Category Archives: Diseases

Superinfection exclusion

Alpha

Beta

Gamma

Delta 

The majority of readers will identify these as the current circulating variant strains of Covid 1. The World Health Organisation decided upon this naming system as being easier and more practical to discussed (sic) by non-scientific audiences 2.

All viruses vary, and viruses with genomes made from ribonucleic acid (RNA) vary more than those which have deoxyribonucleic acid (DNA) genomes. This is because the enzymes that replicate RNA virus genomes do not have an error correction facility. 

The virus that causes Covid-19 is an RNA virus and so is Deformed wing virus (DWV), probably the most significant virus to bees and beekeepers (other than those who have Covid that is).

Virus variation

Why does virus variation matter? 

Or, asking the same question in a more roundabout way, why don’t RNA viruses evolve error correcting enzymes (after all, the DNA viruses have these … can it be that difficult?).

If the enzyme makes errors then that’s surely a bad thing?

Actually … for the particular ‘lifestyle’ that these viruses practice, errors and variation are a good thing.

They benefit the virus.

But you know all this already, even if you don’t think you do.

Covid cases caused by the delta variant

The SARS-Cov2 delta variant now accounts for at least 99% of cases of Covid-19 in the UK. It accounted for just 0.1% of cases in late February.

The delta variant is much more transmissible. It carries errors, or mutations as they’re more correctly known, that – for whatever reason – means it can be passed from person to person much more efficiently 3.

These mutations benefit the virus and allow it to spread further and faster 4.

If the mutations (‘errors’) that the virus acquires are beneficial – by increasing transmission, by expanding the cell, tissue or host range, by helping evade the immune response in a partially vaccinated population (!) for example – then the virus will successfully replicate and produce more viruses carrying the same mutation.

And a bunch of additional ones as well … acquired during the last round of replication.

Strains and types

At some point a virus acquires sufficient mutations from an earlier incarnation that it’s identified as a distinct strain.

For example, SARS-Cov2 is ~82% identical at the genome level to SARS-Cov1 which caused the SARS pandemic in 2003 5. SARS-Cov2 did not evolve from SARS-Cov1, but they share a common ancestor. They are different strains or types of coronavirus.

There are no hard and fast rules that define when a virus is considered a different strain or type. Often it’s historical, reflecting the geographic origin, or the source from which the virus was isolated. Different strains may exhibit different phenotypes – host range, transmission, disease etc. – but don’t have to.

For example (before we get back to honey bee viruses) there are three ‘types’ of poliovirus that are about 80% identical at the genetic level. They all cause exactly the same disease (poliomyelitis) and they replicate in an identical manner – same cell, tissue and host range for example. However, to the human immune system they ‘look’ different. The immune response to poliovirus type 1 will not protect you from infection with poliovirus type 3. That’s why the poliovirus vaccine contains a mixture of all three types, to protect you from all polioviruses.

You can even get infected with two types of poliovirus simultaneously, and the virus can replicate in the same cells in your gut … or, if you’re unfortunate, your brain.

I’ll return to dual infections shortly as it’s an important topic … and related to the study I’m going to discuss.

Deformed wing virus

There are two types of DWV, designated type A and type B 6.

Originally these had names that reflected their original isolation.

DWV type A was termed Deformed wing virus and was isolated from honey bees displaying the characteristic symptoms of developmental deformities shown in the image below.

DWV type B was termed Varroa destructor virus type 1 and was isolated from the ectoparasitic mite Varroa destructor.

It “does what is says on the tin” … DWV symptoms in a recently emerged worker

These viruses are very similar to each other. They are something like 85% identical at the level of the RNA genome. More importantly than this genetic identity (or perhaps similarity would be a better term to use here) is the fact that they appear to cause very similar diseases in honey bees.

Although early studies suggested there were some differences in their virulence, more recent work from Prof. Rob Paxton in Germany, from my lab, and from Dr. Eugene Ryabov and colleagues in the USA suggests these two types of DWV are actually very similar, at least in pathogenesis.

There do appear to be some differences, with the suggestion that type A does not replicate in Varroa whereas (full disclosure, the following study is from my lab) type B does. Undoubtedly, the other genetic differences between the types will confer some subtle variation in phenotype (effectively what they ‘do’), but – as far as beekeeping is concerned – they should probably be considered the same.

A protective, non-lethal type A DWV? 

All of which made a 2015 colony-level study 7 of DWV infection rather intriguing.

This reported the survival of colonies that were infected with a “non-lethal” type B strain which were protected from infection with the “lethal” type A strain 8. The authors summarised the significance of this study like this:

We propose that this novel stable host-pathogen relationship prevents the accumulation of lethal variants, suggesting that this interaction could be exploited for the development of an effective treatment that minimises colony losses in the future.

At the time there was a flurry of excitement and discussion about this 9

Superinfection exclusion

They proposed that the mechanism that prevented the infection with the type A strain was superinfection exclusion.

Virologists love mechanisms … 🙂 

Which brings me back to virus variation. 

Imagine a population of variant viruses trying to infect a new host … like a bee.

Survival of the fittest – selection for better replicating viruses from a mixed population

In mixed infections, a virus that has an advantage over the others in the population ends up ‘winning’ the competition for the resources of the host. They therefore make more progeny viruses.

One of the advantages could be that the virus simply replicates faster

Another – more subtle, but the same outcome – is that the virus prevents other viruses from infecting the same cell (and, by extension, host).

By excluding competing viruses it effectively ‘wins’ the competition.

Superinfection exclusion – one virus (type) can prevent infection by related but different viruses

And some viruses do exactly this using a variety of cellular mechanisms.

For example, many viruses turn off the expression of the cellular receptor (think of this as the door) they use to enter the cell. If there’s no receptor (no door) then another virus cannot enter.

With no other viruses to compete with in the same cell there can only be one type of virus produced 10.

There are other mechanisms as well, but we’ll stick with the receptor one as it’s easy to comprehend.

Superinfection of a cell containing a virus that has the ability to turn off the cellular receptor it uses will effectively exclude the second virus from replicating … hence superinfection exclusion.

What don’t we know about DWV?

A lot 🙁

We don’t know how it gets into cells. We don’t know a huge amount about its replication and we know precious little about the way it interacts with the cellular machinery (the ‘stuff’ in the cell that the virus hijacks to make more viruses) of the host. 

However, since 2015 we do know that all the types of DWV that have been carefully studied appear to be more or less equally virulent. None appear to be ‘non-lethal’ as claimed for the type A virus in the superinfection exclusion paper.

This prompted us to look in a bit more detail at the consequences of dual or sequential infections with DWV in the laboratory. 

Is there a precedence at work?

In mixed infections, does one virus always ‘win’ and predominate in the new virus population?

In sequential infections, does it matter the order in which the viruses are acquired?

And mixed infections are pretty much the norm for DWV infection. All bees, whether previously exposed to Varroa or in Varroa-free regions, appear to have low levels of DWV already present. If parasitised by the mite, these bees must experience a mixed infection.

In addition, studies we published several years ago 11 showed that recombinant viruses – essentially hybrids between type A and type B DWV – often predominated in heavily Varroa-infested colonies 12.

If mixed infections cannot occur, how do such hybrids form?

Some of the answers to these questions are in our recently paper published in the ISME Journal. Gusachenko, O. et al., (2021) First come, first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition. ISME J (2021). https://doi.org/10.1038/s41396-021-01043-4

Mixed DWV infections

I don’t propose to give a pupa-by-pupa account of the studies we conducted. You can read the paper – it’s open access and (because Olesya ‘Alex’ Gusachenko, the lead author, did most of the writing) relatively easy to comprehend 😉

But here are a few highlights.

Over the last few years we have produced reagents that allow us to produce almost ‘pure’ stocks of type A, type B or hybrid type A/B 13 strains of DWV.

At least 99.99% of these stocks are of one DWV type. Note that there will still be variation within this population as the replication errors probably generate one mutation per virus in the population. We therefore refer to these virus stocks as near clonal.

Injection of honey bee pupae with any of these viruses resulted in very similar levels and kinetics of replication – all the viruses replicate as far and as fast as each other.

In mixed injections, when two viruses were administered simultaneously, both replicated to equivalent levels. 

We therefore found no evidence for the dominance of one strain over another.

Sequential DWV infections

But it got more interesting when we did sequential injections. We did these by injecting with one virus, waiting 24 hours and then injecting with a second virus.

Using type A and type B DWV both viruses had replicated to similar high levels (billions of virus per pupa) within 48 hours, irrespective of the order of addition. 

If superinfection exclusion was operating we would have perhaps expected type A to have prevented or reduced the replication of type B. However, that didn’t appear to be the case.

Competition between sequential infecting DWV isolates. VVV is type B, VDD is type A and VVD is a hybrid between them.

But, when we looked at sequential infections between type A and a type A/B hybrid we did see that replication of the second virus was delayed.

Delayed, but not stopped altogether.

It would take a complete post to describe the figure above 🙁 . We’ve quantified the virus present 5-7 days after sequential injections with type A (VDD), type B (VVV) or a hybrid virus (VVD) 14.

The columns labelled VDD→VVV or VVV→VDD show the viruses and order of addition. The dots represent the amount of virus present at 5 or 7 days post injection. When the viruses were more similar to each other – for example, the VVV→VVD or VVD→VVV pairs on the right – there was a greater impact on the replication of the virus added second.

The same but different

We extended these studies to look at sequential infections with two viruses that only differed by 4 nucleotides (the building blocks) of the 10,140 nucleotides in the RNA genome of DWV i.e. 99.6% identical.

Cunningly, these four differences allowed us to unambiguously identify which virus was replicating.

In this part of the study the virus added second did not replicate to detectable levels. 

So … our data clearly demonstrates that viruses that were more similar to each other were more likely to inhibit replication during sequential infections.

In addition, no individual virus type was dominant over any others. 

This didn’t look much like classic superinfection exclusion to us.

Red or green viruses

Not content with generating graphs and tables we went on to take photographs of virus infected pupae. 

You can’t beat a nice colour image when trying to impress the peer reviewers 😉 .

Remember that DWV is too small to see with even the most powerful light microscope. You could fit several billion on the head of a pin.

We therefore engineered the virus genome to ‘show’ us where it was replicating.

Green bees

We did this by introducing an additional gene that fluoresced green or red when under UV light. I’ve discussed green viruses before … the red version uses similar technology, but using a different fluorescent reporter gene.

DWV replication (showed by green fluorescent signal) in the head, wing and abdomen of honey bee pupae

Using the red or green viruses we showed very similar results to those described above. When we superinfected with a genetically similar virus, its replication was inhibited. When it was genetically more divergent it could replicate (and we could visualise it as red or green foci of infection in a variety of tissues of the developing pupa).

Red and green viruses

We also infected bee with the red and green viruses simultaneously. Most of the fluorescent foci of infection were red or green, but a small number were both red and green. 

Green (EGFP) and red (mCherry) expressing DWV coinfecting a honey bee pupa. Arrow indicates dually infected cells.

The most likely explanation for having both colours overlapping in the photograph was because the virus were replicating in the same cells in the honey bee pupa.

Since this was exactly the sort of situation that was needed to generate recombinants (hybrids or chimeras) between the two different DWV viruses we specifically looked for them 15.

And there were lots and lots of recombinants …

Recombination between DWV viruses. The size and position of ‘bubbles’ indicate the location and number of junctions.

The bubble plot above shows the location and frequency of junctions. One virus is plotted on the horizontal and one on the vertical axis. It’s a sort of two-dimensional map of the virus. Think of a junction as where one virus ends and the other starts. They are located throughout the DWV genome – hundreds of them.

This suggests that pupae infected with both type A and type B DWV will act as ‘factories’ for the production of thousands more different hybrid variants between the two viruses.

Most of these hybrids will grow poorly.

Many will be uncompetitive.

But some – like the delta variant of SARS-Cov2 – might be more transmissible.

And some could be more pathogenic.

Or – the nightmare scenario – both 😯 .

What’s this got to do with practical beekeeping?

Every time a beekeeper moves bees about s/he is also moving viruses about.

This happens when you move bees to out apiary, when selling a nuc or when importing a queen.

Double brood ...

Moving viruses (and bees) to a new apiary …

This will contribute to the constant mixing of DWV variants that occurs when bees drift between hives, when drones mate with queens, when phoretic Varroa jumps onto a bee that is robbing a collapsing colony.

There’s a difference of course.

All those bee-driven mixing events are local and small scale … a few bees and a few miles.

But if you import a nuc from Greece via Northern Ireland both the distance and number of bees (and hence number of viruses) is much greater.

Of course, most of this mixing will just generate more mixtures of viruses.

It will also generate more recombinants.

But there’s always the possibility it might throw up a highly virulent, highly transmissible variant.

Which would not be a ‘good thing’.

And if it does, a ‘non-lethal type A strain’ (should such a thing actually exist) is not going to help prevent infection by the mechanism of superinfection exclusion I’m afraid 🙁 .

Without doubt the best way to prevent infection is to minimise the mite numbers in your colonies. This is a subject I’ll be tackling again in a couple of weeks.

But, before I go, do we understand how the more closely related strains of DWV prevent superinfection? 

Yes … probably, and it’s all to do with the immune response of the bee

I’ll discuss this in the future as it’s a mechanism that could be exploited to produce bees immune to the ravages of DWV.


 

More from the fun guy

Great fleas have little fleas upon their backs to bite ’em,
And little fleas have lesser fleas, and so ad infinitum.

Augustus de Morgan’s quote from A Budget of Paradoxes (1872) 1 really means that everything is preyed upon by something, which in turn has something preying on it.

The Flea, engraving from Robert Hooke’s Micrographia (1665)

As a virologist I’m well aware of this.

There are viruses that parasitise every living thing.

Whales have viruses and so do unicellular diatoms. All the ~30,000 named bacteria have viruses. It’s likely that the remaining 95% of bacteria that are unnamed also have viruses.

There are even viruses that parasitise viruses. The huge Mimivirus that infects amoebae 2 is itself parasitised by a small virophage (a fancy name for a virus that infects viruses) termed Sputnik.

Whether these interactions are detrimental depends upon your perspective.

The host may suffer deleterious effects while the parasite flourishes.

It’s good for the latter, but not the former.

Whether these interactions are detrimental for humans 3 also depends upon your perspective.

The deliberate introduction of rabbit haemorrhagic disease virus to Australia benefitted sheep farmers who were plagued with rabbits … but it was bad news for rabbit farmers 4.

Biocontrol

Beneficial parasitism, particularly when humans use a pathogen to control an unwanted pest, is often termed biocontrol, a convenient abbreviation for biological pest control.

There are numerous examples; one of the first and best known is control of greenhouse whitefly infestations with the parasitoid wasp Encarsia formosa.

Tomato leaf with whitefly nymphs (white) parasitized by E. formosa (black).

One of the benefits of biocontrol is its self-limiting nature. The wasp will stop replicating once it runs out of whitefly to parasitise.

A second benefit is the specificity of the interaction between the host and whatever is administered to control it; by careful selection of the biocontrol agent you can target what you want to eradicate without lots of collateral damage.

Finally, unlike toxic chemicals such as DDT, the parasitoid wasp – and, more generally, other biocontrol agents – do not accumulate in the environment and cause problems for the future.

And, with all those benefits, it’s unsurprising to discover that scientists have investigated biocontrol strategies to reduce Varroa mite infestation of honey bee colonies.

