Tag Archives: resistance

Apistan redux†

I’ve discussed Apistan, a pyrethroid treatment for Varroa, in two recent posts. In these I explained in some detail its molecular mechanism of action. I also explained the two major problems associated with Apistan (and the related tau-fluvalinates ) – the widespread resistance of Varroa to Apistan and the residues it leaves in wax.

In this final post I’m going to revisit just how useful Apistan could be if it was used in a more rational manner. I’m going to concentrate on resistance and you’ll probably need to read the previous post on this topic to provide necessary the background. I’ll only really touch on the residues in wax at the end – I’ve already discussed how these can be minimised if you consider them an issue.

This is (another) long post. It draws together the concepts described in previous articles and links the science of Varroa control to potential strategies to benefit practical beekeeping.

How good is Apistan if Varroa are not resistant?

Apistan

Apistan

Exceptionally good. Pyrethroids are some of the most widely used pesticides. They are widely used because they are very effective. Apistan is no exception. When used to treat Varroa populations that are not already resistant it kills over 98% of the mites in the colony when used according to the manufacturers instructions. 98% … that reduces the National Bee Units’ recommended maximum mite load of 1000 to just 20.

Just how effective is emphasised by a quote from the Apidologie paper cited above. “In treated hives, worker pupae and adult bee infestations decreased from 14.2 ± 7.3% to zero and from 15.7 ± 7.3% to zero, respectively. Whereas, in the two control hives, during the first 6 weeks, the average worker pupae infestation increased from 15.9 ± 2.9% to 19.7 ± 3.5%”.

Most mite mortality occurred during the first 4 weeks of treatment and the level of Apistan present at the beginning and end of treatment remained at about 10% i.e. it should be as active at the end of the treatment period as at the beginning.

How good is Apistan in reality

Resistance was first demonstrated in 2002 and is now widespread in the UK. In a recent paper, Ratneiks and colleagues (University of Sussex) demonstrated that Apistan was significantly less effective at killing Varroa when used for a second treatment, four months after the first. In this study they showed only 33% of mites were killed at the second treatment, whereas 58% were killed in colonies treated for the ‘first time in five years’.

This isn’t rocket science … if there are some resistant mites in a population then Apistan will preferentially allow these to survive. Consequently they will make up a greater proportion of the mite population when re-treated.

Since we know the molecular basis of resistance to Apistan it would now be possible to determine – without doing the treatment and counting the corpses – what proportion of mites were resistant in a population before treatment. It would therefore be easy to determine whether treatment would be likely to work.

Equally, it would be possible to determine whether the colonies ‘not treated with Apistan for five years’ still maintained significant levels of Apistan resistant mites. As will become clear, there are studies that contradict this, and the definitive test – the presence of absence of the mutation that confers resistance – was not done in the Sussex study.

Apistan resistance and fitness costs

Mutations, such as the one that confers resistance to Apistan, can – in broad terms – exert three different effects:

  1. Beneficial – the presence of the mutation favours the organism (a fitness benefit), the mutation will be selected for and it’s presence in the population is likely to increase.
  2. Detrimental – the mutations causes a fitness cost and organisms that carry it are likely to reproduce less well, resulting in it being lost from the population.
  3. Neutral – the mutation is neither beneficial nor detrimental.

In the presence of Apistan, the Leucine to Valine mutation at residue 925 (L925V) of the voltage gated sodium channel (VGSC; please see the previous article on the molecular basis of resistance), is a beneficial mutation. Any mites that carry it will not be killed and will be able to reproduce, so increasing it’s prevalence in the population. The same reasoning applies to other Apistan resistance mutations.

The VGSC of Varroa evolved over eons in the absence of Apistan. The mutation is in a part of the protein critical for its function (that’s why Apistan binding blocks function). It’s therefore perhaps unsurprising that in the absence of Apistan selection there is evidence that the L925V mutation is detrimental. In simple terms the VGSC works less well with a Valine at position 925 than a Leucine unless Apistan is present. Where’s the data that supports this?

