen 4 agitator is specifically designed for use with Dawn detergent, as well as for beekeepers to be able to build their own from off-the-shelf components. This model utilizes a sturdier screw-top jar, and we’ve validated 99% mite recovery in 60 seconds. It includes a pull-out magnifying mirror and holder for rapid counting of mites. I intend to publish the plans in an upcoming article, and may even make up kits for assembly.

Figure 2 shows the agitator sitting on a pull-out table that I built in the back of my Honda CRV, set up for us to rapidly get going when we pull into a yard. With these portable agitators, we can perform mite washes (including opening the hive, taking the sample, closing the hive, agitation and counting, and writing the mite count on top of the hive) at the rate of 3-4 man-minutes per hive (a crew of three of us can easily determine the mite count for every colony in a yard of 48 hives in less than an hour). At $25 per hour, that works out to about a buck and a half labor cost to determine each colony’s infestation rate (that’s less than the cost of some treatments). And we can make an immediate management decision for each colony.

Practical application: Without such portable agitators, performing the number of mite washes necessary for our breeding program wouldn’t be feasible. We find that our savings from not spending money on unneeded mite treatments, as well as the identification of colonies that need more than “the usual” treatment (or that have other issues), more than pays for the cost of labor involved in performing the mite washes.

The resistant colonies

The thing that keeps us going with this demo experiment is the beauty of the colonies that we’ve identified as being mite-resistant — which, if we hadn’t performed mite washes on them, we wouldn’t have known even existed in our operation! (Figure 3).

Fig. 3 The index card on this booming double-deep colony after almonds (with an undisturbed green drone frame), shows the counts for mites in samples of a half cup of bees, taken over the course of a year. (We allowed the mites to build up from the starting nuc in April until the first wash in July.) It’s not unusual for the count to spike in November as the colonies briefly go broodless. Even the count of 7 is only about a 2% infestation rate as the bees go into winter. In the case of this colony, the count expectedly dropped again by the March assessment, after the colony had returned from pollination, at which time most of the mites were back in the brood. For what it’s worth, although it needed no treatments, this colony would not have passed the bar to make it as a breeder.

Of our “potential breeder” pool of resistant colonies each year, to stay in the running, a colony must not only maintain a mite count of near zero for the course of an entire year, but also be gentle, healthy, build up quickly, and be productive as far as honey. That subsequently eliminates a goodly portion of those colonies marked as “potential breeders” at the first assessment.

We take most all of our potential breeders to almond pollination. When I take their mite counts in March, any that are not busting at the seams with bees, or that did not put on plenty of honey in the orchards, are no longer considered as breeders.

Practical application: The remaining breeder pool that we actually graft from (around 30) are the queens of colonies that not only completely controlled varroa, but that any beekeeper would be delighted to have in their own operation, as evidenced by the responses of the several large professional beekeepers who have assisted me in breeder selection (Figure 4).

Fig. 4 This colony laughed at varroa. Brooke said it all one day when she quipped “Zeroes are heroes.” Note the lack of a November spike in count. We didn’t recover a single mite in any of the four samples taken from this colony. If its count was still 1 or less in March, it would be considered as a breeder (at low mite infestation rates, there’s virtually no statistically-significant difference between a count of 1 or 0).

Practical application: The mite counts of this colony are impressive, since a non-resistant untreated colony under our management system will typically reach a mite count of well over 50 by September. Unfortunately, we’ve found that the mite resistance of a colony as a whole does not necessarily predict the performance of daughter colonies. For most of these multiple-zero colonies, daughters bred from their queens only exhibit the breeding population average for resistance. So far we haven’t seen a strong degree of heritability for the trait.

The above frustrating fact may be about to change, since the daughters of a couple of last year’s queens exhibited strong heritability, with up to 50% of their daughter colonies showing resistance. You may have guessed that we’ll be breeding heavily from them this season! In any case, we’re continually impressed by how well our Zero Heroes perform.

PROOF OF CONCEPT: Our mite-resistant colonies are often the strongest and most productive colonies in a yard. Many ask me whether we sacrifice gentleness for mite resistance (Figure 5).

Fig. 5 Eric snapped a shot of me as I performed August mite washes in hot weather. I often knock out 50 mite washes in an afternoon. It’s easy to perform the bioassay for gentleness — if I start getting stung, that colony doesn’t make grade.

So what’s our progress so far?

In a recent project by De la Mora [[i]], working with the Ontario Queen Breeders Association, they appeared to obtain a substantial increase in mite resistance after only two generations, using a selection and breeding method similar to mine. That rate of success is better than that of any other breeding program that I’m aware of, so I hope that their good luck continues! For the rest of us, it’s a much slower slog. My sons and I are going into our sixth year of very strong selection.

I recently realized that we may have been inadvertently kicking a proportion of potentially-resistant colonies out of the program at our first mite wash assessment in June or July, when we go ahead and treat any colony with a count of more than 1 mite (we rejected anything above zero in our first years). At that low of an infestation rate (1 mite per 315 bees equals a 0.317% infestation rate — that’s less than a third of a percent), we can calculate the probability of getting different mite wash counts (Figure 6).

[i] De la Mora A, et al. (2020) Selective breeding for low and high Varroa destructor growth in honey bee (Apis mellifera) colonies: Initial results of two generations. Insects 11(12):864.

Fig. 6 I used the binomial calculator at stattrek.com to work the figures. At an average mite infestation rate of a 1 mite per 315 bees (probability of 0.00317), the chances of getting a wash count of zero is 37%; of seeing exactly 1 mite also 37%; leaving a 26% chance of getting 2 or more mites in a wash. That’s a 26% chance of rejecting a potential breeder right off the bat!

Since our records indicate that roughly half the colonies with mite counts of 1 or 0 in June maintain counts below 7 by the next spring, that means that we’ve likely been excluding (and treating) a lot of what were actually resistant colonies, since we’ve been rejecting a very large number of colonies in June or July that had counts of 2 (Figure 7).

Fig. 7 Out of curiosity, I didn’t treat a few colonies that had mite counts of 2 in early July last year to see where they’d go. The above tag answered that question for this colony! My guess is that we’ve been overlooking a large number of colonies that were actually resistant.

Practical application: It’s a goodly amount of work to perform the first washes on well over 1000 hives in June or July. We’ve applied severe and unsparing selective pressure at the first wash, in order to reduce the number of hives to follow up on. But as with Dr. John Kefuss’ experience, we’ve also observed that resistant colonies are often able to take mite counts back down on their own. So it boils down to how many potential breeders you want to track with additional washes. Luckily, even those resistant but rejected (and thus treated) colonies will still wind up producing drones for next year’s matings.

So where do we stand? Last year was a tough year for our bees. They got hit by severe drought, forest fires, flooding, snow and falling tree damage, bear damage, and then a cold snowstorm just after they started ramping up broodrearing prior to almond pollination. We lose very few colonies to mites, but last year the environment was devastating to our bees. Eric and Ian were barely able to fill our pollination contracts for almonds (although those that went graded well).

That said, of the thousand-plus colonies returning from almonds, we still had 126 tracked (and untreated) potential breeders that exhibited mite wash counts in March of less than 7 (less a 2% infestation rate after an entire year without treatment). Figuring in weather, fire, and bear losses of marked potential breeders, and factoring in our over-culling at our first summer wash, I estimate that between 15% to 20% of our 2021 colonies exhibited strong mite resistance (Figure 8).

Fig. 8 The above chart reflects my “best guess” proportions of varroa-resistant colonies in our operation over the years. And in one yard this year, we hit 50%. We’ve still got a ways to go until we get to the 95% level at which I might start claiming that our stock is “mite resistant,” but our progress to date is encouraging. Will we slowly make linear progress (the solid red line), or will our results continue to track exponentially (the dashed red line)? I’ll return to this question in my next article.

Practical application: Compared to selective breeding programs to improve the productivity of agricultural crops or animals, which involve changing the morphology, physiology, and energy efficiency of the organisms, to breed for mite resistance, we may only need to flip a couple of behavioral switches. Maybe someone snuck some cannabis leaf into my smoker, but I’m crossing my fingers that our progress will continue to track the dashed line!

A built-in lag inherent in open mating programs

In a breeding program, one can perform either positive selection or negative selection (or a combination of the two). In our own breeding population, in which 99%+ of the colonies would be considered as being “gentle,” it’s easy to practice negative selection against the occasional queen whose colony turns out to be a bit spicy. On the other hand, if we were breeding solely for honey production, we might positively select as breeders only the queens from the few colonies that put on exceptional amounts of honey. In the case of breeding for varroa resistance, we’re doing both — breeding only from the queens whose colonies were exceptionally good at keeping mites in check (positive selection), while simultaneously removing from the breeding population any queens whose colonies appeared to require treatment or didn’t make much honey (negative selection). But unfortunately, there’s a built-in two-year lag for such negative selection, which acts as a drag against the rate of progress of our program.

Understand that the genetics of drones come only from their mothers (whose alleles in turn consist of a mixture of those from their own mothers and fathers). Therefore, since we breed only a single generation of queens each year, the alleles of the drone pool that they will mate with will come from those drones’ grandfathers and grandmothers — reflecting the genetics of the breeding population from two years earlier. Thus our “directed evolution” of the genetics of the drone pool will always lag behind that of the genetics of the breeder queens selected each year. The genetics of the females of the resulting colonies come 50% from the chosen breeder queens of that year, and 50% from the drones whose genetics reflect that of their grandparents. (Yes, go ahead and reread and digest this paragraph.)

Practical application: In a natural breeding population, this genetic conservation of alleles from several generations back helps to prevent dangerous genetic bottlenecking in the case of a sharp reduction in the size of a breeding population of bees due to environmental events such as severe drought or other weather event, landscape-scale fire, or a devastating disease (the stored mixture of spermatozoa in any surviving queen’s spermatheca conserves the local population’s genetic diversity). Unfortunately, this built-in conservation of alleles also slows down the progress of any human-directed selective breeding program that uses open mating, since the alleles of each year’s drone pool reflects the alleles of the population from two years earlier.

Exhibit A: Mite-count tracking for our 2022 breeders

So after taking mite washes after almonds, I needed to select this year’s breeders. I’m guessing that you might like to see how their mite counts tracked over the course of the year (Figure 9).

Fig. 9 In 2021, in many of our yards mite counts didn’t get high enough for selection until July or even August, and some didn’t even get checked until November. Note that over a third of this season’s breeders exhibited mite counts of zero after a full year without treatment (their prior count histories indicate that those zero counts in March weren’t flukes).

The yard abbreviations indicate different queen bloodlines, so in order to maintain genetic diversity, I included breeders from a diversity of yards, which required me to include a few with March counts of above 1. We will again requeen our entire operation this year with daughters from these queens, especially those from Yard V (in which 24 of the 48 colonies exhibited strong resistance). I’m especially interested in that bloodline, which I plan to use for some isolated-yard inbreeding later this season.

Coming

Next month I’ll take a dive into our resistant colonies’ mechanism for resistance and the genetics involved.

References

[1] Le Cont, Y, et al. (2020) Geographical distribution and selection of European honey bees resistant to Varroa destructor. Insects 11: 873. doi:10.3390/insects11120873

[2] Ayres, J & D Schneider (2008) Two ways to survive an infection: what resistance and tolerance can teach us about treatments for infectious diseases. Nat Rev Immunol. 8(11): 889–895.

[3] Ewald, P (2004) Evolution of virulence. Infectious Disease Clinics 18(1): 1-15.