It’s too early for an aside, but I’ll make one anyway … I’ve discussed the potential antiviral activity of certain fungi a couple of years ago. That wasn’t really biocontrol. It was a fungal extract that appeared to show some activity against the virus. Although that story has gone a bit quiet, one of the authors – Paul Stamets – is also a co-author of the Varroa control paper discussed below.

Biocontrol of Varroa using entomopathogenic fungi

Entomopathogenic means insect killing 5. There are several studies on the use of insect killing fungi to control Varroa 6, with the most promising results obtained with a variety of species belonging to the genus Metarhizium

Metarhizium produces asexual spores termed mitospores. The miticidal activity is due to the adhesion of these mitospores to Varroa, germination of the spore and penetration by fungal hyphae 7 through the exoskeleton of the mite and proliferation within the internal tissues.

A gruesome end no doubt.

And thoroughly deserved 🙂

Although Metarhizium is entomopathogenic it has a much greater impact on Varroa than it does on honey bees. This is the specificity issue discussed earlier.

It is for this reason that scientists have continued to explore ways in which Metarhizium could be used for biocontrol of Varroa.

But there’s a problem …

Although dozens of strains of Metarhizium have been screened, the viability – and therefore activity – of the mitospores is significantly reduced by the relatively high temperatures within the colony.

The spores would be administered, they’d show some activity and some Varroa would be slaughtered. However, over time treatment efficacy would reduce as spores – either administered at the start of the study, or resulting from subsequent replication and sporulation of Metarhizium on Varroa – were inactivated.

As beekeepers you’ll be familiar with the limitation this would impose on effective control of mites.

Varroa spend well over half of their life cycle capped in a cell while it feeds on developing pupae. Anything added to kill mites must be present for extended periods to ensure emerging mites are also exposed and killed.

This is why Apiguard involves two sequential treatments of a fortnight each, or why Apivar strips must be left in a hive for more than 6 weeks.

In an attempt to overcome these limitations, scientists are using directed evolution and repetitive selection to derive strains of Metarhizium that are better able to survive within the hive, and so better able to control Varroa than the strains they were derived from.

Good news and bad news

Like many scientific papers on honey bees 8 those with even a whiff of ‘saving the bees’ get a lot of positive press coverage.

This often implies that the Varroa ‘problem’ is now almost solved, that whatever tiny, incremental advance is described in the paper represents a new paradigm in bee health.

This is both understandable and disappointing in equal measure.

It’s understandable because people (not just beekeepers) like bees. News publishers want ‘good news’ stories to intersperse with the usual never-ending menu of woe they serve up.

It’s disappointing because it’s a variant of “crying wolf”. We want the good news story to describe how the impact of Varroa can now be easily mitigated.

It gets our hopes up.

Unfortunately, reality suggests most of these ‘magic bullets’ are a decade away from any sort of commercial product.

They will probably get mired in licensing problems.

And they may not be any better than what we currently use.

You finally end up as cynical as I am. This might even force you to read the original manuscript, rather than the Gung ho press release or the same thing regurgitated on a news website.

And, if you do that, you’ll better understand some of the clever approaches that scientists are applying to the development of effective biocontrol for Varroa.

We’re not there yet, but progress is being made.

V e r y   s l o w l y.

The paper I’m going to discuss below is Han, J.O., et al. (2021) Directed evolution of Metarhizium fungus improves its biocontrol efficacy against Varroa mites in honey bee colonies. Sci Rep 11, 10582.

It’s freely available should you want to read the bits I get wrong 😉

Solving the temperature-sensitivity problem of mitospores

The strain of Metarhizium chosen for these studies was M. brunneum F52. This had previously been demonstrated to have some efficacy against Varroa. Almost as important, it can be genetically manipulated and there was some preliminary evidence that its pathogenicity for Varroa – and hence control potential – could be improved.

Genetic manipulation covers a multitude of sins. It could mean anything from selection of pre-existing variants from a population to engineered introduction of a toxin gene for destruction of the parasitised host.

In this study the authors used directed evolution of a population of Metarhizium to select for strains with more heat tolerant spores.

Directed evolution of Metarhizium to select mitospores with increased thermotolerance

This is not genetic engineering. They grew spores under stressful conditions and increasing temperatures. Hydrogen peroxide (H2O2) , a mild mutagen, was added in some cases. Nutritional stress also increases population variation. Spores selected using nutritional stress are better able to withstand UV and heat stress.

The optimal growth temperature for the strain of Metarhizium they started with was 27°C. By repeated selection cycles at increasing temperatures they derived spores that grew at 35°C, the temperature within a colony.

Ladders and snakes

A well known phenomena of repeated selection in vitro (i.e. in a test tube in the laboratory, though you actually grow Metarhizium on agar plates) is that a pathogen becomes less pathogenic.

It was therefore unsurprising that – when they eventually tested the thermotolerant spores – only about 3% of the Varroa that died did so due to Metarhizium infection.

Field selection after directed evolution of Metarhizium in the laboratory

They therefore modified the repetitive selection, but this time did it on Varroa-infested colonies in the apiary. Mites that died from Metarhizium mycoses 9 were used as a source to cultivate more Metarhizium.

They were therefore selecting for both thermotolerant (because the experiments were being conducted in hives at 35°C) and pathogenic fungi, because they only cultivated mitospores from Varroa that had died from mycoses.

And it worked …

Amplification of Varroa mycoses by Metarhizium. Black arrows indicate the treatment dates.

After four rounds of selection over 60% of the mites that died did so because they were infected with Metarhizium.

All very encouraging … but note I was very careful with my choice of words in that last sentence. I’ll return to this point shortly.

Before that, here’s the ‘proof’ that the strain selected by directed evolution (which they termed JH1078) possessed more thermostable spores.

Thermostable spores

They measured this by recording the percentage that germinated. At 35°C ~70% of JH11078 spores germinated compared to only ~45% of the M. brunneum F52 strain they started with.

But it’s not all good news

My carefully chosen “60% of the mites that died” neatly obscures the fact that you could get a significant increase in mites dying of Metarhizium, but still have almost all the mites in the hive surviving unscathed.

The authors continued repeated Metarhizium monthly treatments for a full season after the selection experiments described above. The apiary contained 48 colonies, 24 received Metarhizium JH1078 and the remainder received no treatment.

Did Metarhizium treatment stop the well documented increase in Varroa levels observed in colonies not treated with miticides?

Varroa levels in Metarhizium treated and untreated (control) colonies.

Er … no.

They describe this data (above) as showing a ‘delay’ in the exponential increase in Varroa … but acknowledge that it ‘did not totally prevent it’.

Hmmm … looking at the error bars in the last few timepoints I’d be hard pressed to make the case that there was any significant difference in Varroa increase caused by treatment.

And while we’re here look at the mite infestation rate … 10-25 mites per 100 bees.

These are catastrophically high numbers and, unsurprisingly, 42 (~88%) of the 48 colonies – whether treated or untreated – died by the end of 2018, succumbing to “Varroa, pathogen pressure and intense yellow jacket predation”

There was some evidence that colonies receiving Metarhizium treatment survived a bit longer than the untreated controls, but the end results were the same.

Almost every colony perished.

Metarhizium vs. oxalic acid

Typically a paper on a potential improved biocontrol method for Varroa would do a side-by-side comparison with a widely used, currently licensed treatment.

There’s only one comparative experiment between Metarhizium and dribbled oxalic acid treatment. It’s buried at the end of the Supplementary Data 10. In it they show ‘no significant difference’ between the two treatments.

Frankly this was a pretty meaningless experiment … it was conducted in June 2020 when colonies would have been bulging with brood. Consequently 90% of the mites would have been hidden under the cappings. They assayed mite levels only 18 days after a single application of Metarhizium or oxalic acid.

Although it showed ‘no significant difference’ – like the “60% of the mites that died” quote – it obscures the fact that most mites were almost certainly completely untouched by either treatment.

What does this study show?

This study involved a large amount of work.

The directed evolution in the laboratory is a very nice example of how the combination of phenotypic selection and natural variation can rapidly yield new strains with desirable characteristics.

Combination of this with in vivo selection for enhanced pathogenesis successfully produced a novel strain of Metarhizium with some of the features desirable for biocontrol of Varroa.

However, in the apiary-based studies the majority of the colonies, whether treated or not, died.

This shows that, although scientists might have made a promising start, they are still a very long way from having an effective biocontrol solution for Varroa.

Unmanaged Varroa replicates to unmanageable levels

One of the Supplementary Data figures illustrated the Varroa drop per month from colonies in the research apiary.

Cumulative mite drop per colony for the month of July 2018

This is relatively late in the study, July 2018. These hives were established from commercial packages of bees in April 2017. They were either treated with the experimental Metarhizium spores or were untreated controls for the 15 months between April 2017 and July 2018 11.

Look at those Varroa numbers!

This is the mite drop just after the peak of the season. Brood levels would be close to maximum in their short, warm summer 12. The majority of the mite population would have been safely tucked away feasting on developing brood.

This is not the mite drop after miticide treatment … it’s just the drop due to bee grooming, natural mite mortality and the general ‘friction’ in the hive.

The average is 2866 mites dropped per month per hive 😥

Maybe nothing could have saved hives as heavily infested as these? 13

Don’t wait for Metarhizium … be vigilant now

These numbers – of mites and dead colonies – are a stark warning of the replication potential of Varroa and the damage is causes our bees.

Left untreated, Varroa will replicate to very high levels.

Colony mortality – either directly due to the mite and viruses, or indirectly due to the weakened colonies succumbing to robbing – is a near-inevitable consequence.

I’ve discussed the importance of Varroa management repeatedly over the years. It’s a topic I’ll be returning to again – probably in August when it starts to become a necessity.

In the meantime, keep an eye on the mite levels in your own colonies as they get stronger during the season.

While you’re doing that think of the scientists who are looking for practical, effective and environmentally-friendly strategies to control Varroa. Understand that these studies are time-consuming, progress is glacial incremental … and they might not work anyway.

Of course, if we finally manage to develop a suitable Metarhizium-based mite control strategy then bees and beekeepers will not be the only beneficiaries.

Metarhizium has its own parasites. Some of the best characterised of these are small RNA viruses.

If beekeepers are sprinkling billions of Metarhizium spores over their colonies every year then these viruses will be having a great time 😉


 

Winter losses

I lost 10% of my colonies this winter.

It’s always disappointing losing colonies, but it’s sometimes unavoidable.

I suspect the two I lost were unavoidable … though, as you’ll see, they weren’t completely lost.

April showers frosts

Late April may seem like mid-season for many beekeepers based in southern England. While they were adding their second super, the bees here in Scotland were only just starting to take their first few tentative flights of the year.

This April has been significantly cooler in Fife 1 than any year ‘since records began’.

However, the records I’m referring to are from the excellent Auchtermuchty weather report 2 which only date back to about 2013 … I like it because it’s local, not because it’s historically comprehensive 😉

The average April temperate has only been 5.5°C with 15 nights with frost in the first three weeks of the month. In contrast, the same month in 2019 and 2020 averaged over 9°C with only 3-4 nights with frosts 3

In both 2019 and 2020 swarming started at the end of April. Several colonies had queen cells when I first inspected them and I hived my first swarm (not lost from one of my colonies 😉 ) on the last day of the month.

First inspections and winter losses

Unsurprisingly, with appreciably lower temperatures, things are less well advanced this season. None of the colonies I inspected on the 19th were making swarm preparations. Instead, most were 2-4 frames of brood down on the strength I’d expect them to have before they started thinking about swarming.

Nevertheless, most were busy on a lovely spring day … lots of pollen (mainly gorse and some late willow by the looks of things) being delivered by heavily-laden foragers, and fresh nectar in some of the brood frames.

Fresh nectar glistening in a brood frame

The first inspection of the season is an opportunity to not only check on the strength and behaviour of the colony, but also to do some ‘housekeeping’. This includes:

  • swapping out old, dark brood frames (now emptied of stores) and replacing them with new foundationless frames
  • removing excess stores to make space for brood rearing
  • removing the first sealed drone brood in the colony to help hold back Varroa replication

And, as the winter is now clearly over, it’s the time at which the overall number of winter losses can be finally assessed.

Winter losses

Winter losses generally occur for one of four reasons:

  • disease – in particular caused by deformed wing virus (DWV) vectored by high levels of Varroa in the hive. DWV reduces the longevity of the diutinus winter bees, meaning the colony shrinks in size and falls below a threshold for viability. There are too few bees to thermoregulate the colony and too few bees to help the queen rear new larvae. The colony either freezes to death, dwindles to the size of an orange, or starves to death because the cluster cannot reach the stores 4.
  • queen failure – for a variety of reasons queens can fail. They stop laying altogether or they only lay drone brood. Whatever the reason, a queen that doesn’t lay means the colony is doomed.
  • natural disasters – this is a bit of a catch-all category. It includes things like flooded apiaries, falling trees and stampeding livestock. Although these things might be avoidable – don’t site apiaries in flood risk areas, under trees or on grazing land – these lessons are often learnt the hard way 5.
  • unnatural disasters – these are avoidable and generally result from inexperienced, or bad 6 , beekeeping. I’d include providing insufficient stores for winter in this category, or leaving the queen excluder in place resulting in the isolation of the queen, or allowing the entrance to be blocked. These are the things that the beekeeper alone has control over. 

The BBKA run an annual survey of winter losses in the UK. This is usually published in midsummer, so the graph below is from 2020.

BBKA winter survival survey

Over the 13 years of the survey the average losses were 18.2% 7. Long or particularly hard winters result in higher levels of losses.

Lies, damn lies and statistics

I’ve no idea how accurate these winter loss surveys are.

About 10% of the BBKA membership reported their losses, and the BBKA membership is probably a bit over 50% of UK beekeepers. 

I would expect, with precious little evidence to back it up, that the BBKA generally represents the more ‘engaged’ beekeepers in the UK 8. It also probably represents a significant proportion of new beekeepers who were encouraged to join while training.

So, like Amazon reviews, I treat the results of the survey with quite a bit of caution. I suspect beekeepers who have low losses complete it enthusiastically to ‘brag’ about their success (despite its anonymity), while those with large losses either keep quiet or are happy to share their grief. 

Unlike Amazon reviews, I’d be surprised if there are many fake submissions to the BBKA and I’m not aware there’s a living to be made from selling fake colony survival reviews in bulk online.

For comparison, the Bee Informed Partnership in the USA runs a similar survey every year.

Bee Informed Partnership loss and management survey

This survey covers about 10% of the colonies in the USA. Again it is voluntary and likely subject to the same inherent biases that may affect the BBKA survey.

The USA winter colony losses average ~28% over the same 13 year survey period.

Are US beekeepers less good at keeping their colonies alive than beekeepers in the UK?

Perhaps the US climate is less suited to honey bees?

Or, possibly, US beekeepers are simply more honest than their UK counterparts?

I doubt it 9.

Running on empty

My two colony losses were due to queen failures.

Old winter bees and no brood

In the first colony there was no evidence the queen had laid any brood since the previous autumn. There were about 6 seams of bees in the hive, but the outer 2-3 frames were solid with untouched winter stores.

Unused winter stores

This is usually a dead giveaway … literally. The colony hasn’t used the stores because they’ve not had any hungry mouths to feed. With no new brood the colony is doomed.

This queen appears to have simply run out of sperm and stopped laying. She was present (a 2019 marked queen and the same one I’d seen in August last year) and ambling around the frame, but she wasn’t even going through the pretence of inspecting cells before laying.

