Category Archives: Science

Apivar (amitraz) resistance

Apivar is a widely used acaricide (a pesticide that kills mites and ticks) used to control Varroa.

The active ingredient of Apivar 1 is amitraz, a synthetic chemical discovered and developed almost 50 years ago.

Amitraz

Amitraz …

Amitraz has multiple molecular targets. I previously discussed the mechanism of action and summed it up with the words:

Essentially, amitraz binds and activates receptors that are critically important in a range of important aspects of the Varroa activity and behaviour … amitraz changes [this] behaviour and so exhibits miticidal activity. It has additional activities as well … these multiple routes of action may explain why resistance to amitraz is slow to develop.

I made the point in a subsequent post that amitraz resistance was very well documented … in cattle ticks 2 but that there was only anecdotal or incompletely documented evidence of resistance in Varroa in the USA, Argentina and Europe.

Apivar strip – fit and (don’t) forget

Amitraz has been used for mite control in honey bees for over twenty years. Considering its widespread use, the concentrations it is used at, and the relatively high replication rate of Varroa it is surprising that there has not been better evidence of resistance.

But that is no longer the case 🙁

Do you want the good news or the bad news first?

The bad news

A very recent paper 3 has clearly documented amitraz resistant Varroa in several commercial beekeeping operations in the USA.

I’ll discuss the key results of this paper first and then make some general comments on the implications for beekeepers and beekeeping.

The study had three components:

  1. Determine the sensitivity of Varroa never treated with amitraz to the chemical. This forms the baseline sensitivity against which field samples from commercial beekeepers could be tested.
  2. Screen Varroa from hives maintained by commercial beekeepers (with a multi-year history of Apivar usage) for amitraz resistance.
  3. Validate that the reduced efficacy of Apivar correlates with the observed amitraz resistance.

Essentially it involved harvesting live Varroa from colonies by a large-scale dusting with icing sugar 4. The Varroa were then tested to determine whether they showed resistance to amitraz, and the sensitivity was compared with the baseline sample of mites from colonies never treated.

Finally, an Apivar sensitivity test was conducted to determine the proportion of mites killed in a standardised assay in a set time period, again compared with the control (baseline sample).

The results of the study

You should refer to the paper for the primary data if needed.

Not all the apiaries tested yielded sufficient mites to screen for Apivar resistance. This is part of ‘the good news’ which I’ll get to shortly … but first the science.

Of those apiaries that did, Apivar resistance (determined by LC50 – the Lethal Concentration required to kill 50% of the mites) ranged from similar to that seen in the baseline samples to ~20-fold greater than the controls.

Two apiaries had an over 10-fold increase of the resistance ratio (the observed LC50 divided by the baseline LC50), with some individual colonies having high levels of Varroa infestation despite an active application of amitraz.

Apivar kills mites very quickly. Using a known number of mites trapped in a cage with a single small square of Apivar it is possible to ‘count the corpses’ and plot a kill curve over time. Sensitive mites from the control colonies were all killed within 3 hours.

Time course of Apivar efficacy in amitraz-susceptible Varroa

Using this as the baseline control it was then possible to determine the efficacy of Apivar in killing the mites (in the same 3 hour timeframe) from apiaries exhibiting resistance.

Apivar efficacy in commercial beekeeping apiaries.

Two apiaries (B and C, above) contained mites that exhibited high levels of resistance to Apivar, reflected in a low level of Apivar efficacy (above). In these apiaries, an average of less than 80% of mites were killed within the 3 hour assay.

Finally, the author demonstrated a correlation between Apivar efficacy and amitraz resistance. Unsurprising, but a necessary concluding point for the experimental data.

Within apiary variation

It was interesting that the author notes that the range of Apivar efficacy was much greater in colonies from apiaries with clear evidence of amitraz resistance.

For example, apiary B exhibited a range of Apivar efficacy in colonies from 28% to 97%, with an average (plotted above) of 68%. Whilst this is clearly an unacceptably low level, it is interesting that some of the colonies within the same apiary had mites killed at an efficacy similar or better (>90%) to apiaries A2 and A4 in the graph above.

I’ve re-plotted the primary data of Apivar efficacy vs. mite counts from individual colonies to emphasise this point.

Variation of Apivar efficacy vs mite infestation levels in individual colonies from commercial apiaries

Apiaries B and C (red markers) could be considered as ‘failing apiaries’ as the average Apivar efficacy of each was below 80% (see bar chart). Together the average mite load and Apivar efficacy for these two apiaries was 6.75 mites/100 bees and 72% respectively.