[4] 2017 plan: https://scientificbeekeeping.com/the-varroa-problem-part-6a/

2018 update: https://scientificbeekeeping.com/selective-breeding-for-mite-resistance-1000-hives-100-hours/

2019 update: https://scientificbeekeeping.com/selective-breeding-for-mite-resistance-walking-the-walk/

[5] The Varroa Problem: Part 10-Smokin’-Hot Mite Washin’ – Scientific Beekeeping

[6] De la Mora A, et al. (2020) Selective breeding for low and high Varroa destructor growth in honey bee (Apis mellifera) colonies: Initial results of two generations. Insects 11(12):864.

2022 Extended-release Oxalic Update Part 3

Contents

The slow effect of OaE. 1

Mite turnover 3

A bothersome question. 5

You don’t need to kill a single mite in order to control varroa! 5

Varroa’s world: Olfaction, taste, and touch. 7

Disruption of mite sensory perception. 9

“Blinding” or irritant?. 9

A relevant question. 9

Wrap Up. 10

Citations and notes 11

2022 Extended-release Oxalic Update

Part 3

First published in ABJ May 2022

Randy Oliver
ScientificBeekeeping.com

Although I’ve worked extensively with extended-release oxalic acid (OAE) for the past several years, there’s still plenty more to learn about this potentially game-changing treatment for varroa.

I’ve been amazed by how a single application of a pad containing OA dissolved in glycerin, given to a colony badly infested with varroa, can with time completely zero out its mite count, with no apparent adverse effects upon the colony. This observation raises the questions of exactly how the treatment works (mode of action), and why it takes so long.

The slow effect of OaE

There’s no question that oxalic acid (OA) is able to kill mites. An application by dribble (OAD) or vapor (OAV) can cause a rapid spike in varroa mortality, as evidenced by the drop of dead and dying mites onto a stickyboard. But that effect largely wears off after three days. On the other hand, an application of extended-release OA in glycerin (OAE) takes a little longer to ramp up the mite drop, and that elevated drop rate then extends for about a month. Let’s look a little deeper into this difference.

I’ve received data sets of mite drop counts after OAV from a number of beekeepers, Donald Aiken’s being representative (Figure 1).

Fig. 1 Aiken recorded the daily counts of fallen mites following six OAV applications. Note that the daily mite drop immediately spikes upward in the first 24 hours after treatment, and then quickly tapers off over the next three days — despite the continual emergence of mites from the brood each day. This pattern is typical, and indicates a short-term residual action of OAV.

Compare the above to data to that for OAE strips (Figure 2).

Fig. 2 Dehaibes [[i]] applied Aluen CAP extended-release OA strips to 19 colonies. The graph above shows the average daily mite drop [[ii]]. Unlike the short-term spike observed immediately following an OAV or dribble, an application of OAE takes a few days to exhibit full effect, but then continues to show residual activity for up to a month.

Practical application and question: My own limited data (not shown) closely reflect that of Dehaibes, including the two bumps roughly ten days post-application. (I’m currently working on modeling to figure out why this occurs.) However, with the lower-glycerin ratio that I use, the proportional increase in mite drop is lower than that from the strips that they used. But the big question is, if increased mite drop occurs for only a month, then why does it take two months to attain full efficacy?

Mite turnover

When we look into the exposure of mites in the hive to the applied oxalic acid, we must take into account the daily “turnover” of mites as they rotate into the brood for reproduction, and then emerge ~12 days later to disperse (the “phoretic” phase). So let’s put the above mite drop graphs into perspective by looking at some numbers for a high-mite colony (much higher than Dehaibes’ hives) in mid-July, estimated by using my mite model [[iii]] (Table 1).

[i] Dehaibes, S, et (2020) Control of Varroa destructor development in Africanized Apis mellifera honeybees using Aluen Cap (oxalic acid formulation). International Journal of Acarology. 46(6): 405-408.

[ii] I scaled the numbers off Dehaibes’ graph, and took the liberty of using the counts from their Control group as a baseline value.

[iii] From https://scientificbeekeeping.com/randys-varroa-model/

Practical application: The above figures indicate that in a high-mite late-summer colony, there is a turnover of around 650 mites emerging from the brood each day. This helps to explain why an oxalic vaporization treatment can cause a very high mite drop for a few days, since those emerging mites are being newly exposed to the treatment. But the residual activity of the vaporization treatment quickly wears off. Compared to OAV, the slow release of OAE would be expected to cause a lower initial mite drop, but result in an longer-term elevated rate of mite drop as mites emerge from the brood day after day.

The data from Dehaibes and Maggi [[i]] indicate that only around 70% of the mites in a hive drop during the first two weeks of treatment with OAE, despite the fact that any mites in the brood would have rotated out onto the adult bees during that period. Curious, I graphed out the effect upon a colony’s total mite population from applying a treatment that killed a certain percentage of only the ever-rotating population of “phoretic” mites each day (Figure 3).

^[i] Maggi, M, et al (2015) A new formulation of oxalic acid for Varroa destructor control applied in Apis mellifera colonies in the presence of brood. Apidologie 47(4): 596-605.

Fig. 3 With zero kill of the phoretic mites, the overall varroa population would double in a month. Killing 5% of the phoretic mites each day arrests varroa population increase. But it takes killing 25% of the temporarily phoretic mites each day to eliminate most of a colony’s mite population within a month. Note that the 25% kill rate plot closely matches the 70% reduction at two weeks recorded by Dehaibes.

Practical application: With the “gentler” and slower-acting 1:1 ratio of OA to glycerin that I’ve mostly tested, the kill rate appears to be lower, since it takes at least two full months to attain full mite reduction. The tradeoff is that a high-glycerin ratio appears to cause a more rapid mite reduction, but may have more adverse effects upon the bees and brood (as well as being messier to handle).

The above kill calculations appear to explain everything, other than one bothersome question …

A bothersome question

This brings me back again to my observation that the infestation rate (as measured by alcohol wash) of the adult bees actually increases during the first month of treatment, despite the elevated rate of mite mortality (as indicated by mite drop). If the treatment were actually killing 25% of the exposed mites every day, we’d expect to see the infestation rate of the adult bees quickly decline, since a daily 25% kill far exceeds the expected 7% daily emergence of replacement mites from the brood.

This makes me wonder whether OAE is affecting the mites in some manner that prevents them from successfully reentering brood cells to reproduce. And this brings us to the concept that:

You don’t need to kill a single mite in order to control varroa!

Practical application: All mites will eventually die of old age. Unless their reproductive rate is greater than their rate of natural mortality, their population cannot increase.

To control varroa, all that you need to do is to halve their success at reproduction (Figure 4).

Fig. 4 I ran two simulations, with identical inputs, other than reducing the average reproductive success of foundress mites in worker brood cells (indicated in the green text boxes). Note that in the lower simulation, in which I reduced the mites’ reproductive success by half, varroa never became an issue.

Note: Although the mite population in the lower half-rate simulation did end up higher than the starting count by the end of the year, additional runs indicated that at the lower rate of reproduction, it would take 3-4 years for the mite population to build up high enough to cause colony death.

Practical application: It may be constructive to not limit ourselves to treatments that result in the immediate killing of adult mites, and consider long-term treatments that hamper the mites’ ability to successfully reproduce.

Could OAE, due to affecting the mites’ sensory papillae, be hampering them from reentering the safety of brood cells, which would perhaps result in (1) more chance of exposure to a fatal dose of OA, and/or (2) lessen their ability to successfully reproduce? Could those combined effects then be the reason that OAE (1) takes so long to reduce the infestation rate on the adult bees, but (2) is able to eventually zero it out completely?

So how might this work?

Varroa’s world: Olfaction, taste, and touch

Varroa mites are adapted to live in total darkness, and lack eyes. They are completely dependent upon their senses of smell (olfactory), taste (gustatory), and touch (tactile/mechano-reception), as well as for temperature and humidity (thermo- and hydro-reception) in order to not only identify suitable adult bees to catch a ride on, but also for each step involved in the critical recognition of a suitable cell containing a 5th-instar larva to hop into for reproduction.

But mites also lack antennae. Instead, they employ only six of their eight legs for walking, and use their two longer front legs similar to how insects use their antennae. On the tips of those front legs are delicate sensory papillae, through which they experience their world (Figure 5). They have additional sensory papillae on their pedipalps, mouthparts, and other places on their bodies.

Glossary: papilla — a small protuberance; if hair-shaped, called a sensillum.

Fig. 5 I snapped this shot of the underside of a mite’s front leg. The balloon-like inflatable and sticky empodium is to the upper right. Below that, at the tip of the leg, is the “sensory pit organ,” consisting of nine internal papillae, with nine longer hair sensilla surrounding the organ. Some of the sensilla are “wall-pore sensilla,” presumably for the perception of volatiles. Other sensilla are “non-pore sensilla” which serve as tactile, hygro- or thermo-receptors, whereas the morphology of a third type indicates a gustatory function [[i]^,[ii]]. You can view far more detailed scanning electron micrographs of these organs at [[iii]^{, [iv], [v]}].

Practical application: If a treatment affects the function of these sensitive papillae, it would be like blinding a mite and holding its nose, leaving it relatively helpless.

Adult mites outside of a brood cell face a dangerous environment. A mite needs to quickly identify a nurse bee [[vi]], so that it can feed on the nurse’s well-developed fat bodies while it hitches a ride, in order to develop its own ovary and first egg (this ride also allows time for the sperm that the mite received from her brother to mature). A mite can rapidly scamper from bee to bee, but must avoid being bitten or groomed off, so it must somehow locate a space between the abdominal sternites of its unwilling mount, where it can dig in and safely spend its time while it enjoys a meal.

But it can’t stay dug in forever — once her ovary is developed, the mite needs to reproduce, so as the nurse bee carries her from cell to cell as she is smelling and feeding larvae, the mite is simultaneously “sniffing” for the kairomonal signal indicating whether the cell contains a 5th-instar larva calling to be capped over. The mite must then quickly jump off the nurse and onto its new “reproductive host” (soon to be a pupa). Thus in general, a mite is generally opportunistic, waiting for the nurse that it’s riding on to stick her head into a cell containing a 5th-instar larva. However, a mite may on occasion dismount from its ride to actively seek a cell (questing behavior). The way that it finds its way around is well described by Dillier [[vii]]:

Varroa uses its two front legs in the same way as insects use their antennae. These legs are only rarely used for movement and are more frequently displayed in the air.

During many hours of observation of brood frames to collect infested , we repeatedly observed mites leaving nurse bees and running several centimetres over the comb surface before disappearing. [Another researcher] regularly saw Varroa mites walking on undisturbed comb surfaces, … walking along open cell rims, often dipping into the lumen of a cell and then alternate to the other side to dip into the lumen of the next cell and so on.

Practical application: Any beekeeper who’s spent much time looking at combs of mite-infested colonies, soon realizes how rare it is to actually observe a mite walking over a comb surface (or even on the back of a bee). Most all the mites in a hive are either hidden on the bellies of nurse bees, or safely ensconced in a brood cell. It’s when they are forced to move about in the colony that they are most vulnerable.

The mite also has another challenge. If at some point the nurse bee that it’s riding on transitions to mid-age or foraging behavior, the bee’s cuticular hydrocarbon smell changes, which cues the mite that it must then identify a new nurse, and then take the risk of moving from the bee that it’s riding on to a new (and again unwilling) ride in order to have a chance of being carried into a brood cell.

Practical application: Does treatment with OAE somehow interfere with the mites’ ability to identify the right age of bees to hitch rides on, or the right cells to invade?