I removed the queen and united what remained of the colony over a nearby strong colony.

Strong colony ready for uniting

Assuming the queen stopped laying at the end of year all the bees in the hive – and there were a good number – were old, winter bees. These won’t survive long, but will provide a temporary boost to the colony I united them with. 

Every little bit helps 🙂

Even more valuable than the bees were the frames they were on.

Most of the comb in the colony with the failed queen was relatively new. By uniting them I can quickly swap out the old comb (from the stronger hive #34) when I next inspect the hive. At the same time I’ll rescue the frames of sealed stores for use when making up nucs during queen rearing.

Drone laying queen

The second failure was a drone laying queen (DLQ).

These are usually unmistakeable … the brood is clustered, with drone pupae occupying worker brood cells. If the queen has been drone laying for some time there may be lots of undersized ‘runt’ drones present in the hive as well.

Drone laying queen ...

Drone laying queen …

Again, this colony was doomed. With no new queens available and a lot of pretty old bees in the hive they could not be restored to a functioning colony.

However, many of the bees could be saved …

The colony wasn’t overrun with drones. Going by the amount of stores consumed it had probably been rearing worker brood since the winter solstice.

The queen was unmarked and unclipped. I strongly suspect she was a late-season supersedure queen who was very poorly mated.

The 3-4 weeks of drone brood rearing 10 had wrecked quite a few of the frames, but the bees were worth saving.

Under these circumstances I decided to shake the colony out.

When I do this I like to move the original hive and the stand it’s on. If you don’t move the stand the displaced bees tend to cluster near the original hive entrance, festooned from the hive stand. 

In poor weather, or late in the afternoon, this can lead to lots of bees unnecessarily perishing.

However, the stand was shared with two other colonies, so couldn’t be removed. It was also late morning and the weather was excellent.

I moved the hive away and shook the bees out. 

Sure enough … they returned to their original location.

They then marched along the hive stand to the entrance of the adjacent hive.

This way sisters!

And, by the time I left the apiary in mid-afternoon there were only a few diehard bees clustered near where the original hive entrance was.

Why didn’t I just unite them as I’d done with the other failed queen?

Drone brood is a Varroa magnet

Varroa replicate when feeding on developing pupae. The longer development time of drone pupae (when compared with worker pupae) means that you get ~50% more Varroa from drone brood 11

Unsurprisingly perhaps (or not, because that’s the way evolution works) Varroa have therefore evolved to preferentially infest drone brood. When given the choice between a drone or worker pupa to infest, Varroa choose the drone about 10 times more frequently than the worker.

And that ~10:1 ‘preference ratio’ increases when drone brood is limiting … as it is early in the season.

What this means is that the first burst of drone brood production in a colony is very attractive to Varroa.

Unless there are compelling reasons to keep this very early drone brood – for example, a colony with stellar genetics I’d like to contribute as much as possible to the local gene pool – I often try and remove it.

Drone-worker-drone

Drone-worker-drone …

If you use foundationless frames this is often as easy as simply cutting out a single panel of drone brood.

But, in the case of this drone laying queen, it meant that the logical action was to discard all of the drone brood to ensure I discarded the majority of the Varroa also present in the colony 12.

Which is why I shook the colony out, rather than uniting them 🙂

Boxes of bees

Several colonies in one apiary went into the winter on double brood colonies. Inevitably, with the loss of bees during the winter months, the colony contracts and the queen almost invariably ends up laying in the upper box.

The first inspection of the season is often a good time to remove the lower box. It can be removed altogether, or replaced (above the other box) for a Bailey comb change if the weather is suitable.

At this stage of the year the lower box is often reasonably empty of bees and totally empty of brood. 

Emptying a box of bees

If the comb in the lower box is old and dark (see the picture above) I place the upper box on the original floor and add an empty super on top. I then go through the lower box, shaking the bees into the empty super. Good frames are retained, the rest are destined for the wax extractor and firelighters.

Using an empty super helps ‘funnel’ the bees into the brood box.

Sometimes the queen has already laid up a frame or so in the lower box. Under these circumstances – particularly if the comb is relatively new – I’ll simply reverse the boxes, placing the lower box on top of the upper one. This results in the queen quite quickly moving up and laying up the space in the upper brood chamber.

It’s then time to add a queen excluder and the first super.

The beekeeping season has definitely started 🙂


Notes

I commented a fortnight ago about the apparent lateness of the 2021 spring. I’m adding this final note on the afternoon of the 23rd and have still yet to see or hear either cuckoo or chiffchaff on the west coast. Last year they were here in the middle of the month. This, combined with the temperature data (see above) show that everything is a week or two behind events last year.

Which means I can expect to start doing some sort of swarm prevention and control in the next fortnight.

Brexit and beekeeping

The ‘oven ready’ deal the government struck with the EU in the dying hours of 2020 was a bit less à la carte and a bit more table d’hôte.

The worst of the predictions of empty supermarket shelves and the conversion of Essex into a 3500 km2 lorry park have not materialised 1.

But there are other things that haven’t or won’t appear.

And one of those things is bees.

Bee imports

There is a long history of bee imports into the UK, dating back at least a century. In recent years the number of imports has markedly increased, at least partially reflecting the increasing popularity of beekeeping. 

Going up! Imports of queens, nucs and packages to the UK, 2007-2020 (National Bee Unit data)

Queens are imported in cages, usually with a few attendant workers to keep them company. Nucs are small sized colonies, containing a queen, bees and brood on frames. 

Packages are the ‘new kid on the block’ (in the UK) with up to 2500 per year being imported after 2013. Packages are queenless boxes of bees, containing no frames or brood.

Empty boxes after installing packages of bees

They are usually supplied in a mesh-sided box together with a queen. The bees are placed into a hive with frames of foundation and the queen is added in an introduction cage. They are fed with a gallon to two of syrup to encourage them to draw comb.

Installing a package of bees

It’s a very convenient way to purchase bees and avoids at least some of the risk of importing diseases 2. It’s also less expensive. This presumably reflects both the absence of frame/foundation and the need for a box to contain the frames.

But, post-Brexit, importation of packages or nucs from EU countries is no longer allowed. You are also not allowed to import full colonies (small numbers of these were imported each year, but insufficient to justify adding them to the graph above).

Queen imports are still allowed.

Why are were so many bees imported?

The simple answer is ‘demand’.

Bees can be reared inexpensively in warmer climates, such as southern Italy or Greece. The earlier start to the season in these regions means that queens, nucs or packages can be ready in March to meet the early season demand by UK beekeepers.

If you want a nuc with a laying queen in March or April in the UK you have two choices; a) buy imported bees, or b) prepare or purchase an overwintered nuc.

I don’t have data for the month by month breakdown of queen imports. I suspect many of these are also to meet the early season demand, either by adding them to an imported package (see above) or for adding to workers/brood reared and overwintered in a UK hive that’s split early in the season to create nucleus colonies.

Some importers would sell the latter on as ‘locally reared bees’. They are … sort of. Except for the queen who of course determines the properties of all the bees in the subsequent brood 🙁

An example of being “economical with the truth” perhaps?

Imported queens were also available throughout the season to replace those lost for any number of reasons (swarming, poor mating, failed supersedure, DLQ’s, or – my speciality – ham-fisted beekeeping) or to make increase.

And to put these imports into numerical context … there are about 45,000 ‘hobby’ beekeepers in the UK and perhaps 200+ bee farmers. Of the ~250,000 hives in the UK, about 40,000 are managed by bee farmers.

What are the likely consequences of the import ban?

I think there are likely to be at least four consequences from the ban on the importation of nucs and packages to the UK from the EU:

  1. Early season nucs (whatever the source) will be more expensive than in previous years. At the very least there will be a shortfall of ~2000 nucs or packages. Assuming demand remains the same – and there seems no reason that it won’t, and a realistic chance that it will actually increase – then this will push up the price of overwintered nucs, and the price of nucs assembled from an imported queen and some ‘local’ bees. I’ve seen lots of nucs offered in the £250-300 range already this year.
  2. An increase in imports from New Zealand. KBS (and perhaps others) have imported New Zealand queens for several years. If economically viable this trade could increase 3.
  3. Some importers may try and bypass the ban by importing to Northern Ireland, ‘staging’ the bees there and then importing them onwards to the UK. The legality of this appears dubious, though the fact it was being considered reflects that this part of the ‘oven ready’ Brexit deal was not even table d’hôte and more like good old-fashioned fudge.
  4. Potentially, a post-Covid increase in bee smuggling. This has probably always gone on in a limited way. Presumably, with contacts in France or Italy, it would be easy enough to smuggle across a couple of nucs in the boot of the car. However, with increased border checks and potential delays, I (thankfully) don’t see a way that this could be economically viable on a large scale.

Is that all?

There may be other consequences, but those are the ones that first came to mind.

Of the four, I expect #1 is a nailed-on certainty, #2 is a possibility, #3 is an outside possibility but is already banned under the terms of the Northern Ireland Protocol which specifically prohibits using Northern Ireland as a backdoor from Europe, and #4 happens and will continue, but is small-scale.

Of course, some, all or none of this ban may be revised as the EU and UK continue to wrangle over the details of the post-Withdrawal Agreement. Even as I write this the UK has extended the grace period for Irish sea border checks (or ‘broken international law’ according to the EU). 

This website is supposed to be a politics-free zone 4 … so let’s get back to safer territory.

Why is early season demand so high?

It seems likely that there are three reasons for this early season demand:

  1. Commercial beekeepers needing to increase colony numbers to provide pollination services or for honey production. Despite commercials comprising only ~0.4% of UK beekeepers, they manage ~16% of UK hives. On average a commercial operation runs 200 hives in comparison to less than 5 for hobby beekeepers. For some, their business model may have relied upon the (relatively) inexpensive supply of early-season bees.
  2. Replacing winter losses by either commercial or amateur beekeepers. The three hives you had in the autumn have been slashed to one, through poor Varroa management, lousy queen mating or a flood of biblical proportions. With just one remaining hive you need lots of things to go right to repopulate your apiary. Or you could just buy them in.
  3. New beekeepers, desperate to start beekeeping after attending training courses through the long, dark, cold, wet winter. And who can blame them? 

For the rest of the post I’m going to focus on amateur or hobby beekeeping. I don’t know enough about how commercial operations work. Whilst I have considerable sympathy if this change in the law prevents bee farmers fulfilling pollination or honey production contracts, I also question how sensible it is to depend upon imports as the UK extricates itself from the European Union.

Whatever arrangement we finally reached it was always going to be somewhere in between the Armageddon predicted by ‘Project Fear’ and the ‘Unicorns and sunlit uplands’ promised by the Brexiteers.

Where are those sunlit uplands?

And that had been obvious for years.

I have less sympathy for those who sell on imported bees to meet demand from existing or new beekeepers. This is because I think beekeeping (at least at the hobbyist level) can, and should, be sustainable.

Sustainable beekeeping

I would define sustainable beekeeping as the self-sufficiency that is achieved by:

  • Managing your stocks in a way to minimise winter losses
  • Rearing queens during the season to requeen your own colonies when needed (because colonies with young queens produce brood later into the autumn, so maximising winter bee production) and to …
  • Overwinter nucleus colonies to make up for any winter losses, or for sale in the following spring

All of these things make sound economic sense. 

More importantly, I think achieving this level of self-sufficiency involves learning a few basic skills as a beekeeper that not only improve your beekeeping but are also interesting and enjoyable.

I’ve previously discussed the Goldilocks Principle and beekeeping, the optimum number of colonies to keep considering your interest and enthusiasm for bees and the time you have available for your beekeeping.

It’s somewhere between 2 and a very large number. 

For me, it’s a dozen or so, though for years I’ve run up to double that number for our research, and for spares, and because I’ve reached the point where it’s easy to generate more colonies (and because I’m a lousy judge of the limited time I have available 🙁 ).

Two is better than one, because one colony can dwindle, can misbehave or can go awry, and without a colony to compare it with you might be none the wiser that nothing is wrong. Two colonies also means you can always use larvae from one to rescue the other if it goes queenless.

And with just two colonies you can easily practise sustainable beekeeping. You are no longer dependent on an importer having a £30 mass-produced queen spare.

What’s wrong with imported bees?

The usual reason given by beekeepers opposed to imports is the risk of also importing pathogens.

Varroa is cited as an example of what has happened. 

Tropilaelaps or small hive beetle are given as reasons for what might happen.

And then there are usually some vague statements about ‘viruses’. 

There’s good scientific evidence that the current global distribution of DWV is a result of beekeepers moving colonies about.

More recently, we have collaborated on a study that has demonstrated an association between honey bee queen imports and outbreaks of chronic bee paralysis virus (CBPV). An important point to emphasise here is that the direction of CBPV transmission is not yet clear from our studies. The imported queens might be bringing CBPV in with them. Alternatively, the ‘clean’ imported queens (and their progeny) may be very susceptible to CBPV circulating in ‘dirty’ UK bees. Time will tell.

However, whilst the international trade in plants and animals has regularly, albeit inadvertently, introduced devastating diseases e.g. Hymenoscyphus fraxineus (ash dieback), I think there are two even more compelling reasons why importation of bees is detrimental.

  1. Local bees are better adapted to the environment in which they were reared and consequently have increased overwintering success rates.
  2. I believe that inexpensive imported bees are detrimental to the quality of UK beekeeping.

I’ve discussed both these topics previously. However, I intend to return to them again this year. This is partly because in this brave new post-Brexit world we now inhabit the landscape has changed.

At least some imports are no longer allowed. The price of nucs will increase. Some/many of these available early in the season will be thrown together from overwintered UK colonies and an imported queen.

These are not local bees and they will not provide the benefits that local bees should bring.

Bad beekeeping and bee imports

If imported queens cost £500 each 5 there would be hundreds of reasons to learn how to rear your own queens. 

But most beekeepers don’t …

Although many beekeepers practise ‘passive’ queen rearing e.g. during swarm control, it offers little flexibility or opportunity to rear queens outside the normal swarming season, or to improve your stocks.

In contrast, ‘active’ queen rearing i.e. selection of the best colonies to rear several queens from, is probably practised by less than 20% of beekeepers.

This does not need to involve grafting, instrumental insemination or rows of brightly coloured mini-nucs. It does not need any large financial outlay, or huge numbers of colonies to start with.

But it does need attention to detail, an understanding of – or a willingness to learn – the development cycle of queens, and an ability to judge the qualities of your bees.

Essentially what it involves is slightly better beekeeping.

But, the availability of Italian, Greek or Maltese queens for £20 each acts as a disincentive.

Why learn all that difficult ‘stuff’ if you can simply enter your credit card details and wait for the postie?

Overwintering 5 frame poly nuc

Overwintering 5 frame poly nuc

And similar arguments apply to overwintering nucleus colonies. This requires careful judgement of colony strength through late summer, and the weight of the nuc over the winter.

It’s not rocket science or brain surgery or Fermat’s Last Theorem … but it does require a little application and attention.

But, why bother if you can simply wield your “flexible friend” 6 in March and replace any lost colonies with imported packages for £125 each?

Rant over

Actually, it wasn’t really a rant. 

My own beekeeping has been sustainable for a decade. I’ve bought in queens or nucs of dark native or near-native bees from specialist UK breeders a few times. I have used these to improve my stocks and sold or gifted spare/excess nucs to beginners.

I’ve caught a lot of swarms in bait hives and used the best to improve my bees, and the remainder to strengthen other colonies.