However, of the 16 colonies screened from these two apiaries (8 from each):

  • One colony had insufficient detectable mites to be included in the the full analysis.
  • Eight dropped less than 3 mites/100 bees during the sugar dusting analysis (the average over the 63 colonies screened was 5.33 mites/100 bees).
  • Four colonies exhibited ≥90% Apivar efficacy.
  • One colony from apiary B was a clear outlier, with >50 mites/100 bees ( 😯 ) and only ~28% Apivar efficacy. Inevitably this sample skews the averages …

Clearly the average figures presented in the bar chart above hides a very significant level of within-apiary variation.

Weird

I commented recently on the variation in mite levels during midwinter treatment of colonies with OA/Api-Bioxal. I attributed this – with little supporting evidence (!) – to different rates of late-season brood production. Colonies brooding late into the autumn were expected to have higher midwinter mite levels.

However, the variation seen here is different.

With the exception of that one heavily infested colony from apiary B, the mite levels in the ‘failing apiaries’ (B and C) are actually less than the average of the remainder of the study group (3.88 vs. 4.85).

What differs is the efficacy of Apivar treatment, not the resulting mite levels.

Frankly, this is a bit weird … on two counts:

  1. If Apivar treatment had been failing for a long time in apiaries B and C I would have expected much higher than average mite levels.
  2. Considering the amount of drifting and robbing that goes on between juxtaposed colonies I would have also expected Apivar-resistant mites to be very widely distributed within the ‘failing apiaries’.

Caveat on the mite counts – Apiaries in Louisiana, New York and South Dakota were analysed in this study. Louisiana apiaries were sampled in April, the others in July and August. I don’t know enough about the climate or mite-replication kinetics in these states to know how much this would have influenced the mite infestation levels (or prior or ongoing treatment regimes, which would also influence mite numbers). Unfortunately, the locations of the apiaries (A, B, C etc.) are not provided, other than the control apiary which is in Baton Rouge, LA. If the study had been done in the UK mite drops in April and August would have been wildly different depending upon the location.

Idle speculation

Apivar resistance does appear to have arisen in some of these colonies, but it does not appear to have become widely distributed within the apiary.

Why not?

I don’t actually think we have enough information to work with. The paper contains almost no additional background details – Apivar treatment history, use of other treatments, colony loss data etc.

But that won’t stop me speculating a little bit 😉

Do Apivar-resistant mites stop bees from drifting? Probably not, but it would explain why resistance was not widespread in the apiary 5.

More sensibly, perhaps Apivar resistance is detrimental in the absence of selection.

In the colonies in which resistance evolves it gives the mites a significant advantage. The ongoing infestation could encourage prolonged or repeated treatment, so selecting for yet more resistant mites. Eventually the colony succumbs to the resulting high viral load.

In other colonies, treatment is withdrawn (or forgotten … remember, we have zero information here!) and the Apivar-resistant mites are then at a disadvantage to their sisters.

This isn’t unheard of.

Apistan resistance appears to be detrimental in the absence of selection. There are some relatively straightforward molecular explanations for this type of phenotype.

You would have to assume differential colony treatments within apiaries B and C for this to be part of the explanation (and to account for drifting). Let’s hope the colony records are less shambolic than mine many beekeepers keep 😉

Until a clearer picture emerges of the management history of these colonies all we’re left with is the slightly (or very) confusing observation that Apivar resistance is a hive-specific phenomenon.

As the author states:

This colony level resolution suggests that each colony may act an island of resistance with its own distinct Varroa population. Beekeepers have reported inconsistency in amitraz treatment efficacy among colonies within an apiary and this variation seems to support those anecdotal observations.

And the good news?

I think there are two ‘encouraging’ observations in this paper (though of course I’d be happier if there was no resistance).

  1. About half of the commercial apiaries surveyed (5 of 11 that had a long history of Apivar usage) had too few mites detectable to screen for amitraz resistance. Clearly Apivar works, and often works very well indeed.
  2. Apivar resistance is not widespread in the apiaries within which it had arisen. For whatever reason, resistant mite populations appear restricted to individual colonies.

And these, in turn, have implications for practical beekeeping.

Implications for practical beekeeping

How does Apivar resistance evolve? Classically, misuse or overuse of treatments results in their eventual failure. Antibiotics are a good example of this.