Disruption of mite sensory perception

I’m hardly the only one who’s thought of this. It’s also occurred to other researchers, notably Dr. Victoria Soroker and her associates, that we might be able to come up with treatments that disrupt the mites’ sensory perception, and thus their ability to successfully get around and reproduce [[viii]]:

Hive volatiles, mainly from adult bees and brood, play a crucial role in the parasite’s life cycle, by guiding host finding, selection and regulating its reproduction, suggesting that the mite’s olfaction may be an important target for new specific control agents … Inhibition of host sensing leads to incorrect Varroa host selection or reduction in mite’s ability to reach a host.

Practical application: If we could find a sustained-release treatment that hampered the ability of a mite to get around in the hive and locate 5th-instar larvae, this might be the Achilles’ heel of varroa that we’ve been looking for.

But wouldn’t olfactory disruption also affect the bees also living within the darkness of the hive? Perhaps not. Mites are not closely related to insects, and use some different “chemosensory or odor-binding proteins” than do honey bees [[ix]^{, [x]}].

Practical application: There’s a chance that we might identify treatments that affect varroa odor-binding proteins, but not those of the bees. This is a question when we apply treatments, such as thymol, formic, or oxalic acids, that affect cell membranes. Do these strong chemicals affect the sensory perception of mites (which would help us) or the bees (which may be harm the colony), and if so, do they affect one more than the other?

“Blinding” or irritant?

Let’s get back to oxalic acid. It can clearly exhibit acute toxicity to varroa. It’s possible that a sublethal dose might affect mite olfaction. But could it perhaps exhibit a simple irritant effect upon the mite that disrupts their movement?

Could the light “dressing” of OA onto bees’ bodies from either OAV or OAE (which doesn’t appear to be bothersome to bees) irritate the mite’s delicate footpads (empodia)? Could it be that having a tiny amount of OA on a bee’s body “hairs” thus make a mite hesitant to come out of its “safe spot” between the abdominal plates to walk about?

Practical application: Varroa mites must routinely “rotate” between the “phoretic” phase on adult bees, to the “reproductive” phase in capped brood, as well as rotating off its ride as she ages. Could OAE somehow disrupt this rotation? We could use more research along this avenue!

A relevant question

Beekeeper Jeff Steenbergen asked me whether “the presence OA crystals has an off-gassing type effect on mite reproduction, or if the bees really need to move it around the hive?” I started to answer that that OA wouldn’t evaporate at hive temperature. But then I remembered that (to my surprise) I previously found that Apivar strips unexpectedly appear to exhibit vapor action [[xi]].

So I duplicated the setup that I used to test for vapor action of amitraz, by placing some mite-infested bees into two cup cages, one with a layer of OA crystals separated from the bees (and mites) by a screen, and placed them in an incubator at 80°F, 65% RH, in darkness, for three days, feeding 1:1 sucrose (Figure 6).

[i] Rosenkranz, P, et al (2010) “Biology and control of Varroa destructor.” Journal of Invertebrate Pathology 103: 96-119.

[ii] Singh, N, et al (2016) Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction. Insect Mol. Biol. 25: 181-190.

[iii] Dillier, F-X, et al (2006) Review of the orientation behaviour in the bee parasitic mite Varroa destructor: Sensory equipment and cell invasion behaviour. Revue suisse de zoologie; annales de la Société zoologique suisse et du Muséum d’histoire naturelle de Genève 113(4):857-877

[iv] Soler, MD, et al (2005) Scanning electron microscopy of foreleg tarsal sense organs of the poultry red mite, Dermanyssus gallinae (DeGeer) (Acari : Dermanyssidae). Micron 36(5):415-21.

[v] Iovinella, I, et al (2018) Proteomic analysis of chemosensory organs in the honey bee parasite Varroa destructor: A comprehensive examination of the potential carriers for Semiochemicals. Journal of Proteomics 181: 131-141.

[vi] Xie, X, et al (2016) Why do Varroa mites prefer nurse bees? Scientific Reports 6: 28228.

[vii] Dillier, F-X, et al (2006) op cit.

[viii] Soroker, V, et al (2019) Olfaction as a target for control of honeybee parasite mite Varroa destructor. In: Picimbon, JF. (eds) Olfactory Concepts of Insect Control – Alternative to insecticides. https://doi.org/10.1007/978-3-030-05060-3_6

[ix] Iovinella (2018) op cit.

[x] Eliash, N, et al (2019) Varroa chemosensory proteins: some are conserved across Arthropoda but others are arachnid specific. Insect Mol Biol. 28(3): 321-341.

[xi] Questions on Amitraz. https://scientificbeekeeping.com/6501-2/

Fig. 6 You can see the OA crystals on the bottom of the Test cup to the left. After 3 days, zero mites had dropped in either cage. A mite wash afterwards confirmed that there were still live mites on the bees in both Test and Control cups. This non-replicated “quick and dirty” test didn’t directly answer whether reproduction of the mites might be affected by vapor action, but presence of OA near the bees certainly didn’t cause the mites to fall off.

Practical application: The above quickie test (performed during winter when it was difficult to find an infested colony) suggests that OA crystals don’t exhibit vapor action upon adult mites. However, it’s possible that the oxalic esters formed when the acid is dissolved in glycerin, or the trace amounts of OA that I can titrate off the outside of bees’ bodies, exhibit some sort of olfactory-disruptive, or foot-irritant properties to the mites. I’ll leave it to some postdoc to run a proper study to determine whether OAE might affect mite reproductive success.

Wrap-Up

As far as I’m concerned, we’re still low on the learning curve on how best to apply and utilize OAE, and in understanding its actual mode(s) of action. I appreciate feedback from other researchers and permitted beekeepers working with it!

News flash: EPA’s position on experimental use of EOA

I have received numerous requests from “citizen-science” beekeepers across the country, wanting to run their own field trials of extended-release oxalic acid in their particular environments.  For my research, I obtain a “Pesticide Research Authorization” from my own State Lead Agency each year, but other beekeepers have reported that their respective SLA refers them to EPA to obtain an “Experimental Use Permit” (EUP).

Gina Burnett, Senior Regulatory Advisor for the EPA’s Biochemical Pesticides Branch, was gracious enough to go over the regulations with me (relevant verbiage highlighted):

§ 172.3 Scope of requirement.

(a) An experimental use permit (EUP) is generally required for testing of any unregistered pesticide or any registered pesticide being tested for an unregistered use. However, as described in paragraph (b) of this section, certain of such tests are presumed not to involve unreasonable adverse effects and, therefore, do not require an EUP.

(b) Except as provided in subpart C of this part or as specifically determined by the Environmental Protection Agency (EPA), it may be presumed that EUPs are not required when:

(1) The experimental use of the pesticide is limited to:

(i) Laboratory or greenhouse tests,

(ii) Limited replicated field trials as described in paragraph (c) of this section to confirm such tests, or

(iii) Other tests as described in paragraph (c) of this section whose purpose is only to assess the pesticide’s potential efficacy, toxicity, or other properties.

(2) The producer, applicator, or any other person conducting the test does not expect to receive any benefit in pest control from the pesticide’s use.

(c) For purposes of paragraphs (b)(1)(ii) and (b)(1)(iii) of this section, the following types of experimental tests are presumed not to need an EUP:

(1) A small-scale test involving use of a particular pesticide that is conducted on a cumulative total of no more than 10 acres of land per pest, except that:

(i) When testing for more than one target pest occurs at the same time and in the same locality, the 10 acre limitation shall encompass all of the target pests.

(ii) Any food or feed crops involved in, or affected by, such tests (including, but not limited to, crops subsequently grown on such land which may reasonably be expected to contain residues of the tested pesticides) shall be destroyed or consumed only by experimental animals unless an appropriate tolerance or exemption from a tolerance has been established under the Federal Food, Drug, and Cosmetic Act (FFDCA) for residues of the pesticide.

Since the FDA has ruled that “Residues of oxalic acid in or on honey and honeycomb are exempted from the requirement of a tolerance when oxalic acid is used as a miticide in honeybee hives,” the EPA does not have any restrictions as to whether the honey can be harvested and consumed.

Bottom line: Unless your State has more restrictive requirements, you would not need to obtain an EUP from EPA to run small-scale trials with oxalic acid.  Be clear that this only applies to use for experimental testing!

Citations and notes

[1] Dehaibes, S, et (2020) Control of Varroa destructor development in Africanized Apis mellifera honeybees using Aluen Cap (oxalic acid formulation). International Journal of Acarology. 46(6): 405-408.

[2] I scaled the numbers off Dehaibes’ graph, and took the liberty of using the counts from their Control group as a baseline value.

3] From https://scientificbeekeeping.com/randys-varroa-model/

^[4] Maggi, M, et al (2015) A new formulation of oxalic acid for Varroa destructor control applied in Apis mellifera colonies in the presence of brood. Apidologie 47(4): 596-605.

[5] Rosenkranz, P, et al (2010) “Biology and control of Varroa destructor.” Journal of Invertebrate Pathology 103: 96-119.

[6] Singh, N, et al (2016) Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction. Insect Mol. Biol. 25: 181-190.

[7] Dillier, F-X, et al (2006) Review of the orientation behaviour in the bee parasitic mite Varroa destructor: Sensory equipment and cell invasion behaviour. Revue suisse de zoologie; annales de la Société zoologique suisse et du Muséum d’histoire naturelle de Genève 113(4):857-877

[8] Soler, MD, et al (2005) Scanning electron microscopy of foreleg tarsal sense organs of the poultry red mite, Dermanyssus gallinae (DeGeer) (Acari : Dermanyssidae). Micron 36(5):415-21.

[9] Iovinella, I, et al (2018) Proteomic analysis of chemosensory organs in the honey bee parasite Varroa destructor: A comprehensive examination of the potential carriers for Semiochemicals. Journal of Proteomics 181: 131-141.

[10] Xie, X, et al (2016) Why do Varroa mites prefer nurse bees? Scientific Reports 6: 28228.

[11] Dillier, F-X, et al (2006) op cit.

[12] Soroker, V, et al (2019) Olfaction as a target for control of honeybee parasite mite Varroa destructor. In: Picimbon, JF. (eds) Olfactory Concepts of Insect Control – Alternative to insecticides. https://doi.org/10.1007/978-3-030-05060-3_6

[13] Iovinella (2018) op cit.

[14] Eliash, N, et al (2019) Varroa chemosensory proteins: some are conserved across Arthropoda but others are arachnid specific. Insect Mol Biol. 28(3): 321-341.

[15] Questions on Amitraz. https://scientificbeekeeping.com/6501-2/

2022 Extended-release Oxalic (OAE) Update Part 2

Contents

An additional test of matrices and OA:gly ratios. 1

Cardboard Strips vs. Sponges. 3

Compostability of spent sponges. 3

A winter field trial 4

Methods. 5

Results. 9

Discussion. 11

The Elephant in the Room.. 12

Reality Check. 12

A possible easy solution for getting us legal 13

Citations and notes. 15

2022 Extended-release Oxalic (OAE) Update

Part 2

First published in ABJ April 2022

Randy Oliver
ScientificBeekeeping.com

I continue with my research on various application methods of extended-release oxalic acid (OAE), and my efforts to get the method registered with EPA.

An additional test of matrices and OA:gly ratios

I get suggestions from all over the world for absorbent matrices to test. So come autumn, I rustled up enough “leftover” colonies with elevated mite counts to run a small comparative trial. I wanted to compare Maximizer strips to ShamWow towels, and to New Zealand chipboard (cardboard) hung strips. In addition, since New Zealanders typically use a higher-glycerin 1:1.5 ratio, I gave that ratio a try on both the hung chipboard and the Maximizer strips.