The influence of prior treatment on Varroa genotype

Table 1. Apistan resistance mutations in Varroa from treated and untreated colonies

Table 1. Apistan resistance mutations in Varroa from treated and untreated colonies

The table above needs a little explanation. Colonies from Henlow and Shillington were treated with Apistan and tested one month later. Colonies from Harpenden, Bishop Stortford, St. Albans and Peterborough had no history of Apistan treatment in the recent past. Unfortunately, the paper does not make clear when the last treatment was, with the exception of a sample from Harpenden which had not been treated for at least 3 years.

Varroa is diploid i.e. there are two copies of the gene for the VGSC. The S and R heading the columns SS, SR, RR, indicates whether the Apistan resistant mutation is absent (S = sensitive) or present (R=present). SR indicates that the mite was heterozygous, one resistant copy and one sensitive. Whether these mites have lower resistance than RR mites has not been determined – for the purpose of the remaining discussion I’m going to lump the SR mites with the RR mites and assume they are resistant§.

Of 279 mites tested, 40 were from Apistan-treated and 329 from -untreated colonies. Of the 40 mites from Apistan-treated colonies, all contained the mutation conferring resistance to the fluvalinate. Of the 239 mites from colonies not recently treated with Apistan, 215 were sensitive and only 25 were resistant.

This suggests that in the absence of Apistan, Varroa sensitive to the fluvalinate replicate better.

Is this a surprise?

No. Partly for the reasons explained above … the Leucine at position 925 is likely to stop the VGSC working as well. More compellingly though is the wealth of data suggesting that insecticide resistance is associated with fitness costs in a range of other insects.

Colorado beetle

Colorado beetle

For example, pyrethroid resistant Myzus persicae (peach-potato aphid) exhibit fitness effects in overwintering survival, response to aphid alarm pheromone and vulnerability to parasitoids; pyrethroid-resistant Cydia pomonella (codling moth) have reduced fecundity, body mass of instars, adult male longevity and larval development; finally, pyrethroid-resitant mutants of the snappily-named Leptinotarsa decemlineata (which you of course know as the stripy-attired Colorado beetle) have reduced fertility and fecundity.

Google will find relevant reference on all the above examples or you can refer to a concise mini-review by Kliot and Ghanim Fitness costs associated with insecticide resistance published in Pest Management Science (2012) 68:1431-37.

Before discussing implications for practical beekeeping I should add that the rate at which the loss of the L925V mutation, and other mutations associated with Apistan resistance, needs to be accurately determined. If, as looks likely, a period of 3+ years results in selection for the sensitive variant of the VGSC, it might be possible to develop rational Varroa treatments that exploit this.

Apistan resistance, rational Varroa control and practical beekeeping

For the sake of discussion, let’s accept the following statement:

  • Apistan is devastatingly effective on sensitive mite populations.
  • Apistan is much less effective (or almost completely useless) on resistant mite populations.
  • Resistance by Varroa is acquired rapidly and lost over the subsequent 2-3 years in the absence of selection.

An effective and rational Varroa control strategy would only use Apistan once every 3-4 years, alternating it with other treatments. To mitigate the transfer of Apistan-resistant mites between colonies due to drifting and robbing, or due to the movement, sale and/or relocation of hives during the season, Apistan use would have to be coordinated. This coordination would have to be both geographical and temporal. There would be no point in the Fife beekeepers using it one year if the Angus beekeepers planned to use it the following year.

“Like herding cats” I hear some mutter …

Perhaps, but the benefits would be considerable. How could it be achieved? Perhaps by restricting the sale of Apistan to certain years, in a formulation or package that meant it had to be used quickly or became inactive.

What about the residues in wax?

I’m not sure whether the level Apistan accumulates to in wax is sufficient to be a selective pressure on the mite population. Apistan strips are 10% Apistan. Nothing like that much accumulates in wax. In a recent study fluvalinate levels ranged between 2 and 200,000 parts per billion in wax (mean ~7500 ppb). However, it is a valid concern and so would necessitate a relatively simple experiment to determine the rate at which Apistan resistant mutations are lost in the presence of absence of trace levels of Apistan in comb.

Herd immunity and the responsibility of the individual

There’s a debate in human healthcare about the necessity to vaccinate individuals in a well-vaccinated population. The chance of an infectious disease spreading to the unvaccinated individual in a protected population is very slight. So, why vaccinate?

