Tag Archives: Bayvarol

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.