The colonies were in double deeps, in poor shape due to dearth and mites, with 5-10-frame clusters. With help from visiting beekeepers Jim Veitch, Jennifer Radke, and Catherine Edwards, we applied the test strips and fed each colony a patty of pollen sub. Each hive got four 1¾“ x 7½” strips of Maximizer (lightweight) or ShamWow rayon microfiber cloth, laid across the top bars, or three chipboard strips (the recommended amount) hung over the top bars in each brood chamber (Figure 1). The treatments were assigned in a randomized block design, blocked by starting mite count.

Fig. 1 I used Beequip chipboard strips from New Zealand, soaked in a 1:1.5 ratio of OA:gly, by weight. New Zealand supply houses carry a variety of such precut strips.

I ran the trial for 77 days, taking mite wash counts at start and finish (Table 1).

Practical application: All the tested matrices exhibited good mite reduction, with the Maximizer 1:1 and chipboard 1:1.5 strips having the most ending counts of zero or 1. Although Maximizers at the 1:1 ratio looked best (in either raw or worked data), I would like to test chipboard strips at the 1:1 ratio.

Chipboard Strips vs. Sponges

I’ve been focusing upon absorbent pads that can be quickly laid across the top bars of the lower brood chamber, rather than chipboard strips which need to be inserted between the frames of both brood chambers (which is far more time-consuming). But there are situations (such as in singles) in which there is not enough exposure area to the upper surface of a delivery pad to be effective, so the treatment would then need to be inserted between the frames. Chipboard strips are definitely easier to insert than are soft matrices such as sponges or Maximizers.

Practical application: One would expect that strips hung between the frames would have more contact with the bees’ bodies as they squeeze by, and thus be more effective at distributing the OA/gly onto the workers. Surprisingly, that does not appear to be the case. For full efficacy of OAE with pads laid across the top bars between the brood chambers, it takes about 55 sq in (355 cm²) of matrix surface area, holding 50 g of OA dissolved in 50 g of glycerin, per double-deep hive. For cardboard strips hung over the frames, based upon data from Aluen CAP, Dan Aurell, the New Zealand beekeepers, and my own, it actually requires more surface area when using strips (80-115 sq in/ 520 – 740 cm² per double-deep hive). Go figure!

Compostability of spent sponges

Once an OAE matrix has largely dispensed its OA, it needs to be pulled or scraped out of the hive, since the bees generally don’t remove them. The spent strips still contain some amount of OA, and need to be handled appropriately during disposal. Some pieces invariably fall on the ground, so I favor using biodegradable strips, so that we don’t leave non-biodegradable plastic pollution in our yards (the ShamWows may not fit this bill [[1]]).

If a fallen strip gets hit by rain, the OA gets washed out, and the remaining cellulose gets treated by decomposition organisms similarly to that in a fallen leaf. I wondered whether a spent Swedish sponge still containing OA and glycerin would get decomposed if added to a compost pile. So I buried a few in an active compost pile in my garden (Figure 2).

Fig. 2 After a few weeks, I had trouble locating any remnants of the pink sponges, since they were well along in the process of being decomposed.

Practical application: I abhor the way that we’re polluting our environment with plastic, so prefer using biodegradable cellulose matrices.

A winter field trial

By this time, we’ve gotten a good idea about the amount of OAE pad required for good efficacy against varroa during the summer, when colonies have an active broodnest. I also had preliminary data that it could be efficacious during winter and didn’t appear to harm the colony. So I decided to run a full-scale trial in a yard at an elevation that “normally” gets cold enough to cause a winter brood break (as if there were any weather “normal” any more in California).

There were two questions that I wanted to answer:

• Would treatment with OAE cause any adverse effects upon the long-lived winter bees due to prolonged exposure to OA, or otherwise adversely affect colony buildup prior to almond pollination?
• Could OAE be used to try to zero out mites over winter, giving colonies a clean start for their first rounds of brood rearing?

Methods

In early November, following a rough summer dearth, we graded 45 moderately-strong double-deep colonies for strength and took mite wash counts (the colonies had been previously treated with OAE strips). Most had ceased brood rearing a week or so earlier (due to cool weather and lack of incoming pollen), and had only a small amount of sealed brood (about to emerge) remaining. I assigned treatment in a randomized block design, first blocking by mite infestation rate, and then by colony strength, and then randomly assigning an OAE treatment to one of each matched pair of hives. We applied ½ of a Swedish sponge (saturated with a solution of 25 g each of OA and glycerin [1:1 formulation by weight]) to each hive in the Test group (Figure 3).

Fig. 3 We applied a half OAE sponge to each Test colony, placing it in the front third of the cluster. Although this location resulted in suboptimal exposure of the bees to the treatment, it left room for us to feed pollen sub in the middle of the clusters.

Practical application: Bees won’t consume pollen sub that’s come in contact with an OAE sponge, so one should leave a space between them. Another tip that I’ve received is to insert a ¼-inch shim between the brood chambers in order to give the bees more space above the OAE sponges for them to walk over, but I have not yet tested this.

To our surprise (something that we’re getting used to), the weather unexpectedly warmed up, hitting 70°F on the first of December! Plants responded, the bees started bringing in a little pollen, and the colonies resumed rearing a bit of brood. I’ve laid out the timeline in Figure 4.

Fig. 4 The yard got slammed by a cold snowstorm on December 26, which really set back the “spring turnover” of the colonies. But by mid-January, the temperature rose, the alders started producing pollen, and the colonies resumed brood rearing in earnest.

The surprise subfreezing temperatures and coverage with snow hit the colonies hard, and many had piles of dead bees in front of them once the snow melted (Figure 5). We checked in front of every hive for the presence of dead bees, comparing it to treatment assignment, and did not observe any correlation with whether the colony had received OAE or not.

Practical application: The above observation suggests that OAE treatment did not cause elevated mortality of the aged adult bees.

Fig. 5 I took samples of bees from the nine worst piles, and took them home for microscopic analysis, looking for the presence of nosema spores in a homogenized subsample of 10 bees — three subsamples had no nosema, three had a little, and three were fairly well infected.

Practical application: It didn’t appear that nosema was the main problem, since I would have expected to see nosema in dead older bees following a late-winter pollen flow (since pollen in the gut stimulates nosema reproduction).

Then it warmed up again, and by late January the colonies were full of a new round of sealed brood (Figure 6).

 Fig. 6 I took this photo on the day of final grading. The cluster barely covered three frames, but was rearing as much brood as it could keep warm. There are about 1500 pupae on this side, and only 445 adult workers (yes, I counted ‘em). The 7-frame colonies contained three full frames of brood. It’s easy to see how colonies can grow quickly once pollen becomes available.

Although I would have liked to have performed one more grading after the sealed brood emerged, we needed to move the hives to almond pollination, so performed final grading on January 26.

Results

The raw data is presented in Figure 7.

Fig. 7 None of the colonies had yet rebuilt to their starting strengths. For analysis (including the graphs above), I removed four badly-dwindled outlier colonies from the Control group, so did the same for the OAE group as well.

Rather than the absolute values shown above, it may be more informative to compare the colonies’ relative changes in strength vs. their starting strengths (Figure 8).

Fig. 8 All the colonies were weaker in January than in November, but since they were full of sealed brood, they would be expected to greatly increase their sizes to above starting strength by time of grading in almonds. The OAE-treated group as a whole lost slightly more strength than did the Controls, but the difference was not statistically significant [[2]].

Practical application: So was there an adverse effect from treatment? It’s difficult to tease out from this small data set (with only one replicate).  I now wish that I had prioritized colony strength (over initial mite count) when I assigned treatments, since larger clusters have a distinct advantage over smaller clusters as they initiate brood rearing [[3]]. As you can see in Fig. 7, the Control colonies wound up starting out stronger than the Test group, and thus would have been expected to overwinter and then grow better than the Test colonies. So I’m fairly confident that there was no adverse effect upon colony strength or buildup.

So far I haven’t discussed the possible benefit from treating with OAE in the first place — did it bring down the mite infestation rates? I fully expected over-winter treatment to zero out the varroa infestation rate, but it didn’t (Figure 9).

Fig. 9 The mite wash counts generally grew in the Control colonies over winter, but to my surprise, having an OAE pad in the hive did not zero out the infestation rates of the Treated group.

I was surprised by the lack of efficacy due to treatment after 71 days of exposure. The average mite count did go up by 55% in the Control group, but only decreased by a measly 3% in the treated group (Figure 10).

Fig. 10 I used the Henderson-Tilton formula to calculate average efficacy of the treatment, which worked out to only a disappointing 37%.

Discussion

Practical application: Although OAE appears not to cause harm to a colony over winter, it may not cause much harm to the varroa mites either! Although OAE can be highly efficacious during the summer, it remains to be determined whether it is appropriate for winter treatment.

The Elephant in the Room — our “scofflaw problem”

After the invasion of the varroa mite, researchers around the world published research on how this or that agricultural miticide (e.g., Mavrik, coumaphos, Taktic) could successfully be used to control varroa.  Unfortunately, due to the frustratingly-slow registration process required by the EPA to register a product for use in bee hives, beekeepers, desperate to keep their colonies alive, would start using such treatments prior to approved formulated products being brought to the market [[4]].

Such “off-label” use of miticides is in violation of the law, and even when a legal product was finally brought to the market, many beekeepers continued to use the cheaper homemade treatments. It is no secret that our commercial industry remains widely dependent upon imported, unregistered, illegally-applied amitraz products for mite control.  The fact that beekeepers, who often demand pesticide application compliance by farmers, are often pesticide scofflaws themselves, is a point not lost upon the EPA and its enforcement agents.

Our state apiary inspectors (as well as the EPA state lead agencies) have long turned a blind eye towards the illegal use of homemade miticide treatments for varroa management.

Practical application:  Some inspectors are getting tired of this hypocrisy, and are calling upon the EPA to start enforcement actions.  Not only that, but mites are finally starting to develop resistance to amitraz.  Because of this, commercial beekeepers are looking for another effective way to manage varroa.

It occurs to me that now is a chance to get our industry back into compliance.  One of the most promising miticides to replace amitraz is oxalic acid, for which there is only one currently registered (and high-priced) product, with only three approved application methods.  What I was trying to do was to get the Registrant — USDA-ARS — to get the extended-release (in glycerin) application method added to the label (as well as some other improvements)  Unfortunately, USDA recently decided to cease pursuing new registrations.  A number of beekeepers have told me that they are disappointed in USDA-ARS, which hands out millions of dollars in grants to bee researchers each year, for not helping our industry by getting this additional application method approved while they were still the Registrant.  So I’m back in discussion with EPA and others.

Reality Check

OAE is currently being researched in many countries. A formulated product — Aluen CAP — is registered for use in Argentina, Uruguay and Chile, and should soon be approved in other countries. In other regions, some beekeepers are jumping the gun and mixing their own, and due to its obvious safety, authorities often turn a blind eye to this practice. Generic 99.6% purity OA is readily available, meaning that no matter the law, many beekeepers will likely mix up their own OA treatments, rather than purchasing a pricey formulated registered product. What can we do in order to get us into compliance with the law?

Practical application: There is only a small profit margin of in commercial beekeeping, so professional beekeepers try to avoid any unnecessary costs. This means that there is a strong financial disincentive to purchase high-priced registered miticides. Thus, the use of inexpensive illegal treatments results in an unlevel playing field, giving those who use unapproved treatments an unfair competitive advantage over those professional beekeepers willing to use only approved mite treatments. I don’t want to break the law, but I also don’t want to pay through the nose for a registered oxalic product.