The photographs of packages (above) are of colonies we have used for relatively short-term scientific research. 

I’m going to be doing a lot of queen rearing this season. Assuming that goes well, I then expect to overwinter more nucs than usual next winter. 

I then hope that the bee import ban remains in place for long enough until I can sell all these nucs for an obscene profit which I will use to purchase a queen rearing operation in Malta. 😉

And I’m going to write about it here.


Notes

BBKA statement made a day or two after this post appeared. The BBKA and other national associations are concerned about the potential import of Small Hive Beetle (SHB) into the UK via Northern Ireland. Whilst I still think this breaches the Northern Ireland Protocol, it doesn’t mean it won’t be attempted (and there’s at least one importer offering bees via this route). It’s not clear that the NI authorities have the manpower to inspect thousands of packages.

It’s worth noting that SHB was introduced to southern Italy in 2014 and remains established there. The most recent epidemiological report shows that it was detected as late as October 2020 in sentinel apiaries and is also established in natural colonies.

With a single exception – see below – every country into which SHB has been imported has failed to eradicate it. As I wrote in November 2014:

“Once here it is unlikely that we will be able to eradicate SHB. The USA failed, Hawaii failed, Australia failed, Canada failed and it looks almost certain that Italy has failed.”

And Italy has failed.

The one exception was a single import to a single apiary in the Portugal. Notably, the illegal import was of queens, not nucs or packages. Eradication involved the destruction of the colonies, the ploughing up of the apiary and the entire area being drenched in insecticide.

The Beekeepers Quarterly

This post also appeared in the summer 2021 edition of The Beekeepers Quarterly published by Northern Bee Books.

Oxalic acid (Api Bioxal) preparation

This post updates and replaces one published three years ago (which has now been archived). The registered readership of this site has increased >200% since then and so it will be new to the majority of visitors.

It’s also particularly timely.

I will be treating my own colonies with oxalic acid in the next week or so.

Mites and viruses

Varroa levels in the hive must be controlled for successful overwintering of colonies. If you do not control the mites – and by ‘control’ I mean slaughter 😉 – the viruses they transmit to the overwintering bees will limit the chances of the colony surviving.

The most important virus transmitted by Varroa is deformed wing virus (DWV). At high levels, DWV reduces the lifespan of worker bees.

This is irrelevant in late May – there are huge numbers of workers and they’re only going to live for about 6 weeks anyway.

In contrast, reduced longevity is very significant in the winter where more limited numbers of overwintering bees must survive for months to maintain the colony through to the Spring. If these bees die early (e.g. in weeks, not months), the colony will dwindle to a pathetic little cluster and likely freeze to death on a cold winter night.

Game over. You are now an ex-beekeeper 🙁

To protect the overwintering bees you must reduce mite levels in late summer by applying an appropriate miticide. I’ve discussed this at length previously in When to treat? – the most-read post on this site.

I’d argue that the timing of this late summer treatment is the most important decision about Varroa control that a beekeeper has to make.

However, although the time for that decision is now long-gone, there are still important opportunities for mite control in the coming weeks.

In the bleak midwinter

Miticides are not 100% effective. A proportion of the mites will survive this late summer treatment 1. It’s a percentages game, and the maximum percentage you can hope to kill is 90-95%.

If left unchecked, the surviving mites will replicate in the reducing brood reared between October and the beginning of the following year. That means that your colony will potentially contain more mites in January than it did at the end of the late summer treatment.

Mid September

Late summer mite treatment and no midwinter treatment.

Over several years this is a recipe for disaster. The graph above shows modelled data that indicates the consequences of only treating in late summer. Look at the mite levels (in red, right hand vertical axis) that increase year upon year.

The National Bee Unit states that if mite levels exceed 1000 then immediate treatment is needed to protect the colony. In the modelled data above that’s in the second year 2.

In contrast, here is what happens when you also treat in “midwinter” (I’ll discuss what “midwinter” means shortly).

Two optimal treatments

Two optimal treatments

Mite numbers remain below 1000. This is what you are aiming for.

For the moment ignore the specific timing of the treatment – midwinter, late December etc.

Instead concentrate on the principle that determines when the second treatment should be applied.

During the winter the colony is likely to go through a broodless period 3.

When broodless all the mites in the colony must, by definition, be phoretic.

There’s no brood, so any mites in the colony must be riding around on the backs of workers.

A phoretic mite is an easy mite to kill 4.

A “midwinter” double whammy

A single oxalic acid based treatment applied during the winter broodless period is an ideal way to minimise the mite levels before the start of the following season.

Oxalic acid is easy to administer, relatively inexpensive and well-tolerated by the bees.

The combination – a double whammy – of a late summer treatment with an appropriate miticide and a “midwinter” treatment with oxalic acid should be all that is needed to control mites for the entire season.

However, “midwinter” does not mean midwinter, or shouldn’t.

Historically, winter mite treatments were applied between Christmas and New Year. It’s a convenient time of the year, most beekeepers are on holiday and it’s a good excuse to avoid spending the afternoon scoffing mince pies in front of the TV.

Or with the outlaws inlaws 😉

But by that time of year many colonies will have started brooding again.

With sealed brood, mites have somewhere to hide, so the treatment will be less effective than it might otherwise have been 5.

Why go to all the trouble of treating if it’s going to be less effective than it could be?

The key point is not the timing … it’s the broodlessness of the colony.

If the colony is broodless then it’s an appropriate time to treat. My Fife colonies were broodless this year by mid-October. This is earlier than previous seasons where I usually have waited until the first protracted cold period in the winter – typically the last week in November until the first week in December.

If they remain broodless this week I’ll be treating them. There’s nothing to be gained by waiting.

Oxalic acid (OA) treatment options

In the UK there are several approved oxalic acid-containing treatments. The only one I have experience of is Api-Bioxal, so that’s the only one I’ll discuss.

I also give an overview of the historical method of preparing oxalic acid as it has a bearing on the amount of Api-Bioxal used and will help you (and me) understand the maths.

OA can be delivered by vaporisation (sublimation), or by tricking (dribbling) or spraying a solution of the chemical.

I’ve discussed vaporisation before so won’t rehash things again here.

Trickling has a lot to commend it. It is easy to do, very quick 6 and requires almost no specialised equipment, either for delivery or personal protection (safety).

Trickling is what I always recommend for beginners. It’s what I did for years and is a method I still regularly use.

The process for trickling is very straightforward. You simply trickle a specific strength oxalic acid solution in thin syrup over the bees in the hive.

Beekeepers have used oxalic acid for years as a ‘hive cleaner’, as recommended by the BBKA and a range of other official and semi-official organisations. All that changed when Api-Bioxal was licensed for use by the Veterinary Medicines Directorate (VMD).

Oxalic acid and Api-Bioxal, the same but different

Api-Bioxal is the VMD-approved powdered oxalic acid-containing miticide. It is widely available, relatively inexpensive (when compared to other VMD-approved miticides) and very easy to use.

Spot the difference ...

Spot the difference …

It’s very expensive when compared to oxalic acid purchased in bulk.

Both work equally well as both contain exactly the same active ingredient.

Oxalic acid.

Api-Bioxal has other stuff in it (meaning the oxalic acid content is a fraction below 90% by weight) and these additives make it much less suitable for sublimation. I’ll return to these additives in a minute or two. These additives make the maths a bit more tricky when preparing small volumes at the correct concentration – this is the purpose of this post.

How much and how strong?

To trickle or dribble oxalic acid-containing solutions you’ll need to prepare it at home, store it appropriately and administer it correctly.

I’ve dealt with how to administer OA by trickling previously. This is all about preparation and storage.

The how much is easy.

You’ll need 5ml of oxalic acid-containing solution per seam of bees. In cold weather the colony will be reasonably well clustered and its likely there will be a maximum of no more than 8 or 9 seams of bees, even in a very strong colony.

Hold on … what’s a seam of bees?

Three seams of bees

Looking down on the colony from above, a seam of bees is the row visible between the top bars of the frames.

So, for every hive you need 5ml per seam, perhaps 45ml in total … with an extra 10% to cover inevitable spillages. It’s not that expensive, so don’t risk running out.

And the strength?

The recommended concentration to use oxalic acid at in the UK has – for many years – been 3.2% w/v (weight per volume) in 1:1 syrup. This is less concentrated than is recommended in continental Europe (see comments below on Api-Bioxal).

My advice 7 – as it’s the only concentration I’ve used – is to stick to 3.2%.

Calculators at the ready!

The oxalic acid in Api Bioxal is actually oxalic acid dihydrate. Almost all the powdered oxalic acid you can buy is oxalic acid dihydrate.

The molecular formula of oxalic acid is C2H2O4. This has a molecular weight of 90.03. The dihydrated form of oxalic acid has the formula C2H2O4.2H2O 8 which has a molecular weight of 126.07.

Therefore, in one gram of oxalic acid dihydrate powder (NOT Api Bioxal … I’ll get to Api Bioxal in a minute! Have patience Grasshopper) there is:

90.03/126.07 = 0.714 g of oxalic acid.

Therefore, to make up a 3.2% oxalic acid solution in 1:1 syrup you need to use the following recipe, or scale it up as needed.

  • 100 g tap water
  • 100 g white granulated sugar
  • Mix well
  • 7.5 g of oxalic acid dihydrate

The final volume will be 167 ml i.e. sufficient to treat over 30 seams of bees, or between 3 and 4 strong colonies (including the 10% ‘just in case’).

The final concentration is 3.2% w/v oxalic acid

(7.5 * 0.714)/167 * 100 = 3.2% 9.

Check my maths 😉

Recipe to prepare Api-Bioxal solution for trickling

Warning – the recipe on the side of a packet of Api-Bioxal makes up a much stronger solution of oxalic acid than has historically been used in the UK. Stronger isn’t necessarily better. The recipe provided is 35 g Api-Bioxal to 500 ml of 1:1 syrup. By my calculations this recipe makes sufficient solution at a concentration of 4.4% w/v to treat 11 hives. 

There’s an additional complication when preparing an Api-Bioxal solution for trickling. This is because Api-Bioxal contains two additional ingredients – glucose and powdered silica. These cutting agents account for 11.4% of the weight of the Api-Bioxal. The remaining 88.6% is oxalic acid dihydrate.

Using the same logic as above, 1g of Api-Bioxal therefore contains:

(90.03/126.07) * 0.886 = 0.633 g of oxalic acid.

Therefore, to make up 167 ml of a 3.2% Api-Bioxal solution you need to use the following recipe, or scale up/down appropriately:

  • 100 g tap water
  • 100 g white granulated sugar
  • Mix well
  • 8.46 g of Api-Bioxal

Again, check my maths … you need to add (7.5 / 0.886 = 8.46) grams of Api-Bioxal as only 88.6% of the Api-Bioxal is oxalic acid dihydrate.

Scaling up and down

8.46 g is not straightforward to weigh – though see below – and 167 ml may be too much for the number of hives you have. Here’s a handy table showing the amounts of Api-Bioxal to add to 1:1 syrup to make up the amount required.

Api-Bioxal recipes for 3.2% trickling in 1:1 syrup

The Api-Bioxal powder weights shown in bold represent the three packet sizes that can be purchased.

I don’t indicate the amounts of sugar and water to mix to make the syrup up. I’ll leave that as an exercise for the reader … remember that 100 g of sugar and 100 ml of water make 167 ml of 1:1 (w/v) syrup.

Weighing small amounts of Api-Bioxal

The amount of Api-Bioxal used is important. A few grams here or there matter.

If you are making the mix up for a limited number of hives you will have to weigh just a few grams of Api-Bioxal. You cannot do this on standard digital kitchen scales which work in 5 g increments.

Buy a set of these instead.

Digital scales … perfect for Api-Bioxal (and yeast)

These cost about a tenner and are perfect to weigh out small amounts 10 of Api-Bioxal … or yeast for making pizza dough.

A few words of caution

I don’t want to spoil your fun but please remember to take care when handling or using oxalic acid, either as a powder or when made up as a solution.

Oxalic acid is toxic

  • The lethal dose for humans is reported to be between 15 and 30 g. It causes kidney failure due to precipitation of solid calcium oxalate.
  • Clean up spills of powder or solution immediately.
  • Take care not to inhale the powder.
  • Store in a clearly labelled container out of reach of children.
  • Wear gloves.
  • Do not use containers or utensils you use for food preparation. A well rinsed plastic milk bottle, very clearly labelled, is a good way to store the solution prior to use.

Storage

Storage of oxalic acid syrup at ambient temperatures rapidly results in the acid-mediated breakdown of sugars (particularly fructose) to generate hydroxymethylfurfural (HMF). As this happens the colour of the oxalic acid-containing solution darkens significantly.

This breakdown happens whether you use oxalic acid or Api-Bioxal.

Stored OA solution and colour change

Stored OA solution and colour change …

HMF is toxic to honey bees at high concentrations. Studies from ~40 years ago showed that HMF concentrations below 30 mg/l were safe, but above 150 mg/l were toxic 11.

At 15°C HMF levels in OA solution can reach 150 mg/l in a little over a week. At room temperature this happens much faster, with HMF levels exceeding 150 mg/l in only 2-3 days. In the dark HMF levels build up slightly less quickly … but only slightly 12.

Therefore only make up OA solutions when you need them.

If you must store your oxalic acid-containing syrup for any length of time it should be in the fridge (4°C). Under these conditions HMF levels should remain well below toxic levels for at least one year. However, don’t store it for this long … use it and discard the excess.

Or prepare excess and share it with colleagues in your beekeeping association.

Don’t use discoloured oxalic acid solutions as they’ve been stored incorrectly and may well harm your bees.

Another final few words of caution

I assume you don’t have a fridge dedicated to beekeeping? That being the case please ensure that the bottle containing stored oxalic acid is labelled clearly and kept well out of the reach of children.


Notes

A quick trawl through the Veterinary Medicines Directorate database turns up several oxalic acid-containing solutions for managing Varroa. These include:

  • Oxuvar – approved for trickling or spraying, an aqueous solution of oxalic acid to which you add glucose if you intend to use it for trickling.
  • Oxybee – approved for trickling (and possibly other routes, but the paperwork was a minefield!), contains oxalic acid, glycerol and essential oils and is promoted as having a long shelf life.
  • VarroMed – approved for trickling, contains oxalic acid and formic acid and can be used throughout the year in one way or another.

I’ve not read the documentation provided with these and so don’t know the precise concentration of oxalic acid they contain. It will be listed as an active ingredient. I have not used these products. As with everything else on this site, I only write about methods or products I am familiar with. I therefore cannot comment on their relative efficacy compared to Api-Bioxal, to Apivar or to careful siting of your hives in relation to ley lines … or 5G phone masts.

 

Does DWV infect bumble bees?

Covid (the disease) is caused by a virus called SARS-Cov-2. SARS is an abbreviation of severe acute respiratory syndrome and the suffix ‘Cov’ indicates that it’s a coronavirus. The final digit (2) shows that it’s the second of this type of virus that has caused a pandemic. The first was in 2003, and was caused by a virus we now call SARS-Cov-1. That virus had a case fatality rate of 11%, but only infected ~8500 people worldwide. 

SARS-Cov-2 is not a human virus, by which I mean it’s not a virus that has been present in the human population for a long time. It’s actually a virus that most probably originated in bats.

We’re still not sure how SARS-Cov-2 jumped species from bats to humans.

