Single-Target Acaracide Resistance in Varroa

Are Organic Acids A Resistance-Free Alternative?

Earlier this year, Australia recorded its first case of flumethrin-resistant mites in northern New South Wales, and this same population later tested positive to amitraz a non-pyrethroid acaracide. Does this mean these treatments are ineffective and should no longer be used? No, not at all- the resistance is contained inside parts of Southeast Queensland & the aforementioned Northern NSW – Beekeepers elsewhere should continue with their (proper) mite management plan.

This article discusses the mechanisms of Varroa resistance to the single target agents (e.g. flumethrin, fluvalinate, amitraz & coumaphos), how said resistance could arise, as well as the organic acid alternatives found in formic & oxalic acid administrations. It ends with a discussion on the ways of preventing the development of acaracide resistance. The short answer is rotate treatments each season/year and use according to the label. The long answer? Read on.

Resistance Mechanisms in Varroa destructor

The non-technical reader is advised not to skip this. Read slowly and ask questions

Pyrethroids Resistance Occurs via Changes in Voltage-Gated Sodium Channels

The L925I resistance is an example of a point mutation occurring in the mite’s voltage-gated sodium ion channels, which are the target of pyrethroid chemicals like flumethrin and fluvalinate (sold as Bayvarol & Apistan respectively; reviewed in Field et al., 2017). The mutation L925I is a “knockdown resistance”¹ that reduces the activity of pyrethroids on these sodium channels, and similar point mutations exist which produce the same outcome: reduced pyrethroid efficacy (see Dong et al., 2014). I should clarify that fluvalinate resistance arises when the same 925-residue leucine is turned into valine (i.e L925V; Gonzalez-Cabrera et al., 2013).

Some mutations like the one at 925 are widely distributed, with L925I being the one identified in the apiaries at northern New South Wales. There is also metabolic inhibition of flumethrin by the CYP3002B2 enzyme, and this is elaborated on below.

Amitraz Resistance is a Result of Modified Octopaminergic Signalling at Target Sites

Amitraz binds to the receptors of octopamine and tyramine, both of which are involved as “neurotransmitters” and have high-level functions around behaviour, reproduction, development, fight or flight, and so on. Activation of these receptors leads to “death by overexcitation”; the mite is unable to remain attached to the bee, spasming uncontrollably until her end (the mites we see are female).

One study found that mite resistance to amitraz occurred as a result of two amino acid substitutions in the octopamine receptor Oct β₂R: “A substitution of asparagine 87 to serine (N87S) associated with treatment failures in France, and a substitution of tyrosine 215 to histidine (Y215H) in samples collected across the USA” (Hernandez-Rodriguez et al., 2021).

However, a later study out of Israel suggested that these target-site resistance mechanisms had a “minimal association with the resistance phenotype in this population”, as a genetic sequence of these mites did not show the same resistance mutations mentioned above (Mavridis et al., 2025). Their results found an overexpression in several cuticle genes & the metabolic enzyme cytochrome P3002B2 (CYP3002B2) within the resistant population. The former suggests decreased chemical uptake as a result of reduced exoskeleton penetration, which gives time for the mite’s natural detoxifying chemicals to metabolise the compounds and prevent toxicity.

The latter was assessed in vitro and was found to be a metaboliser of amitraz, transforming it into a non-toxic metabolite “without producing any of the known activated/toxic metabolites associated with amitraz” (Mavridis et al., 2025). This CYP3002B2 protein was also able to metabolise flumethrin into a non-toxic form, and since flumethrin is a different chemical class (pyrethroid vs formamidine [amitraz]), it suggests that a mite’s body will upregulated CYP3002B2 as a generalised detoxifying enzyme… good for mite, not for bee (or me; Mavridis et al., 2025). Nonetheless, the authors suggest further in vivo testing is required to determine the extent of CYP3002B2’s contribution to treatment resistance.