A possible easy solution for getting us legal

New Zealand’s Ministry for Primary Industries came up with a very workable solution. They allow beekeepers to prepare and apply generic oxalic acid to hives that they own, plus take full responsibility for applicator safety (Figure 11).

Fig. 11 I lifted the above snip from the actual document [[5]]. Beekeepers in New Zealand can legally prepare and apply generic oxalic and formic acids to their own colonies. This is a sensible solution, since neither substance poses “any unreasonable risk to man or the environment, or a human dietary risk from residues that result from a use of the pesticide” [[6]]

The Exemption does not allow the sale of any unregistered formulated product, but supply houses can sell the raw ingredients for beekeepers to mix up their own (Figure 12).

Fig. 12 This is a kit of the raw materials for making OAE strips, sold by Beequip.nz in New Zealand. Although a beekeeper is allowed to make them for their own use, selling a formulated product would require registration with New Zealand’s Environmental Protection Authority.

If EPA were to grant an Own Use Exemption, the Agency would be off the hook as far any safety responsibility or regulation of use of generic OA by beekeepers, and only be responsible for the registration and regulation of formulated products. This would be of huge benefit to beekeepers, since they would then be able to legally use inexpensive generic OA, and the State Lead Agencies would no longer need to turn a blind eye toward the practice. This could be a win-win for both the beekeeping industry and the EPA.

Unfortunately, in a recent meeting that I requested, EPA’s Office of Pesticide Programs was not open to granting us an Exemption. I’m not sure whether we’d get any traction by having our national organizations petition them. So I’m currently in discussion with some individual beekeepers about pursuing us becoming a Registrant in order to get the extended-release (in glycerin) application method approved, so that we could provide inexpensive “registered” OA, with the OAE application method on the label, to the beekeeping supply houses. Stay tuned — I may start a GoFundMe fundraiser to allow us beekeepers to legally pursue a solution to our “scofflaw problem.”

To be continued…

Citations and notes

[1] ShamWow is made of a blend of rayon and polypropylene. Rayon is a manufactured regenerated cellulose fiber. Polypropylene is a non-biodegradable plastic.

[2] Mann-Whitney one-tailed U-value is 119. The critical value of U at p < .05 is 102. Therefore, the result is not significant at p < .05. The z-score is 1.10566. The p-value is .1335. The result is not significant at p < .05.

[3] Nolan, WJ (1932) The development of package-bee colonies. USDA Technical Bulletin No. 309.

[4] Delaplane, K (1997) Practical science—research helping beekeepers 3. Varroa.  Bee World 78(4): 155-164.

[5] Advertising and own use guidance for compounds for management of disease in beehives (mpi.govt.nz)

[6] Summary of the Federal Insecticide, Fungicide, and Rodenticide Act | US EPA

The varroa incursion in Australia 4 July 2022

Instructions for extended-release oxalic acid

Extended-release oxalic acid for varroa management

This method of application of oxalic acid is not yet approved by the EPA, and I do not in any way advocate any unapproved use in bee hives.  However, EPA does not require an Experimental Use Permit for a limited number of hives.  Check with your State Lead Agency for your own state’s restrictions.

Safety

Although oxalic acid is not as reactive as stronger mineral acids, it can still cause eye damage, and if left on the skin, tissue damage.  Always wear safety glasses and waterproof gloves during preparation, and be careful to avoid splashing.

After preparation or application, wash your hands and equipment with soap and warm water to remove any acid residues, or better yet, neutralize any acid on hands, hive tools, or smoker with a solution of 10 heaping tablespoons of baking soda per gallon of water.

Dosage and delivery matrices

For extended-release application of oxalic acid, it can be dissolved into glycerin, and applied to the hive by either laying pads across the top bars (if applied between two brood chambers), or by hanging strips over the top bars, extending down into the interspaces between the frames.  The delivery strips or pads must be applied so that bees freely contact the surfaces.

Biodegradable cellulose matrices such as cardboard (chipboard), Swedish sponges, or cotton absorbent fabrics may be used.  For full efficacy, roughly 55 square inches of delivery matrix must be used if applied across the top bars, or 100 square inches if hung between the frames.  The instructions below are for moisturizer-free Swedish sponges, which hold 100 g of 1:1 (weight to weight) solution of oxalic acid dihydrate to glycerin.  Other matrices, or different ratios of acid to glycerin, will require different preparation.

Preparation

For extended-release application, oxalic acid can be dissolved in glycerin, absorbed into any number of absorbent matrices.  Field data suggest that for a double-deep hive, there should be roughly 60 square inches of matrix, holding roughly 100 g of 1:1 OA:glycerin (weight to weight), although other ratios may be used (the higher the ratio of glycerin, the more rapidly the OA is dispersed upon the bees, sometimes with adverse effects).  For cardboard strips to be hung over the top bars, it will require more total surface area to hold the same amount of solution.

To prepare enough Swedish sponge pads to treat 10 full-sized colonies in double deep boxes:

• First prepare the sponges by cutting them in half (into 3½” x 8” pads), each of which will absorb 50 grams of the solution (50 g oxalic acid dose per hive).
• Wear safety glasses and waterproof gloves when preparing the solution. Have a neutralizing solution of 10 heaping tablespoons of baking soda dissolved in 1 gallon of water on hand, to neutralize any spills.
• Place 500g OA dihydrate into a stainless steel pan, then add 500g (400 mL) vegetable glycerin (add the glycerin second in order to avoid splashing of the solution).
• Place the pan over a low/medium heat (preferably using a double boiler), and heat the ingredients while closely monitoring the temperature, not to exceed 160°F (the acid crystals will dissolve at as low as 110°F, and start to bubble if the temperature exceeds 170°F).
• Occasionally stir gently until the acid crystals are completely dissolved and the solution is completely clear. At that point, remove the pan from heat.
• While the solution is still hot, either (A) add twenty (20) (3½” x 8”) absorbent cellulose pads (or fifty (50) 1.25” x 15” cardboard strips) on edge into the pan and allow them to absorb the solution, or (B) place the pads into a separate plastic container and slowly and carefully pour the still-hot solution over them. With either method, you may need to use tongs to carefully turn the pads over to obtain full absorption (which must take place before the solution cools).  Be careful to avoid splashing of the solution.
• If all the solution does not absorb, the excess should be drained off before allowing the pads to completely cool. For easier handling, allow the pads to cool for at least a day before application. The oxalic acid will recrystallize during this time, and make the pads easier to handle and apply, with no dripping of solution (under conditions of high humidity, the pads will absorb moisture and may not “dry out”).
• The pads can be stored in a labeled sealed container for up to 2 months, by which time the cellulose will slowly start to degrade.

Application

• Optimal timing of this treatment in treatment rotation is to apply the pads into the brood chamber at time of placement of the honey supers. This treatment should only be used once while colonies are rearing brood, rotating with miticides with other modes of action, such as formic acid, thymol, amitraz, or fluvalinate.  An oxalic dribble or vaporization can then be used during the winter brood break.
• Wear waterproof gloves.
• Using gloved hands or tongs, apply two pads between the brood chambers, placed so as to be within the cluster (avoid placing under a top feeder where syrup may spill, or directly against pollen substitute). Placement of the pads must allow for movement of the bees over both surfaces.  The pads or cardboard strips can also be hung over the top bars, inserting them spread between the two brood chambers.  For cardboard strips, 3-4 will be needed per brood chamber.
• For optimal efficacy, the pads must remain in the hive for 60-75 days, or until most of the acid has been distributed. After treatment, remove the pads, handling them carefully, since they will still contain acid.
• After removal, place the spent pads into a plastic bag or container for transport, and rinse your hands, hive tool, and smoker with neutralizing solution.
• Dispose of the spent pads in a landfill, or compost them.

2022 Extended-Release Oxalic Update: Part 1

2022 Extended-Release Oxalic Update

Part 1

Randy Oliver
ScientificBeekeeping.com

First published in ABJ March 2022

As more and more beekeepers try to wean themselves off amitraz, there is great interest in oxalic acid.  Unfortunately, a dose of OA by any of the currently-approved application methods exhibits little residual action, so has limited efficacy if a colony is rearing brood.  However, as with other miticides sold in extended-release strips or formulations, oxalic can be applied in a similar manner.

REVIEW OF MY FINDINGS ON EXTENDED-RELEASE OXALIC ACID (OAE) TO DATE

My sons and I have experimented, by permit [[1]], with OAE for some time, testing a range of ratios of oxalic to glycerin, as well as with a variety of delivery matrices.  So far, we’ve determined that:

• We like the handling characteristics and efficacy of the 1:1 (by weight) ratio.
• Swedish sponges make a great delivery matrix, but are pricey.
• OAE has the potential to take mite infestation rates down to zero, but it is a long-term treatment. It doesn’t cause as rapid a reduction of mite levels as do most other treatments (formic is the quickest).  It requires about two months to exhibit full efficacy.
• Very importantly, due to its slow action, highest efficacy is attained in yards where there is little mite immigration.
• Its efficacy does not appear to be affected by temperature or humidity (since broodnest environment is regulated by the bees).
• It’s best applied proactively (early in the season), rather than reactively (waiting until the mite level gets high).
• It’s easy on the bees and brood.
• It’s not a worry as far as contamination of honey or the combs.
• Unfortunately, this application method is not yet approved by EPA, and I in no way encourage or endorse the use of it without a proper permit.

OAE HOLDS GREAT PROMISE

Although this application method of oxalic acid is not yet approved by our slow-moving EPA [[2]], it is clear to me that it is going to be a game changer for varroa management.  My sons and I are impressed by how it has performed in our own authorized experiments. I also get reports from beekeepers in countries where it is approved or allowed [[3]], as well as from beekeepers elsewhere (Figure 1).

Fig. 1 Beekeepers from all over the world send me glowing reports on how well OAE can work.  The photos above are from a professional East Coast beekeeper (whom I assume is permitted), who waxed poetic about how OAE treatment resulted in the strongest, healthiest, and most honey-productive colonies in memory, holding the mite levels in his busting colonies to low levels.  His photos speak for themselves.

Practical application:  I’m not trying to sell or promote this application method (especially since it’s not yet approved).  It’s not a panacea, and is best used proactively.  And as with other treatments, there are always outlier colonies in which mite levels somehow don’t respond, and the treatment may not be able to keep up with heavy mite immigration. 

But because it would fit very well into our own commercial operation (since we eschew comb-contaminating synthetic miticides), my sons and I continue to perform permitted research not only for  the benefit of the beekeeping industry, but also in own interest — and for  that of our poor bees, who beg for relief from this parasite.  In the long run, OAE may help us to stave off varroa until mite-resistant bees become widely available [[4]].

TESTING VARIOUS MATRICES

The concept of OAE is to provide sustained release — from an inert delivery matrix — of the oxalic acid onto the bees.  Glycerin acts as the solvent and carrier of the acid, and also has the advantages of being inexpensive, food-grade, a humectant (which helps the acid to be effective), an adhesive (that helps the acid to stick to the bees), and has the added benefit of being distasteful to the bees (so they’re less likely to consume the acid).  But what’s the best matrix to use?

I’ve scoured catalogs and stores for suitable absorbent sheets or pads (preferably biodegradable), and gotten suggestions from beekeepers everywhere.  This spring I chose seven matrices to compare to the successful Swedish sponges (which work better than do the shop towels and cardboard that I’d previously experimented with).