I’ve been told by commercial beekeepers that some use a half dose of Apivar midseason to knock mite levels back sufficiently for the late season nectar flows. This is a typical example of misuse. It may not result in the development of resistance and it may not be a strategy used by the beefarmers managing apiaries B and C, but it is not the correct way to use Apivar.

What about overuse? Mites still dropping after 6 weeks of Apivar? Go on, slip another couple of strips in for another month or two. An (expensive) example of overuse.

Used Apivar strips

Or what about the Apivar strip found lying on the bottom of the hive at the first spring inspection? Again, overuse as there are likely to be lingering traces of Apivar present in the colony all winter 6.

So the first implication for practical beekeeping is to use Apivar correctly to help avoid the development of resistance. Don’t overdose or underdose, remove after 6-10 weeks, do not leave in over the winter.

Secondly, use alternate treatments to knock back the mite population. This is again a classic strategy to avoid selecting for resistance.

For example, use Apivar in late summer and Api-Bioxal in midwinter.

The mechanism of action of these two treatments is fundamentally different, so resistance to one will not confer resistance to the other (and there are no documented cases of oxalic acid resistance I’m aware of).

If you don’t treat midwinter (and you probably should 7) then use Apiguard one year and Apivar the next. Again, totally different mechanisms of action.

Finally, do not rely on individual colonies within an apiary being indicative of all colonies. I know some beekeepers who only conduct mite drop counts in one colony as a ‘sentinel’ 8.

If the drop is high then treatment is needed.

Or vice versa … no mites, so no treatment needed.

There’s a lot of colony to colony variation so it’s worth monitoring them all 9. And this is probably even more important with the colony level Apivar resistance reported in this paper.

Just something else to worry about … 🙁


 

More local bee goodness?

Before the wind-down to the end of the year and the inevitable review of the season I thought I’d write a final post apparently supporting the benefits of local bees. This is based on a recently published paper from the USA 1 that tests whether local bees perform better than non-local stocks.

However, in my view the study is incomplete and – whilst broadly supportive – needs further work before it can really be seen as an example of better performing local bees. I suspect there’s actually a different explanation for their results … that also demonstrates the benefits of local bees.

This is a follow-up to a post three weeks ago that provided evidence that:

  1. Colonies derived from different geographic regions show physiological adaptations (presumably reflecting underlying genetic differences) that seem pretty logical e.g. bees from Saskatchewan express more proteins involved in heat production, whereas Hawaiian bees show higher levels of protein turnover (which would make sense if they had evolved locally to have high metabolic rates).
  2. In a study by Büchler, European colonies survived better overwinter in their local environment; a fact subsequently attributed to the colonies being stronger going into the winter. In turn, this agrees with a recent study that clearly demonstrates the correlation between overwintering success and colony strength.

I suggest re-reading 2 that post as I’m going to try and avoid too much repetition here.

Strong colonies

Strong colonies overwinter better and – if you’re interested in that sort of thing – are much more likely to generate a profit for your honey sales.

So how can you ensure strong colonies at the end of the season?

What influences colony strength?

One thing is colony health. A healthy colony is much more likely to be a strong colony.

In the ambitious 600-colony Büchler study in Europe they didn’t do any disease management. The colonies were monitored over ~2.5 years during which time 84% of colonies perished, at least half due to the ravages of Varroa.

Clearly this is not sustainable beekeeping and doesn’t properly reflect standard beekeeping practices.

Study details

The recent Burnham study makes a nice comparison to the Büchler study.

It was conducted in New York State using 40 balanced 3 colonies requeened in late May.

Queens were sourced from California (~4000 km west) or Vermont (~200km east in the neighbouring state, and therefore considered ‘local’) and colonies were assigned queens randomly.

Unlike some previous studies the authors did not evidence the genetic differences between queens.

A local queen

A local queen

However, the queens looked dissimilar and the stocks were sourced from colonies established in California or Vermont for at least 10-15 generations. I think we can be reasonably confident that the queens were sufficiently distinct to be relevant for the tests being conducted.

Colonies were maintained using standard beekeeping practices, Varroa levels were managed using formic acid (MAQS for European readers) and the colony weight and productivity (frames of bees) was quantified, as was the pathogen load.

In contrast to the Büchler study, Burnham and colleagues only followed colonies over one beekeeping summer season. This was not a test of overwintering survival, but mid-season development.