Coumaphos Resistance is Due to Decreased CYP450 Enzyme Activity

Coumaphos has a metabolic means of resistance, resulting in reduced bioactivation of the molecule (as opposed to reducing its overall toxicity; Vlogiannitis et al., 2021). This works by decrease the activity of two CYP450 enzymes (encoding genes CYP3012A6 & CYP4EP4) that would have otherwise converted the coumaphos pro-drug (“inactive form”) into the active version, coroxon (“coumaphos-oxon”), which irreversibly inhibits the mite’s acetylcholinesterase enzyme, leading to death via overexcitation.

Instead, the resistant mite keeps the ingested coumaphos in its unmetabolised form, and carries on with the Varroa version of business-as-usual. The authors found no significant changes in the mite’s uptake of the acaracide, general detoxification ability nor modifications to acetylcholine’s binding site (the location on where the coumaphos-oxon molecule acts upon). Interestingly, Varroa’s CYP genome only has 26 full-length sequences (“genes”), making it one of the smallest genomes in arthropods; honey bees have around double the amount of these CYP detoxification genes (Vlogiannitis et al., 2021).

Mites Can’t Develop Resistance to Organic Compounds Because of Multi-Target Action?

Theoretically, resistance to the organic acids is still possible, it’s just that in reality, it’s more difficult for Varroa to develop it because of a multimodal mechanism of action. The pyrethroids target a single site in the mite’s body, and one change to this site renders them less effective. One literature review on oxalic acid by Kosch, Mülling & Emmerich found that there is currently “no resistance” to this compound, however further testing is required since its mechanism of action is not-as-well defined or specific to Varroa (Kosch, Mülling & Emmerich, 2024). The review discussed three major forms of oxalic acid administration:

• Trickling, where oxalic acid is dissolved in a water/sugar syrup and dribbled onto the frames
  • This works by having bees ingest the syrup, transferring the compound to the mite as it feeds on the oxalic-acid containing fat bodies. Alternatively, mite could die from direct contact with the dribbles (which is rather rare – what is a mite doing on the top bars anyway?).
• “Exchange via direct contact”, which is a fancy way of saying “using strips that were saturated with oxalic acid and then placed inside the hive” (akin to how flumethrin is administered).
  • Here the compound adheres to the bees’ body, and is transmitted around the hive as the bees groom themselves/each other. A mite that feeds on a “loaded” bee ingests the oxalic acid and dies
• Vaporisation (I’m hesitant to call it “sublimation” as the acid is still in its liquid form), the evaporation of oxalic acid crystals using a commercial that’ll heat up the crystals and eject them as a “hot vapour” (not gas!), killing any mites in uncapped cells.
  • Straightforward: the hive is blasted with hot oxalic acid vapours, knocking down mites “unprotected” by a capped cell. Repeat this three times every ~8 days and you’ve performed yourself a full treatment, as this is the length of a drone’s lifecycle (recall that workers take 21 days & drones 24 to go from egg to eclosion)

Of the three routes of administration, there was no evidence of resistance in the literature, despite signs of “increased dosage”, which is normally associated with a reduction in efficacy (Kosch, Mülling & Emmerich, 2024). Again, given the mixed reporting and variations in treatment styles/geographies, the results were inconclusive, with a lean towards “no signs of mites developing resistance to oxalic acid treatments”.

Formic Acid has Multiple Mechanisms of Action That Makes it Very, Very, Very (Very, Very) Hard for Mites to Develop Resistance

What of the vapours in formic acid? It’s a volatile gas that “fumigates” hives, killing mites near, far and wherever they are, including those inside capped cells (the fumes diffuse across the wax). Its effects are systemic: the acid vapours penetrate the mite’s exoskeleton, binding to the mitochondrial enzyme cytochrome c oxidase and thereby inhibiting the mitochondrial electron transport chain (affirm: Mitochondria is the Powerhouse of the Cell™). Less respiration means reduced removal of metabolic wastes, and the mite ultimately dies from high acidity (“acidosis”).

Genath and colleagues set out to determine formic acid’s exact mechanism of action on Varroa, and found reductions in several enzymes associated with the electronic transport chain (NADH dehydrogenase, cytochrome c oxidase & ATPases [for those who wanted to know]), which would result in increased reactive oxygen species, themselves causing multiple problems to an organism’s cell (Genath et al., 2021). At the risk of overexplaining, the authors also found a reduction in protein balance, with an increase in proteasome activity alongside a simultaneous inhibition of protein biosynthesis.