SARS-Cov-1 made the same transition from bats and we do have a pretty good idea how this happened. Before the virus was found in bats it was detected in palm civets and raccoon dogs, both of which are farmed for food and sold in live game markets. Neither animal shows any symptoms when infected with SARS-Cov-1.

Bats were also sold in the same live game markets in Guangdong province in China and it seems likely that the virus either crossed directly from bats to humans, or went via a third species such as the palm civet.

Pathogen spillover

It’s likely that SARS-Cov-2 followed the same route. We are entering a second – likely extended – period of geographic lockdown due to Covid. Since SARS-Cov-2 made its cross-species jump from bats to humans it has infected at least 41 million people and killed over 1.13 million.

Pathogens that jump from one species to another can have catastrophic consequences for the recipient population 1 or, as in the case of palm civets, might cause no harm whatsoever.

The term ‘pathogen spillover’ is often used to describe the event when a pathogen – whether viral, bacterial or a parasite – escapes or spills over from its natural host to another species. 

CSI in the apiary … motive, opportunity, means

To make the species jump a number of criteria must occur. The pathogen has to be present at a high enough level to be infectious in the ‘donor’ species. For convenience, let’s consider this as the motive to jump species 2.

Secondly, the pathogen needs to have an opportunity to jump species. For example, because the donor and ‘recipient’ species share the same habitat or regularly come into contact.

Finally, it has to have the means to replicate in the recipient. If it cannot replicate it can never get established in the recipient population or cause disease.

In fact, this is an oversimplification. It also needs to be transmissible between individuals of the recipient species (or it will never spread in the recipient species).

So what has all this to do with the bumble bees in the title of this post?

Honey bees get a bad press from some scientists and environmentalists. They compete with native solitary and other bees and pollinators for environmental resources – like pollen and nectar. Increasingly, particularly in agricultural areas, these can be in limiting supply at certain times of the season. For examples, because all the hedgerows have been grubbed up and the wildflower meadows obliterated.

In addition, there are a number of studies that have suggested that honey bee viruses have spilled over into other pollinators, in particular bumble bees, and that this pathogen spillover has contributed to the decline in free-living bee populations.

Do honey bee viruses have the motive, opportunity and means to cause disease in bumble bees?

Are honey bee viruses responsible for the decline in bumble bee populations?

Correlation and causation

There are numerous studies showing that the most widespread honey bee virus, deformed wing virus (DWV), can be detected in wild-caught bumble bees.

Let me pose a quick question … does detection mean ‘replication’?

Deformed wing virus “does what it says on the tin” in honey bees. When transmitted by Varroa it causes developmental defects in pupae that appear as wing deformities in newly emerged workers.

DWV symptoms

DWV symptoms

There’s also one study that implicates DWV in directly causing disease in bumble bees.

This influential paper, published 15 years ago, demonstrated that some bumble bees had the characteristic crippled wings seen in symptomatic emerging honey bee workers. The ‘smoking gun’ was that DWV was also detected in these bumble bees.

Exhibit A : Evidence for DWV infection in bumble bees – click image for full legend.

This is the only figure in the paper and there have been no substantive follow-up papers. For some reason they showed ‘symptomatic’ Bombus terrestris (the buff-tailed bumble bee; panel A, left) and PCR detection data for B. pascorum (the common carder bee).

Nevertheless, this association between presence and symptoms was sufficient for the authors to conclude “we demonstrated that DWV is pathogenic to at least two bumble bee species … causing wing deformity similar to clinically DWV-infected honey bees”.

Here’s a second important question … does the detection of DWV in bumble bees demonstrate it is responsible for the symptoms observed? 3

As a virologist interested in the evolution, replication and transmission of viruses – and a beekeeper – this seemed like a worthwhile topic to explore in a bit more detail.

There might be a correlation between the presence of DWV in bumble bees, but does this account for causation of the DWV-like symptoms seen in the bumble bees?

Motive and opportunity

Let’s get these two out of the way quickly (though we’ll return to opportunity in the notes at the end).

Remember, viruses don’t want to do anything. I’m using motive as a hideously contrived reference to whether the pathogen, DWV, is present at high and infectious levels in honey bees.

It is.

Healthy, mite-naive (but reared in a hive with Varroa) workers can carry as little as ~1000 DWV viruses. Similar levels of DWV are also present in hives from Varroa-free regions – at least of the UK 4. In contrast, after parasitisation by Varroa, symptomatic or asymptomatic adult worker honey bees regularly have more that 109 (one trillion) viruses coursing through their little bodies. 

This virus is highly infectious. When injected into honey bees, as few as 10 viruses are sufficient to start a new infection and are replicated several million-fold within 24-48 hours.

Let’s agree that they have the ‘motive’ … to spread to other hosts.

They also have the opportunity, at least in the broadest sense of the word. Honey bees and bumble bees share the same environment. They collect nectar and pollen from the same flower species. They can even regularly be seen visiting the same flower simultaneously. In addition, it’s not unusual to see bumble bees trying to access honey bee hives to steal nectar.

I’d argue that the virus appears to have ample opportunity to move from one species to the other.

Does DWV replicate in bumble bees?

This is an important question. The data figure (‘Exhibit A’) shown above does not answer this question. Their assay simply detects the presence of DWV, with no indication of whether the virus is replicating.

And the reason this is important is really explained in the section on motive and opportunity above. DWV is ubiquitous and present at extraordinarily high levels in many honey bees. It’s present in honey bee faeces. It can be detected on flowers that honey bees have visited, or in pollen collected from those flowers.

DWV is absolutely everywhere.

So, if it is everywhere, there’s a good chance it might simply contaminate things … like bumble bees that look a bit sick for other reasons.

However, if DWV is involved in causing disease in bumble bees then the virus must replicate in bumble bees.

Virus replication – select for full size and legend.

I’ve used this figure before. To be certain that the virus is replicating you need to identify the intermediate replication products (the negative strand RNA shown as red arrows above).

Almost none of the large number of papers that have reported “DWV infected bumble bees” have identified these intermediate replication products.

Many studies didn’t even bother looking for these critical intermediate products.

So we did

There are two ways we could have investigated this. We could have collected large numbers of bumble bees from the fields and screened them in the lab for these replication intermediates. The problem with this approach is that DWV might be very rare in bumble bees, or might be common, but only replicate rarely. The additional problem is that it involves going out and collecting bees from the environment – that’s hard work 😉 5

An alternative approach is to buy a nest of bumble bees 6 and to inject a few bumble bee pupae and adults with DWV. This is what we did. In parallel, to ‘prove’ the virus used can replicate in honey bees, we injected a few honey bee pupae.

We injected the bees as it’s the definitive way we have of being sure that the virus was present.

‘Gene jockeys’

I’ve got some gifted molecular virologists in my lab. These are scientists who use genetic engineering or biotechnology techniques to address tricky questions (gene jockeys). They are particularly skilled at manipulating nucleic acids, such as the genomes of viruses.

If DWV is so common (it is – see above) how would we know that the replication intermediates were from the virus we injected into the bumble bees, rather than from a virus that was possibly already present?

To solve this puzzle Alex (the lead author on our recent paper) did two things. She engineered a unique genetic marker into the virus which we could look for (and that knew was absent from other similar viruses that might already be present in bumble bees). In addition, she ensured that the virus she injected into the bumble bees contained none of the negative strand RNA that is produced as the intermediate when the virus replicates. 

And … to cut a long story short, we could detect the negative strand RNA replication intermediate in injected bumble bees. In addition, the virus contained the unique genetic marker Alex had engineered into the virus genome, so we were absolutely certain it was the injected virus that was replicating. All of the controls worked exactly as expected.

DWV does replicate in bumble bees … at least under the conditions used in our experiment.

But it does not replicate very fast

The bumble bees we used for these experiments are commercially produced under very clean conditions. When we tested control bumble bees they contained no DWV at all, even using our most sensitive assays.

In contrast to injected honey bee pupae, DWV replicates rather slowly in bumble bees. In honey bees it will amplify a million-fold in 48 hours. However, in bumble bees, in 48 hours we only observed a 100-10,000 fold increase in DWV levels. It was only when we injected large amounts of DWV to bumble bees that we could recapitulate the virus levels seen in symptomatic honey bees.

This was a little puzzling as we’ve assumed that the very rapid replication of DWV in honey bees contributes to pathogenesis. The virus needs to replicate fast to spread through the pupa and infect the particular tissues and organs that, when damaged, result in the characteristic symptoms seen.

If it only replicates slowly in bumble bees how can it cause symptoms?

But hold on … does it cause symptoms?

Not as far as we can tell. 

We never found any injected bumble bees with deformed wings, or any that looked anything like the symptoms seen with DWV in honey bees. The actual quote from the paper is:

Strikingly, none of the eclosed bumble bees showed any signs of the wing deformities that are characteristic of DWV infection of honey bees”. 

Morbidity of DWV in bumble bees

Injected pupae developed until eclosion and were either non-viable, discoloured or apparently normal in appearance (see (c) above). We observed similar numbers of these three types whether the pupae were injected with DWV or mock-injected with buffer alone (see (b) above). What’s more, of those injected with the virus, the level of the virus was the same irrespective of the appearance (or viability) of the bumble bee (see (a) above).

We know that these bumble bees contain replicating DWV but see no evidence for overt disease. I acknowledge that they may have invisible symptoms. However, we see no evidence for the wing deformities reported in the 2005 paper from Genersch and colleagues (‘Exhibit A’, above).

Heavy going? But I’ve only just started … 

So, let’s briefly return to our “motive, opportunity, means” crime analogy and summarise where we’ve got to so far. 

DWV is present at very high levels in at least some honey bees. In addition, bumble bees are likely to regularly come into contact with DWV in the environment. This could happen through contaminated pollen, when attempting to rob hives, or by direct interaction with honey bees when both visit flowers. Finally, and most tellingly, DWV replicates in bumble bees.

So, if I was Peter Falk as Lieutenant Columbo, I’d argue that DWV has the motive, opportunity and means to potentially cause disease in bumble bees.

But, as is apparent from the figure above, it appears not to actually cause disease.

Which is puzzling. 

Just one more thing 7

There’s a major problem with the experiments I’ve discussed so far.

Like all experiments 8 they were tightly controlled, with single variables and lots of statistical analysis to demonstrate our confidence in their reproducibility.

The problem wasn’t technical, it was how well they recapitulated potential transmission in the environment.

We’re not aware of anything that goes around “injecting” bumble bee pupae or adults 9. They are not parasitised by Varroa and, although there are bumble bee mites (such as Parasitellus), they don’t feed on bumble bees in the same way that Varroa feeds on honey bees.

How else might bumble bees acquire DWV?

The obvious route is orally, while feeding. 

There are a few issues with feeding bees DWV. The method is simple enough … you add the virus to sugar syrup and they slurp it down. However, you have little control over how much individual bees consume. Do some bees feed directly and other acquire food when fed by other bees (trophallaxis)? It gets rather difficult control.

In addition, we knew from our studies of honey bees that they are far less susceptible to infection per os (by mouth) than by injection. And by far less I mean tens of thousands of fold less susceptible, at least as adults.

Feeding bumble bees DWV

We chose to investigate two routes of feeding.

The first was to directly feed individual bees in the laboratory. Using this approach we failed to detect any evidence for infection or DWV replication in fed adult bumble bees, even when fed 100 million DWV viruses.

Not an encouraging start. However, larvae generally have increased susceptibility to pathogens, so we also investigated feeding larvae in the laboratory. In these studies we did manage to establish infection. However, to do so we had to add 100 million DWV viruses to he food and only achieved a 50% infection rate. 

The second approach we used was direct feeding of complete bumble bee colonies maintained in the lab.

Bumble bees are easier to keep in the lab than honey bees as they don’t need to be free-flying. Each colony occupies a 30cm3 perforated plastic box, supplied with pollen and syrup. We fed three colonies 100 million DWV per bee per day for 4-6 weeks 10.

That’s a huge amount of virus for a protracted period. Our reasoning here was straightforward. Perhaps there was a particular developmental stage that had increased susceptibility? Perhaps adult bees feeding larvae would – for whatever reason – reduce their resistance to infection via the oral route?

We screened every egg, larva, pupa and adult bee for replicating DWV at the end of the experiment.

There was none present 🙁 ( or perhaps 🙂 , depending upon your viewpoint )

Summary and conclusions

We generated unequivocal evidence that DWV replicates in bumble bees. Specifically in Bombus terrestris, the buff tailed bumble bee. I’d be surprised if it did not replicate in other Bombus species, but that will need to be investigated. 

Infection of adult bees was only possible by injection, with no evidence for infection during feeding.

Bumble bee larvae can be infected with DWV while feeding, but only when fed very large amounts of virus directly. When larvae were reared by a colony supplied with DWV-laced food for several weeks the larvae did not become infected.

These results were recently published in Scientific Reports (Gusachenko et al., 2020) and are freely available. The title of the paper neatly sums up the study “Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis”

What does this mean in terms of our understanding of pathogen spillover from managed honey bee colonies to free living bees? 

If ecologists and environmental scientists are going to make the case that honey bees are threatening the survival of free-living solitary or bumble bees (due to pathogen spillover) they need to:

  1. formally demonstrate that the honey bee virus replicates in the free-living bee
  2. show that this replication is detrimental to the free-living bee
  3. provide evidence for a natural route of transmission by which infection can occur

In my view, and using legal terms again, it’s case proven for the first point in Bombus terrestris 11. In contrast, if I was the judge I’d throw out the other cases due to lack of evidence.

Monkey puzzles

There are viruses everywhere. Every living species has one or more viruses that infect it. Inevitably, because viruses replicate to very high levels, species other than the natural host may become exposed.

But almost always this has no consequences at all …

And to illustrate that I’ll briefly describe a study of monkey viruses infecting humans in Cameroon from several years ago. HIV is one of a very large group of viruses called immunodeficiency viruses. The ‘H’ stands for human, though the virus originated in chimpanzees and is very closely related to the simian immunodeficiency viruses (SIV).

A study of hunters living in the forests of Cameroon showed evidence for exposure to multiple different SIV isolates in tribespeople who hunted non-human primates or who were involved in butchering them or preparing them for market or cooking. There was no evidence these viruses were spreading in the human population, or that there was any sickness or disease associated with prior exposure. 

Environmental exposure happens all the time, and is relatively easy to detect. That’s exactly what had happened with these monkey viruses.

But evidence for environmental exposure, whilst easy to get, is not sufficient to support a claim that the virus causes disease at the level of the individual, or that it threatens an entire population.

As a virologist I think it’s interesting that DWV replicates in Bombus terrestris. However, I’ve yet to see any convincing evidence that DWV spillover from honey bees is responsible for the decline in wild bee populations.

Circumstantial evidence is not the same as convincing evidence.


Notes

What are the missing experiments that we didn’t do?

A key one is to determine whether long-term infection of bumble bees with DWV results in disease. We didn’t see overt deformed wing disease, but it’s possible that infection for weeks could have caused this or other symptoms. 

Although we fed bumble bee nests for weeks with DWV we saw no evidence of larval infection. This was despite previously demonstrating that larvae could be infected with high levels of DWV orally. One possibility is that DWV is inactivated by adult bees. I think it would be interesting to look at the level of infectious DWV in larval food.

Are there natural routes of exposure to DWV that result in bumble bee infection?

Does DWV ever cause environmental contamination at a high enough level to naturally infect bumble bees? For example, is the level of DWV in honey bee faeces high enough and does it ever contaminate pollen?