Results

The take-home message is that colonies headed by the ‘local’ Vermont queens did better. The colonies got heavier faster and brood levels built up better.

Bigger, faster, stronger …

It’s notable that colony weight built up before any brood would have emerged from the new queen (upper panel) and that brood level in colonies headed by the local queen recovered much better after formic acid treatment (arrow in lower panel).

Nosema levels

However, Nosema levels were significantly different (above) as were the levels of Israeli Acute Paralysis Virus (IAPV; below).

Virus loads (DWV, BQCV and IAPV)

There were no significant differences in the Varroa loads before or after treatment (not shown), or in the levels of DWV or Black Queen Cell Virus (BQCV).

Taken together – bigger, heavier, stronger colonies and lower pathogen loads (at least of some pathogens) – seems good evidence to support the contention that local bees are beneficial.

The benefits are precisely what you want for good overwintering – strong, healthy colonies.

That’s a slam-dunk then?

Case proven?

No.

IAPV is a virus rarely detected in the UK. It causes persistent and systemic infections in honey bees and can be found in every caste (drones, workers, queens) and at every stage of the life cycle.

As IAPV is detectable in eggs and larvae – neither of which are Varroa-exposed – it is assumed to be vertically transmitted from the queen. IAPV is also found in the ovaries of the queen, which is additional evidence for vertical transmission.

At the first timepoint (12 days post requeening) the levels of IAPV are different between the two colony types, but not significantly so. However, by 40 days (T2) the levels are very different. At this later timepoint all the bees in the colony will be have come from the introduced queen.

The authors explain the differences in IAPV levels in terms of local bees being more resistant to ‘local’ pathogens … in much the same way that Pizarro’s 168 conquistadors, being more resistant to smallpox, defeated the might of the Inca Empire with the help of the virus diseases they inadvertently introduced to Peru.

I suspect there’s another explanation.

Perhaps the Californian queens were IAPV infected from the outset?

If this was the case they could introduce a new and virulent strain of IAPV to the research colonies and – over time – the levels would increase as more and more workers in the colony were derived from the new queen. IAPV is present in ~20% of US colonies so it seems perfectly reasonable to suggest it might have been largely absent from the Vermont queens and the test colonies, but present in the queens introduced from California.

How should they have tested that?

The obvious thing to do would be to characterise the IAPV present in the colony. IAPV shows geographic variation across the USA. If the predominant virus was of Californian origin it would suggest it was brought in with the queen. This is a relatively easy test to conduct … a sort of 23andme to determine bee virus provenance.

Alternatively, though less conclusively, you could do the experiment the other way round … ship Vermont queens to California and compare their performance with colonies headed by Californian queens on their own territory. If the Californian queens again performed less well it undermines the ‘local bees do better’ argument and suggests another explanation should be sought.

Nosema is sexually transmitted but it is not vertically transmitted, so the same arguments cannot be made there. Why the Nosema levels drop so convincingly in colonies headed by the local queens is unclear. Nosema was present at the start of the study and was lost over time in the stronger colonies headed by the local queens.

One possibility of course is that the stronger colonies were better fed – more workers, more foragers, more pollen, more nectar. Improved diet leads to a more active and effective immune system and an increased ability to combat pathogens. Simplistic certainly, but it is known that diet influences pathogen resistance and colony performance.

So what does this paper show?

I suspect it doesn’t directly show what the authors claim (in the title) … that local queens head colonies with lower pathogen levels.

This largely reflects the lack of proper or complete controls. However, it does not mean that local bees are not better.

More than anything I think this paper demonstrates the impact queen quality has on colony performance.

Perhaps the Vermont-sourced queens were just better queens. Local certainly (on a USA scale definition of the word local), but not better because they were local, just better because they were better.

However, if my interpretation of the source of the IAPV is correct i.e. introduced from the Californian queens, I think the paper indirectly demonstrates one of the most compelling reasons why local bees are preferable.

If they’re local – your apiary, your neighbours, someone in your association – there is little chance they will be bringing with them some unwanted baggage in the form of an undetected exotic pathogen.

Or a more virulent strain of one already circulating relatively benignly.

Extensive bee movements, whether of queens, packages or full colonies, risks spreading parasites and pathogens.

There is compelling evidence that hosts and pathogens co-evolve to reduce the pathogenicity of the interaction. Naive hosts are always more susceptible to introduced pathogens, or novel strains of pre-existing pathogens. After all, look what happened to the Peruvian Inca when they met the measles- and smallpox-ridden conquistadors.