Well, what if increasing numbers decided not to vaccinate? Once protection in the population falls below a certain level there is a significant chance that an infectious disease will spread widely. We saw this in the UK after the MMR (measles, mumps and rubella) vaccine was falsely claimed to be associated with autism. Vaccination rates dropped from 90+ percent, to low 80’s and – in parts of the country – to only 60%. Unsurprisingly, measles cases increased and – tragically, for the first time in years – there were childhood deaths due to measles infection.

This may seem a million miles away from looking after our bees, but there are parallels. As beekeepers we have responsibility for our own stock. We also have responsibility to the wider community of beekeepers which – because of the way our bees forage and mingle – happily exchange pests and pathogens.

Beekeepers who do not control Varroa (and consequently virus) levels threaten the viability of their own colonies and those of other beekeepers in the area. The same applies to the foulbroods. This is why the bee inspectors try and check all colonies in the vicinity of an outbreak. This is why standstill orders are placed on apiaries where outbreaks occur.

Perhaps this sort of communal responsibility also applies to Varroa treatment using Apistan? Beekeepers who treat without demonstrating very high levels of susceptibility first in their stocks are simply selecting for resistant mites, reducing the efficacy of treatment for themselves, and others, in the future. Indiscriminate or incorrect use of Apistan has resulted in widespread resistance, thereby compromising Varroa control for all beekeepers.

The coordination and control, geographically and temporally, of Apistan usage would benefit beekeeping and beekeepers.

And … it would also benefit those who chose never to treat with Apistan. Treated colonies in the one year in three Apistan was used would have very low mite levels. Fewer mites would be transferred from these colonies by drifting or robbing … what’s not to like?


 Redux, as in the literary term meaning brought back or restored, derived from the Latin reducere (to bring back).

 This is one compelling reason why Apistan strips should not be left in the colony longer than is recommended. It kills the susceptible mites within the first month or so. After that it effectively selects for resistant mites, allowing them to replicate.

 With apologies to any population biologists who were reading this and have now given up in horror.

§ And I’ll save discussion of the influence of the incestuous lifestyle of Varroa and Varroa levels on the ratio of homozygotes to heterozygotes at different stages of the season for a later post. It’s a fascinating and at the same time rather sordid tale …

 Or 4 or 5 – this would need to be determined empirically.

Apistan resistance

Apistan

Apistan

In an earlier article I discussed what Apistan is – a pyrethroid miticide – and the consequences of using it. These include decimation of the mite population if it is susceptible, coupled with the accumulation of long lasting residues in wax. These residues may adversely effect queen and drone development. I also discussed ways to avoid build-up of Apistan residues in comb.

The key phrase in the paragraph above is ‘if it is susceptible’. Unfortunately, resistance to Apistan and the related tau-fluvalinates develops very quickly. To understand why we’ll need to look in a little more detail at how Apistan and other pyrethroids work.

How does Apistan work?

Apistan, like other pyrethroids, works by blocking the activity of voltage gated sodium channels (VGSC) resulting in paralysis because the axonal membrane cannot repolarise.

What on earth does that mean?

Action potential

Action potential

Nerve transmissions – like the signal from the Varroa brain to tell the Varroa legs to move – travel along axons. These are usually very long thin cells. In the adjacent image the ‘brain’ is on the left and the leg muscles on the ‘right’. The nerve impulse (the moving arrow) travels down the axon ‘driven’ by a change in polarity (charge) across the membrane of the axon. In the resting state, when there is no impulse, this is positively charged on the outside and negatively charged on the inside. Sodium – remember the ‘S’ in the acronym VGSC – is positively charged and crosses the membrane (out to in) via a small pore or hole as the impulse passes. This makes the inside of the axon transiently positive. The pore or hole is the VGSC.

Top view of a VGSC

Top view of a VGSC

The VGSC is a transmembrane protein. It actually crosses the membrane multiple times and assembles to form a very narrow channel through which the sodium passes. The cartoon on the right shows the top view of a VGSC, looking “down” the pore into the inside of the axon. The blue bits can move to open or close the pore, allowing sodium to traverse – or not – the membrane into the axon. Apistan binds to the transmembrane protein and prevents the pore from closing. As a consequence, sodium continues to pass from the outside to the inside of the axon, the nerve cannot repolarise and no further impulses can be transmitted. As a consequence, Apistan paralyses the Varroa.