I had purchased a number of different brands and shapes of cotton wipes and pads to try, and decided to use the Q-tips brand, since it had crimped edges.  Because they happened to be round, for comparative testing I measured their surface area (4 in²), and cut rectangular strips of identical surface area from the other matrices.

I prepared six pads of each matrix, using a single batch of OA/gly solution, and then allowed them to come into equilibrium at a fixed temperature and humidity in an incubator.  After weighing the pads, beekeepers Brion and Alice Dunbar helped me to haphazardly place one pad of each matrix into each of six hives  (Fig. 2), and allowed the bees to walk over them for 20 days (7 April to 27 April).

Fig. 2  In order to determine how well each matrix dispersed the OA/gly solution onto the bees, we covered the top bars of the lower brood chambers of six hives with masking tape to prevent absorption, and weighed the saturated strips before and after 20 days in the hive. The yellow-starred matrices looked to be the most promising.

We then collected the pads, again allowed them to come into equilibrium in the incubator, and reweighed them as a group in order to determine the average amount of weight lost per pad (Table 1).

Table 1.  The tested matrices, ordered by weight loss.  None lost as much as the sponges, but some were close. I’ve highlighted the most important cells.

Three of the matrices showed promise, due to their amount of absorption or release, as well as other characteristics (the lowest two had been removed by the bees).  So I made up a batch of them, along with some sponges (Figure 3).

Fig. 3 I prepared the four matrices to be tested with the same batch of OA/gly solution at the 1:1 ratio.  Tip: for full absorption, it’s important to give any matrix enough room to expand.

I then ran a quickie trial of the four, testing them by seeing how much they increased the rate of mite drop after application (in 2 colonies for each matrix, randomly blocked by starting mite count) (Figure 4).

Fig. 4 In my preliminary trial of the four most promising matrices, I was unfortunately only able to track mite drops for five days (16-May was the pretreatment baseline count). The cotton pads caused by far the greatest short-term increase in mite drop.

Wow, cotton pads looked really promising!  I had hoped that the bees might tug the saturated cotton fibers off, and drag them down through the colony (Figure 5).

Fig. 5 The above photo was taken after the pads being in the hive 10 days.  There didn’t appear to be the amount of tugging of the fibers that I’d hoped for.  Note the slight propolization around the edges.

Unfortunately, the facial pads were difficult to separate after preparation, and would require 13 round pads to equal the surface area of a single Swedish sponge.  So I didn’t follow up any more with them this season.

Practical application:  Cotton in some form still remains a very promising candidate (I plan to try terrycloth or other absorbent fabric, and plastic-free feminine-hygiene pads).  I also experimented with adding thymol to the pads, but it didn’t appear to improve performance.  Properties to consider with any matrix are its handling characteristics, including preparation, field application, and ease of removal from the top bars after treatment.

SOME QUESTIONS TO ANSWER

As I reported last year [[5]], we had, in late spring and early summer, applied a quarter-sponge strip of OAE to a large number of colonies growing from nucs in experimental hives in several yards, and found that a minimal-dose treatment largely suppressed increase in mite wash counts for a few months.  So we were curious as to whether such a low-dose treatment would prove to be efficacious in larger low-mite colonies.  And based upon our preliminary experimentation with the alternative matrices above, I thought that we could run a single comparative field trial to answer three questions:

• How do Swedish sponges, Maximizer pads, or acrylic felt compare in efficacy at reducing mite loads?
• Is there a minimal surface area (as opposed to amount of OA) of OAE pads necessary to obtain good efficacy in double deeps?
• Can you dose with OAE proportional to the mite infestation rate? e., can you use fewer strips in a low-mite colony?

For experimentation, we had set aside a bunch of intentionally-untreated colonies with 2nd-year queens, grown from March and April nucs split from colonies returned from almond pollination, in four different yards.  So in late May — as their mite infestation rates climbed — I thought up an experimental design to attempt to answer the questions above.

A LARGE-SCALE FIELD TRIAL

Last year we learned that one half-sponge reduced mite levels substantially, and two half-sponges exhibited very good efficacy.  I figured that if we cut the pads into strips the same size as a quarter of a Swedish sponge, and then apply only a single strip to the lowest-mite hives, going up to 4 strips for the highest-mite ones, that we could perhaps fine-tune the optimal dose.

We started the trial by taking mite wash counts from all the hives in the yards on May 20-21, sorted them by starting mite level, and randomly assigned treatment types accordingly (Table 2).

Table 2. We unfortunately didn’t have many colonies with mite counts above 30, and in retrospect, I should have assigned the 4-strip treatment to more colonies in the 11-30 mite groups.

Scientific note: We randomly assigned the treatments, blocked by starting mite count.  We didn’t run any negative Control (untreated) groups, since we were testing for comparative, rather than absolute efficacy (and it would have taken eight Control groups).  If you look at the dates of all these experiments, you can see that we were performing experiments back-to-back in short order, rushing against the season and mite buildup.  I had to make quick, on-the-spot decisions, and in retrospect sometimes wish that I had done something differently. 

We applied the strips on May 26-27 (Figure 6).  On June 30 we spot checked colonies for strip condition and mite wash counts.  At that time point, the acrylic felt did not appear to be performing, so we removed those colonies from the trial.

Fig. 6 According to the starting mite wash count, we applied from one to four 1¾ “ x 7½ ” strips per hive after smoking the bees off of the top bars.

On July 20 (after 55 days of treatment) we took ending mite wash counts.

RESULTS FOR THE SWEDISH SPONGES

The results are shown in Figures 7 and 8 — the more blue you see relative to red means the more efficacious the treatment.

Fig. 7  Contrary to our observations the previous year, applying only a quarter sponge to low-mite colonies did not do the trick (too many had their infestation rates increase substantially).  But similar to last year’s results, two strips (a half sponge equivalent) reduced the mites, and a full-sponge equivalent  (4-strips) nailed it.  Note that some of the highest-mite hives went to counts of zero.

Practical application:  When I shared the results with my sons, it was clear that a 4-strip equivalent (two half-sponges) would now be our standard OAE treatment for full-strength colonies.

RESULTS FOR THE MAXIMIZER PADS

The results for the Maximizers were quite similar to those for the Sponges (Figure 8).

Fig. 8 It looks as though the Maximizers may be slightly less efficacious than the sponges at low dose.  As with with the sponges, the 4-strip hive zeroed out.

Practical application:  Once a colony’s virus infection gets too high due to having an elevated mite load, it may not be able to be saved by any treatment.  I’ve been surprised that in our previous trials, virtually all high-mite colonies recovered if given an OAE treatment.  In this trial, one of the two highest-mite 4-strip colonies for each matrix type did not make it, but the other one not only survived, but ended up with a mite count of zero!

MITE REDUCTION AND EFFICACY CALCULATIONS

Let’s take a look at the comparative effect of treating with either 1 or 3 strips of the two matrices.  To do so, I simply totaled the mite counts for all the colonies in each treatment group at the start and end points (Figure 9).

Fig. 9 Note that the 1-strip groups started with much lower mite levels, which went up, whereas the 3-strip groups started with much higher mite levels, which then went way down.  The absolute reductions were 91% for the Sponges, 90% for the Maximizers.  (n = 21 for 1-sponge, 12 for 3-sponge, 22 for 1-Maxi, 10 for 3-Maxi).

Those absolute reductions (as opposed to calculated efficacies) are pretty impressive in their own right, but don’t take into account the expected increase that could have been factored in had there been comparative data from legitimate controls.

To legitimately calculate efficacy of a treatment, the Test and Control groups would need to start at the same infestation rate.   But a problem with testing varroa treatments is that with a high starting count, the Control colonies may succumb to the mite and virus load.

Unfortunately, one can’t just start the Control group with lower infestation rates than the Treatment group, since my previous data indicate that high-mite colonies tend to exhibit higher efficacy values than those for colonies with lower starting mite counts [[6]].  So I wondered whether that trend held over the range of starting mite counts for the 22 hives in the two 3-strip test groups combined together (Figure 10).

Fig. 10  Correlations between the starting counts and percent mite reduction from treatment for the 22 colonies in the two 3-strip groups combined.  There was no apparent correlation.

In light of the above, and with the important caveat that any efficacy calculation for this trial cannot be considered legitimate (due to the Control group not starting at the same infestation rate), I feel that it is still informative to run the Henderson-Tilton calculation anyway, using the 1-strip applications as the “controls.”  Doing so, the calculated efficacies are 97% for the Sponges, and 98% for the Maximizers!

Practical application:  The 90% absolute reductions, and admittedly-questionable 97% efficacy values are what we were looking for.  The much-cheaper Maximizers are looking pretty good!

TAKE HOMES:

1. For full efficacy of OAE for mite control by using an absorbent matrix laid across the top bars between the brood chambers, it takes about 55 in² of matrix holding 50 g of OA dissolved in 50 g of glycerin.
2. We don’t yet know whether it helps to divide the matrix, but got good results last year with it divided into two pieces.
3. Maximizer pads (Figure 11) are a lower-cost alternative to Swedish sponges, and are a bit easier to scrape off the top bars after treatment.
4. We do know that strips can alternatively be draped over the top bars down between the combs with good results, but I have not done much research on this method of placement.
5. My preliminary research, substantiated by recent work by Dan Aurell [[7]], indicates that Aluen CAP strips (10 g OA per strip), hung between the frames, requires more than 4 strips for equivalent efficacy in a double deep.
6. Aluen CAP uses a 1:2.5 ratio of OA to glycerin; we like a 1:1 ratio; New Zealanders typically use an intermediate dilution.
7. Higher-glycerin formulations (and perhaps cotton pads) release the OA more quickly, and can potentially cause short-term adverse effects.
8. We’ve learned a lot so far, but need to continue our research!

Fig. 11 A dedicated paper cutter with a built-in clamp allows for quick cutting of matrices.  Here are ½-sponge equivalents of Maximizer lightweight pads, which are a readily-available alternative to Swedish sponges.

Update:  This is only a report on my experimental trials.  I do not make any recommendations, so please do not write me for such.  The Spilltech Maximizer pads above (Item # GPC100S) contain a little bit of plastic fiber, and a flame retardant (that does not appear to be harmful to the bees).  We tested the lightweight version, and will test the heavyweight version this year. 

To be continued…

ACKNOWLEDGEMENTS

Thanks to Brion and Alice Dunbar, my sons and crew of Golden West Bees, and to all the beekeepers who have offered suggestions and observations.

CITATIONS AND NOTES

[1] Obtaining each year a Pesticide Research Authorization from the California Department of Pesticide Regulation.

[2] Me and a few other beekeepers had a meeting with EPA’s Office of Pesticide Programs as I wrote this article in January.  I’ll write about it next month.

[3] New Zealand very reasonably has determined that beekeeper use of the organic acids for mite management poses scant risk to the public or environment, allows beekeepers to use them at their own discretion.  More on this next month…

[4] More on this in an upcoming article.  I just returned from one of our outyards, in which 50% of the colonies have exhibited strong mite resistance this season, and required no treatment at all.

[5] Mite Control While Honey is on the Hive: Part 3 – Scientific Beekeeping

[6] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-2/

[7] Pers. comm. and presented at the 2022 ABRC.

An Online Model for Varroa Management

An Online Model for Varroa Management

Randy Oliver and Trish Harness

First published in ABJ February 2022

Beekeepers worldwide have been using Randy’s Varroa Model with great success since its release in 2017.  But there has been a demand for an online version that did not require Excel, and could be used on a cell phone.  Trish started working on such an app in 2018, and now has it ready for release!