So, when thinking about the claims being made by bee importers (or, for that matter, strong advocates of local bee breeding), it’s worth considering all of the factors at play – queen quality per se, genetic adaptation of the queen to the local environment and the potential for the introduction of novel pathogens with introduced non-local stock.

And that’s before you also consider the benefits to your beekeeping of being self-sufficient and not reliant on others to produce your stocks.

I never said it was simple 😉


 

Bee bombs

The last couple of posts on overwintering survival and local bees have been heavy going 1. So, rather than more of the same, here’s something that is both informative and entertaining 2.

Though it maybe wasn’t at the time.

Six-legged soldiers

I’m currently enjoying reading Six-legged soldiers by Jeffrey A. Lockwood. This is an account of the many and devious applications man has found for employing insects in warfare. Whilst the topic certainly isn’t ‘laugh out loud’ entertaining, the book is written in an engaging style with plenty of graphic descriptions, ample Biblical and historical references, and enough wriggly, stinging, aggressive insects to make “I’m a celebrity, get me out of here!” 3 appear like a walk in the park.

As a beekeeper I’m pleased to see that bees feature significantly in the book.

And as a beekeeper who appreciates the importance of the integrity of the colony to bee survival I also found it a little distressing.

But as a source of all sorts of stories for friends and families over the forthcoming holiday season it probably cannot be beaten.

It’s pretty good on mosquitos as well …

Bombus away

The genus Bombus includes lots of the well known bumble bees e.g. Bombus terrestris (Buff tailed), B. pascuorum (Common carder) and B. hypnorum (Tree) . The generic name Bombus is derived etymologically from the Latin word bombus which means buzzing i.e. the noise a bee makes when it flies.

Bombus lucorum

Etymologically, the word bomb, has a similar origin – via bombe in French, bomba in Spanish, bombo and then the Latin bombus.

Boom also has a similar origin.

But that’s not the only link between bees and bombs.

Mushroom shaped clouds

Have you ever seen anyone drop a full brood box?

It is an amazing sight and one best appreciated from a distance and when wearing a full beesuit.

Bees do not appreciate being knocked, shocked or jarred. When I transport hives between apiaries I always give them several minutes to settle before removing the entrance block. If you don’t they tend to boil out the front spoiling for a fight.

So you can imagine that dropping a brood box from waist height achieves – simultaneously – the sudden jarring of the colony and the release of the bees.

Not so much ‘shock and awe‘ as shock and aargh!

The mushroom-shaped cloud of bees that are released are distinctly agitated.

In the absence of a beesuit you’re likely to get hammered.

Even with a beesuit there can be some uncomfortable moments.

And, since soldiers don’t routinely go into battle wearing camouflage BBwear battledress with an inbuilt fencing-style veil, this neatly brings us to using bees as weapons.

Package bees

These days a ‘package’ is one way to buy bees to start a colony.

But as a weapon, a colony of bees isn’t much use until it’s actually in something.

How do you carry them? How do you use them as a projectile?

Well, man is nothing if not ingenious when it comes to weapons development.

The Tiv people of Nigeria used a specially shaped horn, loaded with angry bees (presumably not so much Africanized as African 😯 ). In the heat of battle these would be fired at the opposition, with the horn-shape ensuring the bees both reached the enemy and were kept at a distance from friendly forces 4.

But then, 9000 years ago, pottery containers started to be used for beekeeping … and it got a whole lot easier to move the bee bombs to the front line and drop them on the opposition.

A big, beautiful wall

A wall seems like an obvious way to defend yourself.

The enemy have to knock the wall down, or go over or under the wall.

And if they choose to tunnel under the wall then they’re going to be less than enthusiastic if the tunnel is filled with bees.

Which is what happened in 908 when the Scandinavians laid siege to Chester. The city’s fortifications were impenetrable, so they tunnelled underneath them. The siege was ended when all the city’s beehives were dumped into the tunnel.

Chester City walls. Originally built in ~100 AD by the Romans.

The Scandinavians appear not to have learnt their lesson as they were again repelled by bees while storming the walled city of Kissingen (Germany) during the Thirty Years War (1618-48). In this instance the bees were dropped from a height onto the Swedish forces.

The troops were heavily armed and armoured, and were unfazed.

Their horses were not.