We are not going to do these studies so it will be interesting to see if others do … or whether simply the presence of the virus (whether replicating or not) will be proof’ that honey bee viruses are responsible for the decline in free-living bee populations.

Weed and feed

Weed and feed is a generic term that describes the treatment of lawns to simultaneously eradicate certain weeds and strengthen the turf.

It seemed an appropriate title for a post on eradicating mites from colonies and feeding the bees up in preparation for the winter ahead.

Arguably these are the two most important activities of the beekeeping year.

Done properly they ensure you’ll still be a beekeeper next year.

Ignored, or done too little and too late, you’ll join the unacceptably large number of beekeepers who lose their colonies during the winter.

They think it’s all over

In Fife, on the east coast of Scotland, my beekeeping season effectively finishes with the midsummer ‘mixed floral’ nectar sources. This is a real mix of lime, blackberry, clover and Heinz nectars 1 … many of which remain to be identified.

There’s no reliable late nectar flow from himalayan balsam around my apiaries are and not enough rosebay willowherb (fireweed) to be worthwhile, though in a good year the bees continue to collect a bit from both into early September.

But by then the honey supers are off and extracted. Anything the bees find after that they’re welcome to.

The contrast with the west of Scotland is very marked. Over there my bees are still out collecting reasonable amounts of late heather nectar, though the peak of the flow is over.

Storing supers

Once the honey supers are extracted they can be returned to the colonies for the bees to clean up prior to storing them overwinter. However, this involves additional trips to the apiary and usually necessitates using the clearer boards again to leave them bee-free before storage.

I used to do this and quite enjoyed the late evening trips back to the apiary with stacks of honey-scented supers. More recently I’ve stopped bothering and instead now store the supers ‘wet’. The main reasons for this are:

  • laziness lack of time
  • unless you’re careful it can encourage robbing, by wasps or bees. You need to return supers to all the colonies in the apiary and if you have the hives open too long it can induce a frenzy of robbing 2
  • the honey-scented supers encourage the bees to move up faster when they’re used the following season

If you do store the supers ‘wet’ make sure the stacked boxes are bee and wasp-tight. Mine go in a shed with a spare roof on the top. If there are any gaps the wasps, bees or ants will find them and it then becomes very messy. 

I know many beekeepers who wrap their supers in clingfilm. Not the 30 cm wide roll you use in the kitchen but the sort of metre wide swathe they used to wrap suitcases in at London Heathrow.

Dated super frames

The drawn super comb is a really valuable resource and can be used again and again, year after year. I usually record the year a frame was built on the top bar. Many are now over a decade old and have probably accommodated at least 80 lb of honey in their lifetime 3.

The timing of late season Varroa management

During the brood rearing season the Varroa levels in the colony will have been rising inexorably. Without intervention the mites will continue to replicate on developing pupae that would otherwise emerge as the all-important overwintering bees. These are critical to get the colony through to the following spring.

When Varroa feeds on a developing pupa it transmits the viruses – primarily deformed wing virus – it acquired from the last bee is fed on. These viruses amplify by about a million-fold within 24-48 hours. Pupae that do not die before eclosion may have developmental defects. Importantly, those that appear normal have a reduced lifespan.

The overwintering bees should live for months, but might only live for weeks if their virus levels are high.

And if enough overwintering bees have high viral loads and die prematurely, the probability is the the colony will perish in the winter.

You therefore need to reduce mite levels before the overwintering bees are exposed to Varroa

The full details and justification are in a previous post logically entitled When to treat?

TL;DR 4late August to early September is the best time to treat to protect the winter bees from the worst of the ravages of mite-transmitted DWV.

Use an appropriate treatment

You need to reduce the mite levels in the colony by at least 90% to protect the winter bees.

To achieve this you need an appropriate miticide used properly. 

I use Apivar

Apivar is an Amitraz-containing miticide. Although there are reports of mite resistance in some commercial apiaries, the pattern is very localised (individual hives within an apiary, which is difficult to understand) and in my view it is currently the best choice.

What are the alternates?

  • MAQS – active ingredient formic acid – poorly tolerated at high temperatures, but can be used with the supers present
  • Apiguard – active ingredient thymol – ineffective at lower temperatures (it needs an ambient temperature of 15°C to work – that’s not going to happen in Scotland in September).
  • Apistan – active ingredient a synthetic pyrethroid – unsuitable as there is widespread resistance in the mite population.

Using Apivar

Apivar treatment is temperature-independent. It cannot be used when the honey supers are present. You simply hang two strips in the hive for 6 to 10 weeks and let them do their work. The bees tolerate it well and, unlike MAQS or Apiguard, I’ve not seen any detrimental effects on the queen who continues to lay … making more of those important winter bees.

Apivar strips

Each strip consists of an amitraz-impregnated piece of plastic tape with a V-shaped tab that can be pushed into the comb to hold it in place. 

This generally works well as the frames are usually not moved much as there’s no need for inspections this late in the season.

Apivar strip pushed into comb

However, the strips can be a little fiddly to remove (or fall off during frame handling) and some of our research colonies will continue to be used for at least another month. I’ve therefore used a short piece of bent wire to hang the strips from in these hives.

Apivar strip on wire hanger

I place the strips in opposite corners of the hive, set two frames in from the sides. 

Apivar, wax and honey contamination

Although Amitraz is not wax soluble 5 there are recent reports on BEE-L that one of its breakdown products are, including one that has some residual miticide activity 6

I therefore try and get all the bees into the brood box before starting treatment (I described nadiring supers with unripe honey last week).

Very rarely I’ll leave the bees with a super of their own unripe honey. Usually this happens when the brood box is already packed with stores and overflowing with bees. In this case I’ll mark the super and melt down the comb next season rather than risking tainting the honey I produce.

I attended a Q&A session by the Scottish Beekeeping Association last month in which the chief bee inspector discussed finding Apivar strips in honey production hives. He described the testing of honey for evidence of miticide contamination and potential subsequent confiscation.

This is clearly something to be avoided.

Remember to record the batch number of Apivar used and note the date in your hive records. I just photograph the packet for convenience. The date is important as the strips must be removed after 6 weeks and before 10 weeks have elapsed. 

It’s finally worth noting that the instructions recommend scraping the strip with a hive tool part way through the period if they are being used for the full ten week course of treatment. The strips usually get propolised into the frame and the scraping ‘reactivates’ them to ensure that the largest possible number of mites are killed off.

And, after all, that’s what they’re being used for.

Apivar is expensive

Well … yes and no.

Yes it feels expensive when walking out of Thorne’s of Newburgh clutching one small foil packet and being £31 poorer. 

But think about it … that packet is sufficient to treat 5 colonies.

Is £6.20 too much to spend on a colony?

My 340 g jars of honey cost more than £6.20 and my productive colonies produce at least one hundred times that amount of honey. 

I don’t think 1% of the honey value is too much to spend on protecting the colony from mites and the viruses they carry.

Mite drop

Varroa killed by the miticide 7 fall to the bottom of the hive. If you have an open mesh floor (OMF) they fall through … onto the ground or the intervening neatly divided Varroa tray, enabling you to easily count them

Varroa trays ...

Varroa trays …

Remember that amitraz, the active ingredient of Apivar, works by direct contact. This is why you place the strips diametrically opposite one another so that as many bees as possible contact them. Unlike Apiguard, it makes no difference whether the Varroa tray is present or not.

It is useful to ‘count the corpses’ to get an idea of the infestation level and the efficacy of the treatment.

I’m going to discuss what you might expect in terms of mite drop in the winter (I need to plot some graphs first). However, this is something you could think about before then … knowing Apivar kills mites in less than three hours after exposure, what do you think the mite drop should look like over the 6-10 weeks of treatment?

Enough weeding, what about feeding?

I treat and feed colonies on the same day.

I also do the final hive inspection of the season. At this I look for evidence of a laying queen, the general health of the colony, the amount of brood present and the level of stores in the brood box. 

If the colony is queenless (how did that happen without me noticing earlier?) I simply unite the colony with a strong, healthy queenright colony. I don’t bother testing it with a frame of eggs … time is of the essence.

It’s too late to get a queen mated (at least in Fife … when I lived in the Midlands I got a few September queen matings but they could not be relied upon) and I rarely, if ever, buy queens.

I only feed with fondant in the autumn.

Convenience food

I described fondant last week as a convenience food

A spade's a spade ...

A spade’s a spade …

I’ve described in detail many of the benefits of fondant in numerous previous posts. Essentially these can be distilled to the following simple points:

  • zero preparation; no syrup spillages in the kitchen, no marital strife.
  • bucket- and feeder-free; no need to carry large volumes of syrup to the apiary and no feeders to store for the remaining 11 months of the year. All you need to feed fondant is a queen excluder and an empty super … and you’ve got those already.
  • easy to store; unopened it keeps for several years 8.
  • super speedy; I can feed a colony, including cutting the block in half, in less than 2 minutes.
  • good for queen and colony; perhaps that’s stretching it a bit. What I mean is that the bees take the fondant down more slowly than syrup, consequently the queen continues to lay uninterrupted as the brood nest does not get backfilled with stores. This is good for the colony as it means the production of more winter bees.
  • an anti-theft device; you can’t spill fondant so there is much less chance of encouraging robbing by neighbouring bees or wasps.
  • useful boxes; the empty boxes are a good size to store or deliver jarred honey in – each will accommodate sixteen 1 lb rounds.

I’ve fed nothing but fondant for about a decade and can see no downsides to its use.

Money, money, money

I’ve never used anything other than commercially purchased “baker’s” fondant … don’t believe the rubbish (about ‘additives’) some of the bee equipment suppliers use to justify their elevated prices.

You should be paying about £1/kg … any more and you’re being robbed. This year (2020) I paid less than 90p/kg.

Do not use the icing fondant sold by supermarkets for Christmas cakes. I’m sure there’s nothing much wrong with it, but – at £2/kg – you’ll soon go bankrupt. 

Tips for feeding fondant

Fondant blocks are easier to slice in half if they are slightly warm.

Use a sharp bread knife and don’t slice your fingers off. 

You can cut the blocks in half in advance in the warmth of your kitchen and then cover the cut faces with clingfilm to prevent them reannealing, but I just do it in the apiary.

Take care with sharp knives … much easier with a slightly warm block of fondant

Alternatively, use a clean spade 9.

Always place the block cut face down on a queen excluder directly over the top bars of the brood frames. With a full block, it’s like opening a book and laying it face down. Do not place it above a crownboard with a hole in it.

You want the bees to have unfettered access to the open face of the fondant block.

Fondant on queen excluder with eke

Ideally, use a framed wire queen excluder.

These are easier to lift off should you need to go into the colony.

Which you don’t 😉

There’s no need to continue inspections this late into the season. Go and enjoy a week or two away in Portugal … or perhaps not 🙁

If you need to store an unused half block of fondant wrap the cut face in clingfilm.

All my colonies get one full block (12.5 kg) and many get a further half block, depending upon my judgement of the level of the stores in the hive.

Insulation

The bees will take the fondant down over 2 – 4 weeks. They do store it, rather than just using it as needed. By late September or early October all that will remain is the blue plastic husk. The photo below is from mid-October. This colony has had a ‘topup’ additional half block after already storing a full block of fondant.

They fancied that fondant

With cooler days and colder nights, you want to reduce heat loss by the colony and minimise the dead space above the bees into which the heat escapes.

Although bees take fondant down at lower temperatures than they do syrup, there’s no point in giving the colony more additional space to heat than they need.

Poly super and fondant ...

Poly super and fondant …

Depending upon the availability of equipment I do one or a combination of the following:

  • use a poly super to provide space for the fondant
  • compress the fondant (use your boot) into as little space as possible and you squeeze it into a 50 mm deep eke, which (conveniently) is the same depth as the rim on my insulated polcarbonate/perspex crownboards 10.
  • use an eke and an inverted perspex crownboard with no need to compress the fondant
  • add a 50 mm thick block of insulation above the crownboard, under the roof (which may also be insulated)

Fondant block under inverted perspex crownboard – insulation block to be added on top is standing at the side

Oh yes … before I forget … completely ignore any advice you might read on using matchsticks to provide ventilation to the hive 11.

They think it’s all over … it is now

That’s the end of the practical beekeeping for the season 🙁

If your colonies are strong and healthy, if the mite levels are low and they have sufficient stores, there’s almost nothing to do now until March 12

Now really is a good time for a beekeeper to take a holiday.

Make a note in your diary on the date you need to remove the Apivar strips

Write up your notes, pour a large glass of Shiraz and make plans for next season 🙂


 

A virus that changes bee behaviour

Particle physicists might not agree, but I think that evolution is the most powerful force in the universe. It is responsible for the fabulous diversity of life, for everything from the 6,000,000 kg Pando clonal colony of quaking aspen covering 43 hectares of the Fishlake National Forest in Utah, to the teeniest of tiniest of viruses.

As a microbiologist I’m acutely aware of the role evolution has played in the genetic arms race between hosts and pathogens. This is what is responsible for the multi-faceted immune system higher organisms carry – the antibodies, the lymphocytes, the complement and interferon responses, and everything else.

In turn, the fast replicating bacteria and viruses have evolved countermeasures to subvert these immune mechanisms, to switch them off entirely or to decoy them into targeting the wrong thing. This ‘arms race’ has gone on since well before the evolution of multicellular organisms (~600 million years ago) … and continues unabated.

Evolution is powerful for one simple reason; if a particular genetic combination 1 ‘works’ it will be passed on to the progeny. If a virus evolves a way to resist the immune response of the host, or to spread between hosts more efficiently, then the trait will be inherited.

Molecular mechanisms and behavioural changes

Some of changes work at the molecular level, invisible without exquisitely sensitive in vitro analysis; protein A binds to protein B and, in doing so, stops protein B from doing whatever it should have be doing. These are important but often very subtle.

Rabies: Slaying a mad dog, 1566 illustration from Wellcome Images

Other changes are far more obvious. Take rabies for example (or don’t, it’s not recommended as it has a near-100% case fatality rate) … the primary host of the rabies virus are carnivorous mammals. Infection causes gross behavioural changes that facilitate virus transmission. The animal becomes bolder and much more aggressive, resulting in virus transmission through biting.

Recent studies have elegantly demonstrated that this (also) is an example of protein A binding to protein B at the molecular level, it’s just that the phenotype 2 is very much more marked.

A protein on the surface of the virus resembles a snake venom toxin and has the ability to bind to nicotinic acetylcholine receptors present in the central nervous system of the mammalian host. These receptors ‘do what they say on the tin’ and bind the neurotransmitter acetylcholine. If the virus protein binds the receptor the response to acetylcholine is blunted and this, in turn, leads to hyperactivity, one of the key behavioural responses caused by rabies viruses.

That’s enough about mad dogs.

If virus-induced behavioural changes are so obvious, why haven’t lots of different examples already been identified and characterised?

Sniffles

Part of the problem is the blurring of distinctions between overt behavioural changes and the direct symptoms induced due to the virus replicating.

Human rhinovirus, the aetiological agent of the common cold, causes upper respiratory tract infections. You get a runny nose and you sneeze a lot.

Gesundheit

Your behaviour changes.

However, it’s generally accepted that the sneezing and runny nose are a result of the physiological response to infection, rather than a virus-induced behavioural response to facilitate transmission.