No single varroa management strategy is appropriate for every beekeeper, since we keep bees in dissimilar climates, with short or long brood breaks, diverse colony population dynamics, various treatment windows of opportunity, preferred mite treatments, and disparate goals and limitations (almond pollination, honey production, avoidance of comb-contaminating miticides or wish to use “organic”methods, labor costs, etc.).

For that reason, with no background whatsoever in modeling or spreadsheets, Randy painstakingly worked for over a year to create his open-source varroa management model, first publishing it in 2017 [[1]].  The logic for the model is laid out in Figure 1.

Fig. 1 The inputs for the model are details that can be measured or estimated by the beekeeper:  the mite wash count, cluster size and amount of brood over the course of the season, the amount of mite immigration, and the expected efficacy of each treatment.

After inputting a few values for the above parameters, the model then indicates the expected impact of treatments, and allows a beekeeper to customize his or her management strategy (Figure 2).

Fig. 2 A beekeeper can run simulations of various management strategies with the goal of avoiding the red crash indicator — it doesn’t cost you anything to lose a colony on the computer!  In this simulation, a 50% efficacious mite treatment in May (when high efficacy is tough to attain, due to the amount of mites in the brood), followed by an 80% efficacious treatment on the first of October, didn’t do the trick, and the colony would be expected to crash.

Note: Since there are far too many variables involved for any model to predict the degree of colony morbidity due to increasing virus transmission due to mite buildup, or colony performance with changing forage or weather, the model assumes that the infestation rate is kept to a low enough level to not change the expected colony structure.  Thus, the “crash” indicator is only a rough approximation, and can be adjusted.

Randy encouraged, and fully expected, bee researchers or professional modelers to take his crude model and improve it, but it’s been mostly beekeepers themselves who have caught errors and offered suggestions.  So over the years he’s updated it continually — the current iteration is Version 19, available for download at [[2]].

To those familiar with using Excel spreadsheets, this varroa model is simple to learn to use, and for those with a deeper understanding of varroa and honey bee biology and reproduction, is fully customizable for any bee stock or conditions anywhere in the world.

However, not every beekeeper has Excel on their computer (or even has a computer), or is comfortable with using spreadsheets, so some asked for a simpler version that they could download to their beloved cell phone.  Not being a programmer himself, Randy put out the word for help…

TRISH STEPS UP TO THE PLATE

In 2018, beekeeper/computer science undergrad Trish Harness recruited an upper-level computer science class at Hiram College to collaborate on developing an online version of the varroa model.  After the class term ended, she continued to work diligently on it herself, going through countless iterations over the years, bouncing ideas and prototypes back and forth between us.  We’ve finally arrived at a version that we’re both happy with.  All you need to do on your cell phone or laptop is to go to chickabuzz.com/model.html and type in a few input values.  So let’s run a few simulations to show how to use it, starting with Figure 3.

Fig. 3 A screenshot of Trish’s model (on a cell phone, best viewed sideways), using the same inputs as for Fig. 2 (the blue cells indicate the inputted treatments).  She also allows the user to choose which parameters they want to have shown in the chart — in this example, all are checked.

For Trish’s simplified app, she wanted to make the varroa infestation stand out.  So the varroa population (red line) is shown at a 10x multiple (topping out at 5000 mites), and the mite wash count for a sample of a half cup of bees tops out at 45 (a 14% infestation rate).  From a management standpoint, hitting either of the top-out limits indicates that immediate treatment is called for.

Compare the above results for the same simulation, but instead of only two treatments, the beekeeper checked the upper grey boxes to apply three moderately-efficacious (80% reduction) treatments in April, August and November (Figure 4).

Fig. 4 Success — no crash!  This treatment regimen prevented the mite population or brood infestation rate from climbing too high, and the colony would be expected to survive the winter.  Note that this treatment regime would be sustainable, since the starting and ending mite counts (100 and 104) are essentially the same.

For another example, let’s show a simulation for a beekeeper who purchased a nuc in early April, with a starting varroa population of only 50 m ites (Figure 5).

Fig. 5 A starting wash of a half cup of bees from this nuc would typically detect zero mites.  In this simulation, instead of indicating total brood, it’s clicked to show only the amount of sealed brood.  The beekeeper applied a 50% efficacious treatment at the first of August, and then a 90% efficacious treatment on November 1^st, as the last of the sealed brood was emerging.

If this happened to be the beekeeper’s first colony, they would be impressed by their low mite counts in April and December, but the colony may still have been suffering from a late-season Deformed Wing Virus infection due to its high mite level in October and November, and might not make it through the winter.  And even if it did, it would begin the season with 157 mites — three times as many as it started with in the previous year.  So this varroa management strategy would not be sustainable.

In the simulation below (Figure 6), the beekeeper proactively applied a formic acid treatment in March, and then applied 5 OAVs every 15 days starting in mid-August.  The model indicates the expected proportion of the mites in the brood at any time (bottom row), which helps you to estimate the efficacy of various treatments.  For example, a 30-second oxalic acid vaporization (OAV), using 1 g per brood chamber may only kill around 80% of the mites on the adult bees, and none under the cappings.  So for the first application, since an estimated 56% of the mites would be in the brood, that would leave 44% out on the bees, for which an 80% kill rate would result in an overall reduction of the mite population of 35% (44% x 80% = 35%, the figure that the beekeeper would enter).  For each subsequent OAV, the expected overall efficacy could again be estimated as shown.

Fig. 6 By proactively hitting the mites in April (and perhaps suppressing the swarm impulse by the brood break induced by a formic treatment), the beekeeper got by with a series of late-summer low-efficacy OAVs while the colony still contained brood, taking it into the winter with a barely-acceptable infestation rate of 8 mites in a wash.  But note that the ending mite population was nearly 4 times that of the start, which would spell trouble the next season.

Note: Randy’s been analyzing data sets from several beekeepers who have taken mite drop counts following repeated OAVs.  It appears that repeated applications of 1 g per brood chamber, applied in a 30-second blast, although “safe” for the colony, may result in relatively low overall efficacy, even if repeated at 4-day intervals, especially if much mite immigration is taking place (there was moderate immigration in the above simulation).  Indeed, mite wash counts might even give the impression of no efficacy (as recently observed by Berry [[3]]).  Note that in the above simulation, the mite wash counts (the numbers on the blue line) over the course of 5 OAV applications did not change, despite reducing the total mite population by two-thirds.

For a last simulation, let’s switch to a Mediterranean California climate, with a nectar and pollen flow in September and October, and with a bit more mite immigration during late summer from surrounding neighbors’ hives (Figure 7).

Fig. 7 This San Francisco beekeeper was also proactive by treating in mid-March, but got only 70% mite reduction due to the large amount of sealed brood.  By mid-July, there was much less brood due to the summer dearth, and she obtained 90% efficacy with a double application of Apiguard (thymol).  But then a new spike in broodrearing, coupled with troublesome mite immigration, allowed the mites to rebound.  Notice how the proportion of brood cells infested by at least one mite (dark red area) built up to a point of no return by late November, causing all her bees to “disappear” while she was distracted by the holidays.  What a bummer of a way to start the New Year!

By running simulations, one can start to better understand mite population dynamics, and response to various treatments.

USING MITE MODELS AS A REALITY CHECK

No model of a complex biological system can be perfect, but these simulations, based upon Randy’s experience in his own operation, as well as feedback from other beekeepers worldwide, indicates that they generally hit the mark.  Randy continually checks the base model against data from others to confirm its accuracy.

The full model requires inputs of only:

• A starting mite infestation rate, estimated by working backwards from an actual mite wash count from a bee sample taken from a frame adjacent to brood at any time point,
• A choice of colony type (or customize for your location by inputting the number of frames covered with bees and amount of brood over the course of your season),
• An estimate of mite immigration (several options given, or customize).
• And any treatments applied (the models list expected efficacies of various treatments).

Trish’s model lacks some of the customization options, but can be used right in the field on your cell phone.

There’s no reason to be surprised by mites unexpectedly getting out of control in your hives.  By running simulations, you can test out various mite management strategies for your colonies in advance, adjusting them for your available treatment windows, working around honey flows, and rotating different treatments to avoid breeding resistant mites.

Practical application:  We’ve found that the most successful strategy is to deal with varroa proactivelyrather than reactively.  Get ahead of the mites as early in the spring as possible, so that you go into the honey flow with an extremely low infestation rate, then harvest your honey as soon as you can after the flow, and knock the mites back before your colonies are producing their last rounds of brood before winter.

A NOTE FROM RANDY

Trish has worked hard and long to create this mobile version of my varroa model, and plans to continue to improve and update it (always check for the latest version).  She doesn’t get paid for this, so if you find it to be useful, please send her a donation.  Her website is Chickabuzz.com [[4]] and her online model can be accessed at chickabuzz.com/model.html

MORE INFORMATION AND DOWNLOADS

[1] https://scientificbeekeeping.com/the-varroa-problem-part-12/

[2] https://scientificbeekeeping.com/randys-varroa-model/

[3] Berry, Jennifer (2021) Multiple applications of vaporized oxalic acid.  Bee Culture Dec. 2021: 70-73.

[4] Trish’s website: https://chickabuzz.com/

Hopguard 3 as a Summer Treatment

HOPGUARD 3 AS A SUMMER TREATMENT

Beekeeper-Funded Research

Randy Oliver, with collaboration by Aris Roberts

First published in ABJ February 2022

In the summer of 2020, I tested using Hopguard 3 to control varroa. Unfortunately, the label was not clear, and I didn’t realize that for summer treatment I was allowed to repeat the application of the strips. So I ran another test.

INTRODUCTION

If varroa starts to get ahead of you while your bees are putting on honey intended for harvest, there are only two currently-approved treatments to get the mites back under control — formic acid (MAQS or Formic Pro), or Hopguard. I’ve already shown my results for Formic Pro [[1]], and have discussed with Hopguard’s manufacturer suggestions for rewriting the label in order to make it more understandable.

The active ingredient of Hopguard — “potassium salts of hops beta acids” — is extracted (using liquified CO₂) from the same type of hops flowers used to add the bitter flavor to beer. Preliminary investigations indicated that bees tolerate hops beta acids about six times better than can varroa [[2]], thus the development of this product as a food-grade “biopesticide” for controlling the mite.

Hopguard has gone through three iterations as a formulated product, each an improvement over the previous. The main limitation for Hopguard and Hopguard II was the brevity of its “residual activity” [[3]^, [4]], meaning that although it caused a quick kill of mites on the adult bees, its action tapered off within a few days (and thus would not affect the remaining mites in the brood as they emerged). The current iteration, Hopguard 3, is an improved formulation that extends the action for several days, and can be very efficacious if applied to colonies lacking brood [[5]]. It also appears to be gentler on the colony, as evidenced by fewer dead bees in front of the hives after application, compared to that from Hopguard II (personal observation).

However, during summer, when at least 50% of the varroa population is hidden under the cappings [[6]], Hopguard must be reapplied in order to obtain good efficacy. Gregorc, after testing it, concluded that three consecutive treatments would be required [[7]]. Luckily, the label allows for Hopguard 3 to be applied up to four times a year, and specifies applying consecutive treatments two weeks apart (although that interval could use further exploration, based upon the research previously cited).

In order to answer the optimal reapplication interval question, I ran a field trial beginning at the end of June. In addition, high-school student Aris Roberts volunteered to independently collect data on daily mite drops following August application of Hopguard 3 strips in his hives in Virginia. Although Aris’ data was collected secondly, it’s worthwhile to look at it first.