The siege collapsed as the cavalry mounts were driven into a frenzy by the bees. Even now, most beekeepers are aware that bees and horses don’t mix well.

Again in the 1600’s, the besieged nuns of Wuppertal (Germany) knocked over all the hives in their apiaries before – wisely – hiding indoors. The maelstrom of bees drove the marauding soldiers away and the town was subsequently renamed Beyenberg (‘bee town’).

Bee boles

A bee bole is a recess in a wall 5 designed to house a hive of bees, which – in the days when they were constructed – was likely a skep. Many castles and fortified town walls have bee boles built into them.

How convenient.

What could be easier than to drop these on the marauding troops trying to scale the ramparts or storm the drawbridge?

Bee boles in Kellie Castle, Fife, Scotland

As an aside, IBRA (The International Bee Research Association) maintain the comprehensive Bee Boles Register which is well worth searching if you are interested in historical beekeeping (or early bomb design).

Avoiding friendly fire

You’ll notice that a lot of these bees were being used in relatively close combat situations.

Having witnessed a brood box being dropped, I can assure you that bees are rather indiscriminate after a “dropped from a great height onto a hard surface” experience.

Far better to use the container housing the bees as a projectile, launching them at the opposition from a safe distance.

Safe in terms of contact with the enemy … and the bees 😉

The Greeks and subsequently the Romans developed and perfected the siege engine, capable of launching all sorts of things up and over defensive walls.

Including beehives.

Illustration of a ballista being loaded and drawn – note BBwear ‘Corinthian helmet’ style beesuit and veil.

The Greek ballista and the Roman onager were torsion powered siege engines developed between 400 BC and 350 AD. Both were capable of firing stones, often wrapped in combustible material set alight, with smaller later models also used as battlefield weapons firing projectiles 500 – 1000 yards.

They’d have barely broken a sweat firing one or more skeps at the enemy.

The Romans were so keen on bee bombs that there was a documented decline in hive numbers during the late Roman Empire.

And this enthusiasm continued … as did the demand for hives to hurl.

By the 14th Century those dastardly weapons designers had developed a windmill-like device capable of launching hive after hive from the end of its rapidly rotating arms.

Bees will not fly over water

But they will in a skep catapulted from a ship.

As the army developed entomological weaponry the navy exploited it.

As early as 330 BC pottery hives were being thrown at enemy ships during naval battles. Cannons and cannonballs eventually superseded 20,000 A. mellifera ligustica in a skep, but there is well-documented use of bees in naval warfare until at least the 1600’s.

Bees would therefore have been carried by warships for hundreds of years. It’s not documented how the colonies were managed or maintained. Perhaps they only fought local battles? However, since that rather defeats the purpose of a highly mobile navy it can be assumed that bees were probably transported long distances by sea … bringing a whole new meaning to the term migratory beekeeping.

Gunpowder and bees

Eventually the development of modern weaponry overtook the use of bees and beehives. Fortunately we don’t have to discuss the aerodynamic benefits of cedar vs. poly hives 6.

Gunpowder and explosives made the Gatling gun-like skep-launching windmill catapult a relic of the good old days of warfare, when the infantry hankered after really cold days when the bees would be torpid and much less aggressive.

But, as a couple of masochists have already demonstratedApis mellifera is pretty tame where stinging is concerned.

Apis dorsata, the giant honey bee of South East Asia, is much bigger than our honey bee, and is reputed to pack more of a punch when stinging 7.

These bees build large exposed nests and the colony may have up to 100,000 bees in it.

Apis dorsata nest, a single exposed comb which may be a metre wide.

Which doesn’t mix too well with gunpowder or, more specifically, a firecracker containing gunpowder.

During the Vietnam War the Viet Cong would attach firecrackers to dorsata nests relocated to the jungle trails used by the enemy. As a patrol passed by they would ‘light the blue touch paper’ and set off the firecracker.

And then stand well back.

And, at about the same time (1960’s), the Americans were developing chemical warfare approaches using isopentyl acetate, the alarm pheromone, with the intention of spraying it onto enemy troops and redirecting the bees to attack them instead.

Six-legged soldiers

There’s lots more in Six-Legged Soldiers … get a copy and enjoy reading it over the Christmas vacation. Jeffrey Lockwood is an entomologist and University of Wyoming Professor. The Sunday Times 2009 review of the book criticised it as ‘scarcely scholarly’, being a mix of myth, legend and historical facts.