But I don’t suppose many beekeepers will feel much sympathy for the mite 😉

Why isn’t the beekeeper paralysed as well?

Nerve impulses in Varroa and humans are transmitted in essentially the same way. We also have VGSC’s that operate in a similar manner. Why doesn’t Apistan also paralyse careless beekeepers? More generally, why are pyrethroids the most widely used insecticides, available in all garden centres and supermarkets?

Two factors are at work here. The first is the specificity of binding. The VGSC is a protein. Proteins are made from building blocks termed amino acids. The precise sequence, or order, of amino acids is usually critical for protein function. However, two proteins with a similar function can exhibit differences in the amino acid sequence. Although the human and mite VGSC have a similar function they have a different amino acid sequence. Apistan binds much better to the mite VGSC than the human VGSC (this also explains why bees aren’t also paralysed by Apistan … the miticide is specific for the mite VGSC and binds poorly to the honey bee VGSC). In addition, many mammalian species have a number of detoxifying enzymes which deactivate pyrethroids, rendering them ineffective. Together, this explains the specificity of Apistan and other pyrethroids, and the low level of toxicity to humans.

So now you know how Apistan works we can address the much more important question …

Does Apistan work?

Unfortunately, usually not. Since the late-1990’s there have been a large number of publications of Apistan- or fluvalinate-resistant mites from many countries, including the USA (1998, 2002), Israel (2000), UK (2002), Spain (2006), Korea (2009) and Poland (2012). The National Bee Unit used to report Varroa resistance test results by geographic region in England and Wales. Resistance was first reported in mites from Cornwall and Devon (in 2001 and 2002). By 2006 resistance was very widely distributed throughout England. By then approximately a third of all mite samples tested were resistant. The number of tests conducted (or at least reported) then dwindled and there have been none reported since 2010. Not no resistance … no tests. Presumably it’s no longer worth reporting as resistance is so widespread.

The most up-to-date map on the distribution of Apistan resistance I could find is in the NBU booklet on Managing Varroa [PDF; page 28 of the 2015 edition], though the data presented is from 2009.

However, bee equipment suppliers continue to sell Apistan (even Vita, the manufacturer, states that resistance is widespread) and beekeepers continue to use it. Many do so without first testing whether the mite population in their colonies is sensitive to the miticide. How should this be done?

Testing for resistance

Vita suggest two tests. Their first (the “rule of thumb test”) is deeply flawed in my view. It suggests simply looking for a drop of 100’s of mites in the first 24 hours after treatment starts as indicative of a sensitive population.

This isn’t good enough. What if there were thousands of mites present? Perhaps 20% of the population are sensitive, with the remainder resistant. 20% of 5000 mites is 1000 … so you might expect a drop of 100-200 (the majority of the phoretic population) within the first 24 hours. Some might consider this drop indicates a sensitive population … it doesn’t.

It’s not sufficient to count the corpses … you need to know how many mites were unaffected by the treatment.

The second Vita-recommended test is a cut-down version of the “Beltsville” pyrethroid resistance test which is fully described in an NBU pamphlet (PDF). This is much more thorough. Essentially this treats ~300 bees with Apistan, counts the mites that are killed in 24 hours and then counts the unaffected mites remaining on the bees. It’s only by knowing the total number of mites at the start and by determining the percentage of mites sensitive that you can be sure that the treatment is effective.

What is the molecular basis of resistance?

We’re almost there … specific pyrethroids, like Apistan, bind to specific parts of the VGSC. The VGSC is a protein made up of a long connecting chain of amino acids. The binding of the pyrethroid requires an interaction with a small number of specific amino acids in the VGSC. If these particular amino acids change – through mutation for example – then the pyrethroid will no longer bind. If the pyrethroid does not bind the VGSC can open and close again, so the axon repolarises and the mite is not paralysed. The mite is resistant and can then go on to rear lots more resistant baby mites … which, in due course, transfer the viruses that kill your bees.

And that’s exactly what happens.