It’s worth noting that all that “stuff” that comes out of your nose contains infectious virus, so it’s perhaps an artificial distinction between the general symptoms of sickness and evolutionarily-selected host behavioural changes caused by the virus.

Which in a roundabout way …

… allows me to finally introduce the topic of bee viruses that cause host behavioural changes involved in their transmission.

Or, rather, one bee virus that does this … though I’m certain that there will be more.

Israeli Acute Paralysis Virus (IAPV) is an RNA virus transmitted horizontally by direct contact between bees, or while feeding on developing pupae, by the parasitic mite Varroa destructor. It was implicated as a causative agent of Colony Collapse Disorder (CCD), though really compelling evidence supporting it as the primary cause never materialised.

It’s a virus UK beekeepers should be aware of, but unworried by, as it is extremely rare in the UK.

A recent paper has shown two behavioural changes in response to IAPV infection in honey bees. One of them – that facilitates horizontal transmission between colonies – is also partially explained at the molecular level.

The paper was published a couple of months ago:

Geffre et al., (2020) Honey bee virus causes context-dependent changes in host social behavior. Proc. Natl. Acad. Sci. USA 117:10406-10413. 3

I’m going to focus on the results, rather than the methods, though the methods are rather cool. They used barcoded bees to allow the automated image analysis of every bee in a colony for some of the studies where they had introduced known IAPV infected individuals.

Responses of nestmates to IAPV infected bees

Imagine watching a few hundred waggle dances and being able to recount the position, distance and response of every bee ‘watching’ 4 the dance, and then being able to summarise the results.

Over five days.

Non-stop.

Including nights (and yes, bees do still waggle dance at night – a subject for the future).

The scientists orally infected groups of 30 bees with a sub-lethal dose of IAPV, marked them and released them into an observation hive. They then recorded their movements around the hive and their interactions with other bees in the colony. In particular, they focussed on trophallaxis interactions where one bee ‘feeds’ another.

Trophallaxis is also considered to be a method of communication in the hive and has been implicated in disease transmission.

The authors love their whisker plots and statistical analysis.

Who doesn’t? 😉

However, they generally make for rather underwhelming images in a bee blog for entertaining reading. Here .. see what I mean …

Number of trophallaxis interactions per hour.

Suffice to say that the results obtained were statistically significant.

They showed that the infected bees in the colony actually moved about the colony more than their nestmates. Conversely, they were engaged in fewer trophallaxis interactions i.e. it appeared as though they were being ‘ignored’ by their nestmates.

Were they really being ignored altogether or did their nestmates approach them, detect something was amiss and move away?

Antennation

Antennation is the mechanism by which bees recognise nestmates. They use the sensitive chemoreceptors on their antenna to detect cuticular hydrocarbons (CHC) which are distinctive between bees from different hives.

Antennation is a precursor to trophallaxis.

After all, bees do not want to feed a foreigner, or exchange chemicals involved in communications, or even potentially risk being exposed to a new pathogen.

Good as the barcoding and camera system is, it’s not good enough to record antennation within the observation hive. To do this they manually 5 recorded antennation events between IAPV-infected bees and nestmates in cages in the laboratory 6.

In these studies IAPV-infected bees were engaged in the same number of antennation events as control bees. This strongly suggests that the nestmate could detect there was something ‘wrong’ with the IAPV-infected individuals. In support of this conclusion, the authors also demonstrated that bees inoculated with a double stranded RNA (dsRNA) stimulator of the honey bee immune response were also also antennated equally, but engaged in less trophallaxis interactions.

Therefore, these studies appear to show that nestmates exhibit a behavioural response to IAPV-infected bees (and bees with elevated immune responses, recapitulating their response to pathogen infection) that is likely to be protective, reducing the transmission of horizontally acquired viruses.

It’s worth noting two things here.

  1. There were no virus transmission studies conducted. It’s assumed that the lack of trophallaxis reduces virus transmission. That still needs to be demonstrated.
  2. This response is not induced by the virus on the host. It’s a response by nestmates of the host to virus infected individuals (or individuals that present as ‘sick’). As such it’s not the same as the rabies example I started this post with.

Virus-induced behavioural responses

But do the IAPV-infected bees behave differently when they come into contact with other bees who are not their nestmates?

After all, IAPV is a pathogenic virus and its continuing presence within a population (not just a single hive) depends upon it being spread from hive to hive.

For a highly pathogenic virus this is very important. If you spread from bee to bee within a hive and kill the lot you also go extinct … this partly explains the mechanism by which highly virulent viruses become less virulent over time.

But back to IAPV. What happens when IAPV-inoculated bees interact with bees from a different hive?

For example, what would happen if they drifted from one hive to another in a densely populated apiary? Drifting is a significant contributor to the spread of bees between adjacent colonies – studies show that 1% of marked bees drift to adjacent hives over a 3 day window. This partially accounts for the genetic mix of workers (up to 40% are unrelated to the queen that heads the colony) in a hive, a fact generally unappreciated by beekeepers.

The authors first showed that IAPV-infected bees could apparently leave and return to the hive with a similar frequency as uninfected foragers. Their flying was not compromised.

They then resorted again to recording interactions in the laboratory between IAPV-infected bees or control dsRNA-inoculated bees and workers from a different hive.

This was where it gets particularly interesting.

Hello stranger

The dsRNA-immunostimulated bees (remember, these induce a generalised immune response characteristic of a ‘sick’ bee) were treated aggressively by unmatched workers from a different hive.

In contrast, the IAPV-infected bees (which were ‘sick’ and would have been undergoing immunestimulation caused by the IAPV infection) experienced significantly less aggression than both uninoculated workers (which induced an intermediate response) and the dsRNA-inoculated.

This strongly suggests that IAPV is somehow able to modulate the appearance or behaviour (and one often determines the other) of the host to make it more acceptable to an unmatched worker.

They extended this study to conduct “field-based assays at the entrances of three normally managed honey bee colonies”, monitoring whether IAPV-infected bees were more likely to be accepted by the guard bees at the entrance of the hive.

They were. The IAPV-infected bees received a less aggressive reception and/or entered the hives much more easily than the controls.

But what about proteins A and B?

Good question.

The behavioural alterations described above must be explainable in terms of the molecular changes that IAPV induces in the bees. By that I mean that the virus must make, or induce the making of, a chemical or protein or other molecule, the presence of which explains their acceptance by the foreign guard bees.

And the obvious candidates are the cuticular hydrocarbons (CHC) that are recognized during antennation, which I introduced earlier.

And here the story leaves us with some tantalising clues, but no definitive answer.

The scientists demonstrate that there were marked differences between the CHC profile of IAPV-infected and control bees. Again, they used their favoured whisker plots to show this, but collated all of the CHC data into an even more difficult to explain scatter plot of linear discriminant analysis.

CHC profiles (relative abundance) shown using linear discriminant analysis.

The key take home message here is that for each of the CHC’s analysed there were differences in both the quality and relative abundance between the control bees, bees immunestimulated with dsRNA and the IAPV-infected bees.

These differences were so marked that you can see distinct clustering of points in the analysis above … these bees ‘look’ 7 different to the guard bees that antennate them.

This is a great story.

It’s as yet incomplete. To complete the understanding we will need to know which of those CHC’s, or which combination, when suppressed (or overrepresented) induce the guard bees to say “Welcome, step this way … “.

We’ll then of course need to find out how IAPV induces the change in CHC profile, which takes us right back to protein A and protein B again.

Ever the pedant

Much as I like this science I’d perhap argue that, again, the virus isn’t directly inducing a behavioural change in the host.

What it’s doing is inducing a behavioural change in the response to the infected host (by the guard bee). So perhaps this again isn’t quite the same as the rabies example we kicked off with.

A behavioural change in the host might include IAPV-infected bees drifting more, or drifting further. Alternatively, perhaps a colony with widespread IAPV infection could more easily indulge in robbing neighbouring colonies as they would experience less aggression from guard bees.

Smaller is better ...

Reduced entrance to prevent robbing …

I can see immediate evolutionary benefits to a virus that induced these types of behavioural changes. It’s not an original idea … the late Ingemar Fries suggested it in a paper two decades ago 8.

I’m also certain that researchers are looking for evidence supporting these types of directly-induced behavioural changes caused by viral pathogens in honey bees.

All religion, my friend, is simply evolved out of fraud, fear, greed, imagination, and poetry

Edgar Allen Poe may or may not have said this.

However, while we’re on the thorny subject of pathogen-induced behavioural changes in the host, it might be worth mentioning a couple of more controversial areas in which it has been proposed.

In the snappily titled paper “Assortative sociality, limited dispersal, infectious disease and the genesis of the global pattern of religion diversity” Fincher and Thornhill argue 9 that the wide diversity of religions in the tropics (compared to temperate regions) is driven by infectious disease selecting for three anti-contagion behaviours; in-group assortative sociality; out-group avoidance; and limited dispersal. It’s an interesting idea and I’m pleased I don’t have to test it experimentally. Their argument is that these three behavioural changes select for fractionation, isolation and diversification of the original culture … and hence the evolution of religions.

Conversely, perhaps microorganisms induce religious behaviours (rather than religion per se) that facilitate their transmission. This is exemplified in the entertainingly titled paper “Midichlorians – the biomeme hypothesis: is there a microbial component to religious rituals?” by Panchin et al., (2014). They argue that microbes – and they are really thinking about the gut microbiota here – might be able to influence their hosts (humans) to gather for religious rituals at which both ideas (memes) and infections are more easily transmitted.

Perhaps something to think about when mindlessly spinning out all that summer honey in the next few weeks?

Party, party

I think it’s fair to say that both the papers in the section above have some way to go until they achieve mainstream acceptance … if they ever do.

Furthermore, the general area in which parasites, bacteria and viruses, induce changes in the behaviour of their hosts’ is really in its infancy. We are aware of a lot of behavioural changes, but few are understood at the molecular level 10. As such, we often don’t know whether the association is correlative or causative.

Evolution is certainly a powerful enough selective force to ensure that even extremely subtle benefits to the pathogen may become a genetically-fixed feature of the complex interaction it has with the host.

Respiratory viruses, such as the common cold, Covid-19 and influenza infect millions of people globally and are readily transmitted by direct or indirect contact.

That’s why most of the readers of this post have a face mask nearby and a bottle of hand sanitizer ‘at the ready’. Or should.

Direct transmission benefits the virus as it does not have to survive on a door handle, milk bottle or petrol filling pump.

But direct transmission requires that people meet and are in close contact.

And a paper 10 years ago demonstrated that infection with influenza virus resulted in increased social interactions in the 48 hours post-exposure, compared with the same period pre-exposure 11.

It’s amazing what viruses can do … or might do … or (just look around you) are doing.


 

Barcoding bees

Every jar of honey I prepare carries a square 20mm label that identifies the apiary, batch, bucket and the date on which is was jarred. The customer can scan it to find out about local honey … and hopefully order some more.

The label looks a bit like this:

Scan me!

This is a QR code.

You’ll find QR codes on many packaged goods in the supermarket, on bus stop adverts, on … well, just about anything these days.  QR codes were first used in 1994 and are now ubiquitous.

QR is an abbreviation of quick response.

It’s a machine-readable two-dimensional barcode that is used to provide information about the thing it’s attached to.

QR codes contain positional and informational content. In the image above the three corners containing large squares allow the orientation to be unambiguously determined.

Within the mass of other, much smaller, black and white squares are several alignment points, an indication of the encoding 1 and the ‘information payload’. 

Large QR codes can contain more information and more error correction (so they can be read if damaged 2 ). Conversely, small QR codes contain reduced amounts of information and less error correction, but can still be used to uniquely identify individual things in a machine-readable manner.

A barcoded bee and barcode diagram.

And those ‘things’ include bees.

I am not a number 3

I had intended to write a post on how pathogens alter honey bee behaviour. This has been known about in general terms for some time, but only at a rather crude or generic level. 

To understand behavioural changes in more detail you need to do two things:

  • observe bees in a ‘natural setting’ (or at least as natural as can be achieved in the laboratory)
  • record hundreds or thousands of interactions between bees to be able to discriminate between normal and abnormal behaviour. 

And that isn’t easy because they tend to all look rather similar.

Lots of bees

How many of the bees above are engaging in trophallaxis?

Does the number increase or decrease over the next five minutes? What about the next hour?

And is it the same bees now and in an hour?

And what is trophallaxis anyway? 

I’ll address the last point after describing the technology that enables these questions to be answered.

And, since it’s the same technology that has been used to monitor the behavioural changes induced by pathogens, I’ll have to return to that topic in a week or two. 

Gene Robinson and colleagues from the University of Illinois at Urbana–Champaign have developed a system for barcoding bees to enable their unique identification 4.

Not just a few bees … not just a couple of dozen bees … every bee in the colony.

Though, admittedly, the colonies are rather small 😉

Each barcode carries a unique number, readable by computer, that can be tracked in real time.

So, unlike Patrick McGoohan, these bees are a number.

bCode

The scientists designed a derivative of the QR code that could be printed small enough to be superglued to the thorax of a worker bee. They termed these mini-QR-like codes bCodes 5. The information content of a bCode was limited by its size and the reference points it had to carry that allowed the orientation of the bee to be determined.

In total the bCode could carry 27 bits of data. Eleven bits (each essentially on or off, indicated by a black or white square) encoded the identification number, allowing up to 2048 bees to be uniquely numbered. The remaining 16 bits were the error-correction parity bits that had to be present to ensure the number could be accurately decoded.

If you’re thinking ahead you’ll realise that the maximum number of bees they could therefore simultaneously study was 2048. That’s about 1/25th of a very strong colony at the peak of the season, or the number of bees covering both sides of a two-thirds full frame of sealed brood.

It’s enough bees to start a one frame nucleus hive, which will behave like a mini-colony 6 and, in due course, expand to be a much larger colony.

And if you’re thinking a long way ahead you’ll realise the every barcode must be affixed to each bee in the same orientation. How otherwise would you determine whether the bees were head to head or abdomen to abdomen?

Labelling bees

This is the easy bit.

Each bCode was 2.1mm square and weighed 0.6mg i.e. ~0.7% of the weight of a worker bee. Honey bees can ‘carry’ a lot more than that. When they gorge themselves before swarming they ingest ~35mg of honey. 

The bCode therefore should not be an encumbrance to the bee (and they confirmed this in an exhaustive series of control studies).

A single frame of sealed brood was incubated and the bees labelled within a few hours of emergence. Typically, two batches of ~700 bees each were labelled from a single frame for a single experiment.

Each bee was anaesthetised by chilling on ice, the bCode glued in place (remember … in the same orientation on every bee) and the bee allowed to recover.

Labelling a single bee took 1-2 minutes.

Labelling 1400 bees takes several people a long time.

I said it was easy.

I didn’t say it was interesting.

Smile for the camera

I’ve not yet discussed the goal of the study that needed barcoded bees. It’s not really important while I’m focusing on the technology. Suffice to say the scientists wanted to observe bees under near natural conditions.

Which means a free-flying colony, on a frame of comb … in the dark.

Free-flying because caged bees do not behave normally.

On a frame of comb because they were interested in the interactions between bees under conditions in which they would normally interact.

And in the dark because that’s what it’s like inside a beehive (and it’s one of the features that scout bees favour when selecting a site for a swarm).

Camera and hive setup.

The scientists used an observation hive with a difference. It had an entrance to allow the bees to fly and forage freely and it contained a single sided, single frame. In front of the frame was a sheet of glass separated by 8mm from the comb. This prevented the bees from clambering over each other, which would have obscured the bCodes 7. Behind the frame was an 850nm infrared lamp to increase contrast, and the front was illuminated by several additional infrared lamps.