It’s not hard to see the consequences of higher protein breakdown and decreased protein production- just another mechanism by which formic acid kills Varroa. The downstream effects are plentiful: nutrient deficiency, hypoxia, generalised stress, hormone imbalance, reproductive impacts… the list goes on. Long story short, it’s highly unlikely, if not “impossible” for Varroa to develop resistance against formic acid, given such multimodal mechanisms of action. Suppose a mite population was able to overcome the mitochondrial inhibition (a rare event in itself, since multiple enzymes are involved) – they would still have to deal with ribosome inhibition, or proteasome activation – survival is slimmer than a pack of Esses.

So Why Isn’t Everyone Using Formic Acid?

It’s because of the factors that affect formic acid’s success:

• Temperature (Formic Pro’s label advises use when ambient temperatures are ~10-30°C),
• Evaporation dynamics (did you apply the treatment correctly & was it stored properly?),
• Colony strength (a weak hive may end up succumbing to the fumes),
  • A strong hive may also succumb if the dose is too high – I learnt this the hard way.
• Hive setup (the treatment is more concentrated in a single box versus three box Langstroth) and,
• Product formulation (absorbent pads and gel formulations versus slow-release products).

When temperatures or dosing exceeds the recommended range, the colony suffers greatly, with the queen being most vulnerable. Tihelka’s 2018 review cites how high concentrations of formic acid vapours can halt oxygen consumption in brood and induce cell death in larval tissues, how it can increase protein breakdown in the worker’s cuticle, reduce the number of sealed broods, and trigger the removal of brood located around the site of treatment placement, culminating with the abscondment/death of the entire hive if doses get too high (Tihelka, 2018).

If you acknowledge the caveats above, there’s no problem with (correctly & cleverly) using formic acid as part of your integrated pest management approach. Ristanic and colleagues found that in addition to killing mites, formic acid also increased the hygienic behaviour of A. mellifera whereas flumethrin and amitraz did not (Ristanic et al., 2025). The benefit here is from the indirect “disease prevention” that comes about from increased hygienic behaviour. Hypothetically, suppose that in setting out to treat Varroa, the formic acid also helps in stopping the spread of European foulbrood or Nosema, as a result of increased cleanliness.

Treatment Failure is Not the Same as Treatment Resistance

Is it really “resistance” that a beekeeper is reporting when they still find Varroa after conducting a chemical treatment, or was the hive unsuccessful with the treatment? Consider that a poor result can suggest:

• Reinvasion by Varroa/mite migration (as in from returning robber bees or drifters from other colonies)- both scenarios attributable to “bad luck”,
• Incorrect dosing, as when two flumethrin strips are used for a full-sized hive instead of four,
• Temperature‑dependent delivery, as with formic acid above,
  • Interesting case with formic acid: if you test for mites soon after treatment, you will get a skewed results with plenty of mites, not because the treatment failed, but rather due to the workers removing dead mites from the cells of newly-emerged bees. Wait a fortnight or so for all the capped brood to emerge and then perform a mite check.
• Expired products, so check the packaging and label.

The ideal “interpretation standard” requires a uniform method of pre- & post-monitoring, as well as uniform bioassays & molecular diagnostics for genes known to confer resistance Varroa– the latter two approaches not easily available to your average beekeeper. A report published by the European animal health panel notes that a standardised & universally-adopted testing system for quantifying Varroa resistance has historically been lacking and will continue lacking due to global differences in beekeeping approaches (I would also add: the difficulties in identifying and assessing the mite loads of wild colonies). For those interested in the full report on this matter, consult the European Food Safety Authority (EFSA) Panel on Animal Health and Welfare; see More et al., 2017. Warning: it’s drier than a three-year old pollen patty; ask me how I know.

How Beekeeper Mismanagement Results in Resistance Development: Pointing Fingers?