ARIS’ MITE DROP COUNTS

Aris painstakingly counted daily mite drop on the stickyboards of six hives every day for 37 days. After establishing baseline counts prior to treatment, he applied Hopguard 3 at the rate of 1 strip per 5 frames of bees, reapplying strips first after 7 days, and then after another 10 days (Figure 1).

Fig. 1 There was an apparent wide range of varroa infestation rates of the monitored colonies, based upon their baseline and immediate post-treatment mite fall counts. Note how for even the highest-mite colonies, three treatments of Hopguard 3 brought daily mite drops down to low levels.

For better comparison, I summed up all the mite drop counts for the six hives each day, and compared them to the baseline count from the day before first application of Hopguard 3 (Figure 2).

Fig. 2 By a month from the start, three repeated treatments with Hopguard 3 had steadily decreased daily mite drop down to a quarter of the baseline counts. In answer to the question of the residual activity of the strips, the effect appears to drop off by around 7 days.  Data from Aris Roberts.

Practical application: Hopguard 3 may have extended the strips’ residual activity, but Aris’ data above suggests that it isn’t doing much after a week. So retreatment should be done within two weeks, in order to minimize the number of mites emerging from, and then reentering brood cells during the interim. Unfortunately, the above data were collected before I wrote the protocol for my own field test in California.

SUGGESTIONS FOR BEEKEEPERS WANTING TO DO RESEARCH

Aris shows us how even a teenage hobby beekeeper can collect very useful data of use to the beekeeping community. For more information on how you or a group can perform meaningful research yourselves, I’ve recently posted the slides for a presentation that I put together on how to perform bee research at [[8]]. I’m generally happy to help beekeepers with experimental design and protocols, and am willing to post good research by others at ScientificBeekeeping.com.

A FIELD TRIAL IN CALIFORNIA

Not yet knowing the optimal retreatment schedule for Hopguard 3, I consulted with Fabiana Ahumada, now at BetaTec Hop Products, who has been continually involved with the development of Hopguard since its early testing at the USDA Tucson Lab. We decided that I should test three applications at either 7- or 14-day intervals.

MATERIALS AND METHODS

I started the trial toward the end of the honey flow in late June, while there was still some incoming nectar and pollen. The weather was very hot and dry (Figure 3). Unfortunately, we had by that time controlled varroa in most of our yards, and the only colonies with mite levels high enough for collecting efficacy data were a group of untreated hives in my home yard, consisting mostly of “potential breeder queens” that had gone without mite treatment for over a year, but now had mite counts going up. I presciently wrote in my notes at the time that “the rate of mite increase in these colonies may be depressed relative to colonies that had not previously exhibited some degree of resistance to mite buildup.” As we’ll see, this concern turned out to be valid.

Fig. 3 The trial was run during hot weather in July and August, with daily highs typically in the 90s F.

All colonies were in double deeps, headed by 2nd-year queens. Colony strength ranged from 15-20 frames covered with bees (they had earlier been reduced in size to prevent swarming), all with brood in both boxes, and plenty of honey in the upper box.

We performed pretreatment mite washes on 27-28 June, taking level ½- cup samples of bees from frames adjacent to brood in the upper brood chamber, mechanically agitated them, and recorded the number of mites. I unfortunately could come up with only 18 hives with high enough mite counts to use for the trial. I assigned treatments in a randomized block design, blocked by starting mite count, with 7 hives in the weekly application group, 7 in the 14-day application group, and 4 hives with midrange counts [[9]] in the Control group. We took follow-up mite wash counts after one and two full bee brood cycles after first application of the strips, and again after 66 days, in order to determine the long-term effect of treatment. The experimental design is shown in Table 1.

We applied the first treatment to all hives the day after taking the first mite counts (Figure 4).

Fig. 4 Application of Hopguard 3 strips. We applied them (generously) as per label instructions of 1 strip per every 5 frames of bees. Most colonies got four strips, but the few with only 15-frame starting strength got only three. We removed the previous strips at each retreatment.

No mite treatment is completely harmless to the bees. The manufacturer cautions to smoke the top bars to cause the queen to move out of the way of a sticky strip as you insert it. If a bee gets rolled by a strip during the insertion process, we’ll find it dead on the landing board the next morning (Figure 5).

Fig. 5 The unfortunate wetted bee in the center of the photo will likely die from being rolled by an inserted strip. However, we’ve applied Hopguard to many hives, and have yet to see a queen get killed.

Application of Hopguard strips initially causes the bees to move broodrearing away from the strips (Figures 6 & 7).

Fig. 6 Colonies tend to shift their brood rearing away from fresh strips.

Fig. 7 But after a couple of weeks, once the strips’ odor had dissipated, the bees will resume broodrearing under them, and eventually start chewing away at the strips to remove them.

I didn’t perform actual measurements, but my visual inspections indicated that there was more brood, especially open brood, in the Controls than in the treated colonies during treatment. This apparent suppression of broodrearing may help the treatment to work against varroa (similar to formic acid, thymol, and amitraz treatments, which also suppress brood rearing to some degree).

The honey flow began tapering off after starting the trial, so colony populations had already peaked. It was hard to tell, but there appeared to be an effect upon the cluster size from treatment. At the three-week assessments, the Control colonies covered all 20 frames in their hives, whereas the clusters in many of the treated colonies had shrunken somewhat. When I reapplied the Hopguard strips to the treated colonies, I often had to move the replacement strips inward to keep them within shrinking clusters (Figure 8).

Fig. 8 The clusters tended to shrink a bit in the treated colonies. On the left is a typical colony immediately after application of the first treatment. In subsequent treatments (right), the clusters often no longer covered the strips. Despite this short-term effect, the colonies recovered quickly.

MY MISTAKE IN USING MITE-RESISTANT COLONIES

Let’s take a look at the mite wash counts for all the hives over the course of the trial (Figure 9).

Fig. 9 Either application regimen reduced the infestation rate of the adult bees in both the short term or the long term. Three treatments with Hopguard 3 definitely knocked the mites back, but neither treatment zeroed out mite wash counts. But why the heck did mite counts also go down in the untreated Controls after that time?

Practical application: By two weeks after treatment, any mites that were originally hidden in the brood would have emerged, after which they generally spend a few days feeding on nurse bees before they reenter a brood cell in order to reproduce. The question then is, would retreatment at 7 days result in higher efficacy than at 14 days? To my surprise, it didn’t appear to make much difference.

It appears that it was a mistake for me to have used rejected potential breeder colonies (those that had mite counts increasing in their second season) for most of the hives in the trial, since all four Control hives eventually started to bring their counts down on their own (some exhibited strong uncapping and VSH behaviors; Figure 10). So this screwed up the expected increase of mite counts in the Control group, which prevented me from performing any meaningful efficacy calcs. Live and learn! This trial needs to be repeated using nothing but demonstrated non-resistant colonies.

Fig. 10 Once their infestation rates got above a certain level, some of the Control colonies exhibited strong uncapping and/or “varroa sensitive hygiene” (VSH) behavior, which apparently was effective at reducing their infestation rates. I’ve seen other resistant colonies do the same — putting it into gear if varroa reaches a certain level.

Practical application: After five years of strong selection for mite resistance, roughly 10% of our colonies require no mite treatment whatsoever. When testing any miticide, I normally use high-mite colonies that obviously show no mite resistance. Unfortunately by the time I started this Hopguard trial, I’d already used up any high-mite colonies for other experiments, and thought that I could use some rejected potential breeders. In retrospect, that was not a good idea!

So perhaps the best interpretation of the data is to look at the percent changes in the average infestation rates for each treatment group over time (Figure 11).

Fig. 11 So, what with the screwy Control group, can we salvage any meaningful information from this field trial? In this graph, I show the percentage of each group’s starting infestation rate (normalized to 100%) at each subsequent time point. At Day 21, by which point the count in the Control group had gone up by 27%, the Hopguard 3-treated groups had gone down by around 80% — not bad at all! [[10]]. Most tellingly, by the end of the trial at Day 66, the Hopguard-treated colonies still had mite infestation rates of around a third of what they started with. (Keep in mind that the trial wrapped up in early September, when mite counts generally rise substantially.)

THE TAKE-HOME

Although I was not able to calculate a legitimate figure for efficacy, I was impressed by Hopguard 3 for being a relatively-gentle mite treatment that could be used during hot weather without concern for queen loss or honey contamination.

Practical application: Similar to formic acid or oxalic dribble, Hopguard is fast-acting and highly efficacious on the “phoretic” mites on the adult bees, which is an important consideration when you want to reduce a high mite infestation rate quickly.

Based upon Aris’ findings that the strips are no longer much active after a week, the Day 66 infestation rates were taken a month to month-and-a-half after the treatments wore off, so the remaining mite populations would have had time to rebuild. These findings suggest that if you need to knock back varroa while you’ve got honey on the hive, two applications of Hopguard 3 two weeks apart provide pretty good control.

A single application of Hopguard 3 may be adequate if a colony is broodless (I’ve successfully used it myself at that time with good success), but it clearly takes at least two applications during the summer if a colony has an active broodnest. The overall efficacy from applying the strips every two weeks, rather than weekly, appeared to be about the same. This comes as a surprise to me, since I would have bet that the three weekly applications would have done a better job. So it looks like three applications might not be worth the cost or effort (it does take a bit of time to apply and remove the strips).

Practical application: Due to my mistake of using colonies with a degree of mite resistance, please do not consider this to be any sort of definitive trial of Hopguard 3. But based on this too-small and confounded test, I’d consider repeated treatment with Hopguard 3 to be a viable non-contaminating, food-grade, and “natural” “knockback” treatment for double-deeps while harvestable honey is on the hive. Since it appears to be relatively gentle on the colony, Hopguard 3 appears to be another valuable tool in our arsenal against varroa.

ACKNOWLEDGEMENTS

Thanks to Brion and Alice Dunbar for their assistance in my field trial.

CITATIONS AND NOTES

[1] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-3/

[2] Rademacher, E., et al (2015). The development of HopGuard® as a winter treatment against Varroa destructor in colonies of Apis mellifera. Apidologie 46(6): 748-759.

[3] DeGrandi-Hoffman, G, et al (2012) The effects of beta acids from hops (Humulus lupulus) on mortality of Varroa destructor (Acari: Varroidae). Experimental and Applied Acarology 58(4): 407-421.

[4] Vandervalk, L, et al. (2014) New miticides for integrated pest management of Varroa destructor (Acari: Varroidae) in honey bee colonies on the Canadian prairies. Journal of economic entomology 107(6): 2030-2036.

[5] Nasr, M, et al (2014) An effective improved application method of HopGuard for varroa control in Canada. Available online.

[6] https://scientificbeekeeping.com/randys-varroa-model/

[7] Gregorc, A, et al. (2018). Toxicity of selected acaricides to honey bees (Apis mellifera) and varroa (Varroa destructor Anderson and Trueman) and their use in controlling varroa within honey bee colonies. Insects, 9(2), 55.

[8] https://scientificbeekeeping.com/tips-for-citizen-scientists/

[9] When testing for efficacy of varroa treatments, using mite washes as the metric, I’ve found that the percent mite reduction is usually substantially greater for higher-mite hives (https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-2/). This presents a problem if Control colonies are chosen randomly, since Controls that start with very high mite counts will often collapse prior to the end of the trial, but if you start with only low-mite Controls, they really can’t be considered to be legitimate controls. So for this trial, I used the four hives with mite counts in the middle as Controls.

[10] If I were trying to sell the product, I would have tossed any subsequent data, since I could have claimed 85% efficacy at that time point.