Bees cannot see light in the infrared range, so they were effectively in the dark.

The camera used (an Allied Vision Prosilica GX6600 … not your typical point and shoot) recorded ~29MP images every second. A typical experiment would involve the collection of about a million images occupying 4-6 terabytes of hard drive space 8.

The recorded images were processed to determine the temporal location of every bee with a visible (and readable) bCode. This was a computationally interesting challenge and involved discarding some data – e.g. barcodes that moved faster than a bee can walk or barcodes that fell to the bottom of the hive and remained motionless for days (i.e. dead bees). About 6% of the data was discarded during this post-processing analysis.

Trophallaxis

Which finally gets us to the point where we can discuss trophallaxis. 

Honey bees and other social insects engage it trophallaxis.

It involves two insects touching each other with their antennae while orally transferring liquid food. It occurs more frequently than would be required for just feeding and it has been implicated in communication and disease transmission

bCoded bees and trophallaxis

So, if you are interested in trophallaxis, how do you determine which bees are engaging in it, and which are just facing each other head to head?

In the image above the two bees in the center horizontally of the insert 9 are engaged in trophallaxis. The others are not, even those immediately adjacent to the central pair.

Image processing to detect trophallaxis – head detection.

This required yet more image processing. The image was screened for bees that were close enough together and aligned correctly. An additional set of custom computer-vision algorithms then determined the shape, size, position and orientation of the bees’ heads. To be defined as trophallaxis the heads had to be connected by thin shapes representing the antennae or proboscis.

And when I say the image … I mean all million or so images.

Bursty behaviour

And after all that the authors weren’t really interested in trophallaxis at all.

What they were really interested in was the characteristics of interactions in social networks, and the consequences of those interactions.

This is getting us into network theory which is defined as “Well out of my depth”

Transmission of things in a network depends upon interactions between the individuals in the network.

Think about pheromones, or honey, or email … or Covid-19.

It’s only when two individuals interact that these can be transmitted between the individuals. And the interaction of individuals is often characterised by intermittency and unpredictable timing. 

Those in the know – and I repeat, I’m not one of them – call this burstiness. 

If you model the spread of ‘stuff’ (information, food, disease) through a bursty human communication network it is slower than expected.

Is this an inherent characteristic of bursty networks?

Are there real bursty networks that can be analysed.

By analysing trophallaxis Gene Robinson and colleagues showed that honey bee communication networks were also bursty (i.e. displayed intermittent and unpredictable interactions), closely resembling those seen in humans.

However, since they had identified every trophallaxis interaction over several days they could follow the spread of ‘stuff’ through the interacting network.

By simply overlaying the real records of millions of interactions over several days of an entire functional community with an event transmitted during trophallaxis they could investigate this spread..  

For example, “infect” (in silico) bee 874 in the initial second and follow the spread of the “infection” from bee to bee through the real network of known interactions.

In doing this they showed that in a real bursty network, interactions between honey bees spread ‘stuff’ about 50% faster than in randomised reference networks. 

Why isn’t entirely clear (certainly to me 10, and seemingly to the authors as well). One obvious possibility is that the topology of the network i.e. the contacts within it, are not random. Another is that the temporal features of a bursty network influence real transmission events. 

Scientists involved in network theory will have to work this out, but at least they have a tractable model to test things on …

… and at a time when some remain in lockdown, when others think it’s all a hoax, when social distancing is 2m 11, when some are wearing masks and when prior infection may not provide protective immunity anyway, you’ll appreciate that ‘how stuff spreads’ through a network is actually rather important.

Stay safe


 

Aristotle’s hairless black thieves

Aristotle not in his beesuit

Almost every article or review on chronic bee paralysis virus 1 starts with a reference to Aristotle describing the small, black, hairless ‘thieves‘, which he observed in the hives of beekeepers on Lesbos over 2300 years ago 2.

Although Aristotle was a great observer of nature, he didn’t get everything right.

And when it came to bees, he got quite a bit wrong.

He appreciated the concept of a ‘ruling’ bee in the hive, but thought that the queen was actually a king 3. He also recognised different castes, though he thought that drones (which he said “is the largest of them all, has no sting and is stupid”) were a different species.

He also reported that bees stored noises in earthenware jars (!) and carried stones on windy days to avoid getting blown away 4.

However, over subsequent millenia, a disease involving black, hairless honey bees has been recognised by beekeepers around the world, so in this instance Aristotle was probably correct.

Little blacks, maladie noire, schwarzsucht

The names given to the symptomatic bees or the disease include little blacks or black robbers in the UK, mal nero in Italy, maladie noire in France or schwarzsucht (black addiction) in Germany. Sensibly, the Americans termed the disease hairless black syndrome. All describe the characteristic appearance of individual diseased bees.

Evidence that the disease had a viral aetiology came from Burnside in the 1940’s who demonstrated the symptoms could be recapitulated in caged bees by injection, feeding or spraying them with bacterial-free extracts of paralysed bees. Twenty years later, Leslie Bailey isolated and characterised the first two viruses from honey bees. One of these, chronic bee paralysis virus (CBPV), caused the characteristic symptoms described first by Aristotle 5.

CBPV causes chronic bee paralysis (CBP), the disease first described by Aristotle.

CBPV infection is reported to present with two different types of symptoms, or syndromes. The first is the hairless, black, often shiny or greasy-looking bees described above 6. The second is more typically abnormal shivering or trembling of the wings, often associated with abdominal bloating 7. These bees are often found on the top bars of the frames during an inspection. Both symptoms can occur in the same hive 8.

CBP onset appears rapid and the first thing many beekeepers know about it is a large pile (literally handfuls) of dead bees beneath the hive entrance.

It’s a distressing sight.

Despite thousands of bees often succumbing to disease, the colony often survives though it may not build up enough again to overwinter successfully.

BeeBase has photographs and videos of the typical symptoms of CBPV infection.

Until recently, CBP was a disease most beekeepers rarely actually encountered.

Emerging and re-emerging disease

I’ve got a few hundred hive year’s worth 9 of beekeeping experience but have only twice seen CBP in a normally-managed colony. One was mine, another was in my association apiary a few years later.

A beekeeper managing 2 to 3 colonies might well never see the disease.

A bee farmer running 2 to 3 hundred (or thousand) colonies is much more likely to have seen the disease.

As will become clear, it is increasingly likely for bee farmers to see CBP in their colonies.

Virologists define viral diseases as emerging if they are new in a population. Covid-19, or more correctly SARS-CoV-2 (the virus), is an emerging virus. They use the term re-emerging if they are known but increasing in incidence.

Ebola is a re-emerging disease. It was first discovered in humans in 1976 and caused a few dozen sporadic outbreaks 10 until the 2013-16 epidemic in West Africa which killed over 11,000 people.

Often the terms are used interchangeably.

Sporadic and rare … but increasing?

Notwithstanding the apparently sporadic and relatively rare incidence of CBP in the UK (and elsewhere; the virus has a global distribution) anecdotal evidence suggested that cases of disease were increasing.

In particular, bee farmers were reporting increasing numbers of hives afflicted with the disease, and academic contacts overseas involved in monitoring bee health also reported increased prevalence.

Something can be rare but definitely increasing if you’re certain about the numbers you are dealing with. If you only have anecdotal evidence to go on you cannot be certain about anything very much.

If the numbers are small but not increasing there are probably other things more important to worry about.

However, if the numbers are small but definitely increasing you might have time to develop strategies to prevent further spread.

Far better you identify and define an increasing threat before it increases too much.

With research grant support from the UKRI/BBSRC (the Biotechnology and Biological Sciences Research Council) to the Universities of Newcastle (Principle Investigator, Prof. Giles Budge) and St Andrews, and additional backing from the BFA (Bee Farmers’ Association), we set out to determine whether CBPV really was increasing and, if so, what the increase correlated with (if anything).

This component of the study, entitled Chronic bee paralysis as a serious emerging threat to honey bees, was published in Nature Communications last Friday (Budge et al., [2020] Nat. Comms. 11:2164 https://doi.org/10.1038/s41467-020-15919-0).

The paper is Open Access and can be downloaded by anyone without charge.

There are additional components of the study involving the biology of CBPV, changes in virus virulence, other factors (e.g.environmental) that contribute to disease and ways to mitigate and potentially treat disease. These are all ongoing and will be published when complete.

Is chronic bee paralysis disease increasing?

Yes.

We ‘mined’ the National Bee Units’ BeeBase database for references to CBPV, or the symptoms associated with CBP disease. The data in BeeBase reflects the thousands of apiary visits, either by call-out or at random, by dedicated (and usually overworked) bee inspectors. In total we reviewed almost 80,000 apiary visits in the period from 2006 to 2017.

There were no cases of CBPV in 2006. In the 11 years from 2007 to 2017 the CBP cases (recorded symptomatically) in BeeBase increased exponentially, with almost twice as much disease reported in commercial apiaries. The majority of this increase in commercial apiaries occured in the last 3 years of data surveyed.

Apiaries recorded with chronic bee paralysis between 2006 and 2017.

BeeBase covers England and Wales only. By 2017 CBPV was being reported in 80% of English and Welsh counties.

During the same period several other countries (the USA, several in Europe and China) have also reported increases in CBPV incidence. This looks like a global trend of increased disease.

But is this disease caused by CBPV?

It should be emphasised that BeeBase records symptoms of disease – black, hairless bees; shaking/shivering bees, piles of bees at the hive entrance etc.

How can we be sure that the reports filed by the many different bee inspectors 11 are actually caused by chronic bee paralysis virus?

Or indeed, any virus?

To do this we asked bee inspectors to collect samples of bees with CBPV-like symptoms during their 2017 apiary visits. We then screened these samples with an exquisitely sensitive and specific qPCR (quantitative polymerase chain reaction) assay.

Almost 90% of colonies that were symptomatically positive for CBP were also found to have very high levels of CBPV present. We are therefore confident that the records of symptoms in the historic BeeBase database really do reflect an exponential increase of chronic bee paralysis disease in England and Wales since 2007.

Interestingly, about 25% of the asymptomatic colonies also tested positive for CBPV. The assay used was very sensitive and specific and allowed the quantity of CBPV to be determined. The amount of virus present in symptomatic bees was 235,000 times higher than those without symptoms.

Further work will be needed to determine whether CBPV is routinely present in similar proportions of ‘healthy’ bees, and whether these go on and develop or transmit disease.

Disease clustering

Using the geospatial and temporal (where and when) data associated with the BeeBase records we investigated whether CBPV symptomatic apiaries were clustered.

For example, in any year were cases more likely to be near other cases?

They were.

Across all years of data analysed together, or for individual years, there was good evidence for spatial clustering of cases.

We also looked at whether cases in one year clustered in the same geographic region in subsequent years.

They did not.

Clustering of CBPV – spatial and temporal analysis.

This was particularly interesting. It appears as though there were increasing numbers of individual clustered outbreaks each year, but that the clusters were not necessarily in the same geographic region as those in previous or subsequent years.

The disease appears somewhere, increases locally and then disappears again.

Apiary-level disease risk factors

The metadata associated with Beebase records is relatively sparse. Details of specific colony management methods are not recorded. Local environmental factors – OSR, borage, June gap etc. – are also missing. Inevitably, some of the factors that may be associated with increased risk are not recorded.

A relatively rare disease that is spatially but not temporally clustered is a tricky problem for which to define risk factors. Steve Rushton, the senior author on the paper, did a sterling job of analysing the data that was available.

The two strongest apiary-level factors that contributed to disease risk were:

  1. Commercial beekeeping – apiaries run by bee farmers had a 1.5 times greater risk of recording CBP disease.
  2. Importing bees – apiaries which had imported bees in the two preceding years had a 1.8 times greater risk of recording CBP disease.

Bee farming is often very different from amateur beekeeping. The colony management strategies are altered for the scale of the operation and for the particular nectar sources being exploited. For example, colonies may already be booming to exploit the early season OSR. This may provide ideal conditions for CBPV transmission which is associated with very strong hives and/or confinement.

Bee imports does not mean disease imports

There are good records of honey bees imported through official channels. This includes queens, packages and nucleus colonies. Between 2007 and 2017 there were over 130,000 imports, 90% of which were queens.

An increased risk of CBP disease in apiaries with imported bees does not mean that the imported bees were the source of the disease.

With the data available it is not possible to distinguish between the following two hypotheses:

  1. imported honey bees are carriers of CBPV or the source of a new more virulent strain(s) of the virus, or
  2. imported honey bees are susceptible to CBPV strain(s) endemic in the UK which they were not exposed to in their native country.

There are ways to tease these two possibilities apart … which is obviously something we are keen to complete.

All publicity is good publicity …

… but not necessarily accurate publicity 🙁

We prepared a press release to coincide with the publication of the paper. Typically this is used verbatim by some reporters whereas others ask for an interview and then include additional quotes.

Some more accurately than others 🙁

The Times, perhaps reflecting the current zeitgeist, seemed to suggest a directionality to the disease that we certainly cannot be sure of:

The Times

Its sister publication, The Sun, “bigged it up” to indicate – again – that bees are being wiped out.

The Sun

And the comments included these references to the current Covid-19 pandemic:

  • “Guess its beevid – 19. I no shocking”
  • “It’s the radiation from 5g..google it”
  • Local honey is supposed to carry antibodies of local virus and colds – it helps humans to eat the stuff or so they say. So it could be that the bees are actually infected by covid. No joke.

All of which I found deeply worrying, on a number of levels.

The Telegraph also used the ‘wiped out’ reference (not a quote, though it looks like one). They combined it with a picture of – why am I not surprised? – a bumble bee. D’oh!

The Telegraph

The Daily Mail (online) had a well-illustrated and pretty extensive article but still slipped in “The lethal condition, which is likely spread from imports of queen bees from overseas …”. The unmoderated comments – 150 and counting – repeatedly refer to the dangers of 5G and EMFs (electric and magnetic fields).

I wonder how many of the comments were posted from a mobile phone on a cellular data or WiFi network?

😉

Conclusions

CBPV is causing increasing incidence of CBP disease in honey bees, both in the UK and abroad. In the UK the risk factors associated with CBP disease are commercial bee farming and bee imports. We do not know whether similar risk factors apply outside the UK.

Knowing that CBP disease is increasing significantly is important. It means that resources – essentially time and money – can be dedicated knowing it is a real issue. It’s felt real to some bee farmers for several years, but we now have a much better idea of the scale of the problem.

We also know that commercial bee farming and bee imports are both somehow involved. How they are involved is the subject of ongoing research.

Practical solutions to mitigate the development of CBP disease can be developed once we understand the disease better.


Full disclosure:

I am an author on the paper discussed here and am the Principle Investigator on one of the two research grants that funds the study. Discussion is restricted to the published study, without too much speculation on broader aspects of the work. I am not going to discuss unpublished or ongoing aspects of the work (including in any answers to comments or questions that are posted). To do so will compromise our ability to publish future studies and, consequently, jeopardise the prospects of the early career researchers in the Universities of St Andrews and Newcastle who are doing all the hard work.

Acknowledgements

This work was funded jointly by BBSRC grants BB/R00482X/1 (Newcastle University) and BB/R00305X/1 (University of St Andrews) in partnership with The Bee Farmers’ Association and the National Bee Unit of the Animal and Plant Health Agency.