No one is pointing fingers at beekeeper mismanagement (yet), because of various reasons: “they weren’t trained well enough”, “there is a lack of treatment varieties on the market”, “the government should be more clear on the guidelines”, or my favourite “he/she has been a beekeeper for XYZ years and know what they’re doing!”² The unofficial consensus is that the average beekeepers (who are often not a part of beekeeping clubs) are not rotating their treatments or are using products off-label.

Take the Australian government’s guidance permit on flumethrin as an example:

• Strips are to be used between combs in the central brood rearing area (in a ten-frame Langstroth it is between frames 3 & 4 / 7 & 8 in ten frame hives).
  • I like to place them between frames 4-7, sue me.
• A 3-6 week treatment is used to “supress” an infestation, whereas a 6-8 week duration is for “control” (“treatment”) of an infestation.
• It is not to applied more frequently than 6-8 weeks
  • If you’ve used it for the control treatments above, then you have to wait another 6-8 weeks, to reapply, i.e. ~16 weeks from the day you first placed in them your hive.
• Not to be used more than nine times per year.
  • If you’re having to treat your hives nine times a year, you’ve got bigger problems.
• Where possible, it should be rotated with other products that have a different mode of action.
  • E.g. flumethrin-oxalic acid or flumethrin-formic acid, but not flumethrin-fluvalinate as they both have the same mechanism of action: overexcitation of the sodium ion channels.

You my dear reader probably find this list to be reasonable and easy to follow, and I do too. Alas, many beekeepers (and people in general) do not think this way. I am continually surprised (and entertained) by the “alternative treatment” crowd, just think of those social media posts advocating for rhubarb leaves (which naturally contain oxalic acid) as a Varroa treatment… oxalic acid from vapours and oxalic acid from vegetables are not the same thing.

Why Resistance Evolves Readily in Varroa

I conclude with comments on the drivers of resistance. A selection pressure arises when the same chemicals are used year after year in the same population. As a result, those mites with the favourable alleles (those genes that confer resistance) are the ones who will survive. Given that Varroa reproduction is incestuous (the reproductive mother mite lays both male and female eggs in the cells, and the females emerge mated to begin the cycle anew), the favourable trait will rapidly spread in a population. You can see this when reading between the lines of the research: “resistant colonies came from areas with historically high chemical XYZ usage”.

Then there is the issue of sublethal exposure and chemical residue persistence which contribute to additional selection. Residues in wax and beebread can persist and apply chronic (but low grade?) selection pressures even after a beekeeper stops applying a product, as recorded by Benito-Murcia and colleagues, who found that residual fluvalinate contamination in the wax comb resulted in the emergence of mite populations with pyrethroid-resistant genes even though the colony was not treated with this compound within the past four years (Benito‑Murcia et al., 2021).

Then you have the poor beekeeping practices alluded to above, and failure to follow the proper techniques. Finally, there’s bad luck: you’ve treated your hive only for them to rob a collapsed colony and become reinfested from that “mite bomb”, or you’ve had infested bees drifted into your colony… bad luck and trouble.

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Footnotes

1. To get technical with kdr mutations, L925I represents the shift from a “leucine” at amino acid residue (“position”) 925 to an I, or “isoleucine”.  Another mutation is methionine 918 to taurine or leucine or valine or isoleucine (M918T/L/V/I), leucine 932 to phenylalanine (L932F)… the list goes on: threonine 929 (T929I/C/V/N), phenylalanine 1534 (F1534C), leucine 1014 (L1014F/H/S/W), and phenylalanine 1538 (F1538I); Karakaya , Arslanhan & Onder, 2024. ↩︎
2. I do consider “not trained well enough” to be a valid reason, when left to our own devices, we would rather perform enjoyable tasks like extracting honey or swarm management. Taking the time to learn about integrated pest management or the basis of chemical Varroa treatments is riveting for someone without a background in science/biology… many such cases!
   Somewhat related is that the that the colonies used in the study of coumaphos-resistant populations mentioned above came from “a beekeeper who used coumaphos almost exclusively for many years” (Vlogiannitis et al., 2021). Not trained well enough, indeed. ↩︎