Leucine

Leucine

A single mutation that causes a substitution of amino acid number 925 in the Varroa VGSC, which is usually a leucine, to either a valine, a methionine or an isoleucine, is sufficient to prevent Apistan and other tau-fluvalinates from binding. At least 98% of mites resistant to Apistan have one of these substitutions. Apistan resistant mites with substitutions at position 925 have been found in the UK, eastern Europe and several sites in South-Eastern USA. It wouldn’t be surprising if the remaining ~2% of resistant mites had a mutation at one of the other amino acids involved in pyrethroid binding. Further studies will confirm this (there are alternative mechanisms that cause resistance, but the one described here is the most frequently seen).

Why aren’t all Varroa mites resistant to tau-fluvalinates?

Apistan resistance has clearly been demonstrated for the last two decades. Resistance is easy to acquire and selection – in the presence of the pyrethroid – is effectively absolute. Without the necessary mutation the mites die, with the mutation they survive.

Bees – and the phoretic mites that are associated with them – are moved around the place all the time, by migratory beekeepers, by importers and through robbing and drifting between colonies.

Why therefore aren’t all Varroa mites now resistant to Apistan and other tau-fluvalinates?

The answer to that is interesting and suggests strategies that could make Apistan an effective treatment again … but I’ll save that for another time.


Only transiently as the charge is reversed shortly afterwards by a similar, though not identical,  mechanism that does not use the VGSC. However, life is simply too short to describe this bit as it’s not needed to understand pyrethroid – or Apistan – activity and resistance.

 The incestuous life cycle of the Varroa mite is important here. This post is already too long to fully elaborate on this but the size of the mite population relative to available open brood (and whether you get single or multiple occupancy of cells) will likely influence the proportion of resistant, partially resistant and sensitive mites in a population.

Credits – the action potential GIF was created by Laurentaylorj from Wikipedia.

 

Apistan and residues

This is the first of two or three posts on Apistan, a widely used yet often ineffective miticide sold for Varroa control. I was originally going to title this post “Don’t do this at home” and restrict discussion to Apistan misuse and resistance in the UK. However, having drafted the article it was clear there was more than could be covered in a single post (or at least comfortably read).

I’ve therefore split it up; the first focuses on what Apistan is, how it’s used and the consequences of use for the hive. Next time – though possibly not next week – I’ll cover the molecular mechanism of activity and mite resistance.

What is Apistan

Apistan

Apistan … tau-fluvalinate

Apistan® is a miticide used to kill Varroa. It is a registered tradename used in the UK and other parts of the world. The active ingredient is a synthetic pyrethroid tau-fluvalinate (or sometimes τ-fluvalinate). Synthetic in this instance means it is not a natural compound, but is produced using a chemical process. Other miticides containing the same active ingredient include Klartan® and Minadox® – precise compositions may vary, but the important component is the tau-fluvalinate. In the UK, Apistan is supplied by Vita (Europe) Ltd. and sold by all the leading beekeeping equipment suppliers. I’ll use the name fluvalinate and Apistan interchangeably in the remaining text.

Instructions for use

Apistan can be used at any time of year but its use is recommended in late summer after the honey harvest. The active ingredient, fluvalinate, is supplied as impregnated polymer strips, two of which are hung vertically in the brood box, between frames 3 & 4 and 7 & 8. It is a contact miticide and needs to be located near the centre of the colony to get trampled through the broodnest. Nucs and weak colonies only should be treated with one strip. The treatment period is 6 to 8 weeks i.e. a minimum of two full brood cycles. The instructions specifically state that it should not be used for less than 6 weeks, or more than 8 weeks. This is to avoid the selection of a resistant mite population. Apistan should not be used when there is a nectar flow.

How effective is Apistan?

On susceptible mite populations Apistan is fantastically effective. Cabras and colleagues in Italy reported greater than 99% efficacy in studies published in 1997.

Fluvalinates and foundation

Importantly, because of its chemical formula, Apistan is fat soluble, meaning it is readily absorbed into or dissolves in fats … like beeswax. It is also a very stable compound. In a relatively recent study by Jeff Pettis and colleagues all 21 samples of commercial foundation tested were contaminated with fluvalinates. This was a US study and I’m not aware of an equivalent analysis of UK foundation suppliers. However, there is an international trade in beeswax and fluvalinates are used globally. I’d be very surprised if any commercially-purchased foundation – perhaps other than the certified organic stuff – was  not contaminated with fluvalinates.