I cannot imagine a better review and it probably explains why it is so entertaining to read 🙂

Mite bombs

These are something altogether different to bee bombs … and for regular beekeepers, much more relevant.

A mite bomb is a heavily mite infested and collapsing colony that liberally spreads Varroa mites around the neighbourhood. Recent evidence suggests that this occurs primarily during late-season robbing of weak (mite infested) colonies by strong colonies.

This is the primary reason late summer miticide treatment should be coordinated over a wide geographic area. What’s the point of treating your strong colonies if they’re going to load up on mites when robbing weak colonies in the adjacent fields?

Which reminds me, and should remind you, that winter mite treatments will be needed in the next few weeks to ensure your bees get the best possible start to the new season.

We’ve had a protracted cold period here in Fife and my colonies will probably be treated in the next 5-7 days before there’s a chance they start brood rearing again.


 

Locally adapted bees

This is a follow up to the last post on Strong hives = live hives which was written in response to the oft-repeated mantra that ‘local bees are better adapted to the local environment’.

In that previous post a study of the overwintering survival of colonies headed by queens from very different locations was discussed. There was no difference whether the queen (and consequently all the workers she subsequently mothered) had come from Vermont or Florida.

Instead, the primary correlate of overwintering success was the strength of the colony 1 going into the winter.

Migratory beekeeping

Despite the size differences between the US and UK (or Europe), the honey bee population structure is actually more distinct on this side of the Atlantic.

In the USA the huge impact of migratory beekeeping causes considerable mixing of the bees on the continent. Those on the east and west coasts are distinct, but those in the north and south, or across smaller geographic scales, are really rather similar.

It’s not only commercial migratory beekeeping that enforces this, it’s also some of the very large-scale queen rearing operations. These ship queens all across the USA ensuring that there is less genetic diversity than you’d expect from the vast geographic (and climatic) differences.

Bee caravan

Bee caravan …

So, perhaps the study I discussed last week was not particularly surprising after all … ? 2

In contrast to the US, beekeeping activities in the UK and Europe are rather more localised.

In the UK we still import thousands of queens, but we don’t move our hives across the continent – often more than once a season.

We might take a dozen hives to the heather moors 150 miles away, but we never take them 2500 miles to pollinate almonds.

‘Local’ bees in Europe

Probably as a consequence of less large-scale migratory beekeeping, and less ‘centralisation’ of commercial queen rearing, there is genetic evidence for ‘local’ strains of bees in Europe.

In addition, there is evidence that these genetic differences result in changes to the individual proteins that the bee expresses … and that these may result in local ecological adaptations.

However, this still doesn’t get us to ‘local bees are better adapted to the local environment (and this explains why local bees survive better)‘ …

Andalucian apiary

Local Andalucian apiary

But there is even some evidence to support this last statement as well.

So let’s look at each of these points in turn 3.

Genetically diverse bees

Biologists use the terms genotype and phenotype to describe the genetic makeup of an organism and its appearance. Most beekeepers are familiar with the different phenotypes of honey bee – the dark ‘native’ bees, carniolans, Buckfast etc.

The phenotype is defined and determined by the genotype, but we don’t necessarily know which genes determine which physical characteristic. Population geneticists therefore often use different genetic features to discriminate between different groups or populations.

Microsatellites are DNA markers that contain variable numbers of short tandem repeat sequences. In honey bees, microsatellites are abundant and highly variable. They are therefore very useful for differentiating between populations or groups of populations, though how this is done is outside the scope of this post.

In 1995 Arnaud Estoup and colleagues reported the microsatellite analysis of 9 populations of honeybees from Africa (intermissa, scutellatacapensis) and Europe (mellifera, ligustica, carnica, cecropia), previously distinguished phenotypically. In their enticingly titled paper Microsatellite Variation in Honey Bee (Apis Mellifera L.) Populations: Hierarchical Genetic Structure and Test of the Infinite Allele and Stepwise Mutation Models 4 they support the earlier morphometric (phenotypic) definition by Ruttner of three distinct evolutionary branches of honey bee.

In a series of particularly impenetrable tables and phylogenetic trees they also demonstrate the the European lineages are genetically distinct and, importantly, that sub-populations could be readily identified 5.

Ecological adaptation of bees

Microsatellites are essentially non-functional genetic markers that we can use for analysis. They are carried alongside the thousands and thousands of genes that encode the proteins that make the wings, eyes, guts, feet etc. of honey bees. Other proteins also influence the behaviour of bees – how and when they swarm, their cold tolerance, there longevity.