Are fluvalinates in wax foundation a problem?

These studies are difficult to conduct using field-realistic levels of miticides. Nevertheless, despite the fact that the absolute toxicity of fluvalinates for honey bees is very low (i.e. a lot is needed to kill the bees – the compound has a high LD50 0%) there is compelling evidence that sub-lethal levels are probably detrimental. Drones reared in fluvalinate-treated hives exhibit increased mortality, reduced bodyweight and decreased sperm production. Similarly, queens reared in treated colonies exhibited lower body weight. More recent studies by Keith Delaplane and colleagues tested emergence weight, memory, learning and longevity of workers exposed to fluvalinates and did not show any significant differences between treated and untreated colonies. In contrast, coumaphos – an organophosphate used for Varroa control – was clearly detrimental in these studies. Perhaps the most significant result in this study was that mite levels in treated and untreated colonies were unaffected … there was no evidence that the Apistan worked. I’ll discuss resistant in a future post.

Avoiding fluvalinate residues in comb

There are a variety of ways to avoid fluvalinates in comb. The first would be to use certified organic wax foundation. Thorne’s sell this for about twice the price of their standard worker brood foundation. This foundation is manufactured from beeswax sourced from New Zealand. Although certified organic, it’s not clear whether the wax has been tested for the presence of fluvalinates (an expensive process … so I’d be surprised if it had been). For reasons that will become clear shortly, just because the colonies used to source the wax had not been treated does not mean that there are no fluvalinates present in the comb from which the wax was rendered. Apistan was licensed for use in New Zealand seventeen years ago, shortly after Varroa was imported to the country.

An obvious way to reduce fluvalinates in comb is to use foundationless frames. Even if commercial foundation contains traces of the chemicals, by using only thin starter strips you can significantly reduce contamination. Perhaps even better, by making your own starter strips from wax recovered from your own brace comb, cappings or foundationless frames, you can exclude the need for commercial foundation – and all the ‘extra goodies’ it contains – completely. I’m also investigating the use of unwaxed wooden starter strips this season, removing any chance of initial contaminants (note that this is not my primary reason for trying these).

And now the bad news …

Unfortunately, avoiding commercial foundation of any sort and letting the bees draw comb directly from unwaxed starter strips still might not prevent the appearance and accumulation of fluvalinates in your hives. In the Delaplane study they used brand new hives and foundationless frames with plastic starter strips. After one year they compared treated and untreated colonies for the presence of fluvalinates in drawn comb. Unsurprisingly, treated colonies contained high levels of residual Apistan. However, untreated colonies also contained statistically significant levels of Apistan, four times higher than their detection limit. Coumaphos was also detectable at significant levels in untreated colonies. The authors suggest that the presence of both Apistan and Coumaphos was due to drifting of bees from treated colonies carrying the miticide into the untreated colonies. Therefore, even if you don’t use Apistan, if your neighbour does you are likely to get low levels of fluvalinates accumulating in comb – even when using foundationless frames.

The Delaplane study appeared in 2013. An earlier article appeared in Bee Culture in 2009 which described the fluvalinate contamination of both commercial foundation and comb supplied by ‘chemical free’ beekeepers. It’s much easier reading than the data-rich Delaplane article.

Conclusion

If used appropriately, at the right time of the season on a susceptible mite population, Apistan is very effective at killing Varroa. If used like this, Apistan levels will accumulate in the beeswax in the colony. This may be detrimental for drones or queens reared in the colony, but current studies indicate is probably has negligible effects on the worker bees.

However, widespread use of Apistan has resulted in the rapid and widespread selection of resistance in the mite population … meaning that Apistan often has negligible effects on Varroa. I’ll discuss this in more detail in another post.


What do you think happens to all the reclaimed beeswax traded with Thorne’s and other companies? It’s recycled into new sheets of foundation. You might not use fluvalinates, but many beekeepers do and this will be generously divided up across all the new sheets of pressed foundation.