We can now measure genetic variation of individual genes easily through so-called ‘next generation sequencing’ of the whole genome of the honey bee. However, the variation we see is one step removed from the variation at the protein level that directly influences how the bee copes in (or is adapted to) different environments.

But, it turns out, we can measure the variation at the protein level as well using a technique termed proteome profiling.

If distinct genetic populations of bees have adapted to particular environments (through selection, either natural or by beekeepers) we would expect the proteins they express – that both make the bee and determine its behaviour – should be different.

For example, simplistically, if a bee had evolved to live in a very windy environment we might expect the proteins forming the flight muscles would be stronger, enabling the bee to fly on windier days 6.

Collect the data, decipher what it all means …

Alternatively, you could turn the analysis around:

  • Identify the differences in the proteins that are expressed
  • Work out (or look up) what those particular proteins do and …
  • Conclude that those adaptive changes are required by that sub-population of bees in a particular ecological environment.

And, using proteome profiling, this is exactly what Robert Parker and colleagues reported in 2010 7. They compared proteins from adult bees sourced from geographically dispersed locations (Canada, New Zealand, Chile, USA).

They then grouped proteins into particular pathways e.g. energy metabolism, and observed significant differences.

Pathway analysis of honey bee midgut proteins across the populations studied.

As far as we’re concerned here – which is evidencing that locally adapted bees are actually different from each other in a meaningful way – the precise differences Robert Parker and colleagues aren’t too important.

But … if you insist.

Cold-adapted bees e.g. those from Saskatchewan (SK1, SK2), exhibited much higher levels of proteins involved in heat production in the mitochondria. In contrast, bees from warmer climates e.g. Hawaii (HI), showed higher levels of proteins involved in biosynthesis/folding and degradation of proteins.

Importantly, distinct populations of bees from geographically-distant regions exhibit differences that, logically, could be expected to make them better adapted to that environment.

But, there’s a bit still missing …

The key phrase in that last sentence is ‘could be expected’.

What was not shown in these two studies is that the differences observed are responsible for the better performance or survival of those bees in those environments.

Which finally brings me to a study by Ralph Büchler entitled The influence of genetic origin and its interaction with environmental effects on the survival of Apis mellifera L. colonies in Europe 8.

Local bees do survive better

This was an ambitious and large scale study of the survival of ~600 colonies in 21 apiaries in Europe. The colonies included 5 sub-species (carnicaligusticamacedonia and mellifera) and 16 different genotypes of bees.

In each of the 21 apiaries a local genotype was tested in parallel with at least two non-local genotypes. The large team of scientists/beekeepers involved used standardised management protocols which excluded any form of disease management e.g. no control of Varroa or other diseases. Consequently (many) colonies were lost to Varroa and were removed from the study once infestation levels had reached 10% (i.e. 1 in 10 workers carried phoretic mites) or bee numbers dropped below 5000.

The study started in autumn 2009 and ended in March 2012. During this ~2.5 years 84% of the colonies perished. Almost half of these losses were attributable to Varroa … not a particular surprise.

There are a lot of variables in this study – sub-species (5), genotypes (16), apiaries (21) – so the statistics and analysis are a bit of a minefield.

Count the corpses

Essentially the researchers ‘counted the corpses’ (i.e. colonies that died). They then looked at the survivors and tried to determine the characteristics they shared.

Unsurprisingly, survival of colonies in different apiaries was not the same. Graphed below is the percentage of colonies that survived (vertical axis) in each of the 21 apiaries against time (horizontal axis).

Trajectories of colony survival for the different locations.

These differences are presumably due to local forage availability, colony management, climate etc. We know that bees do better in some places than others 9.

When survival of different genotypes was compared they were much of a muchness, with two outliers.

Trajectories of colony survival for the 16 different genotypes

But, very significantly, colonies headed by local queens did significantly better than colonies headed by non-local queens.

Trajectories of colony survival for the origin of the queens

Why do local bees survive better?

The differences between the two lines – local and non-local queens – in the Kaplan-Meier survival curve above may not look particular good … they both drop disconcertingly quickly, indicating lots of dead colonies.

But it is.

The authors unequivocally demonstrate this statistically, but for beekeeping purposes it’s perhaps even more convincing to simply state that:

“colonies with local queens survived on average 83 ± 23 days longer than those with non-local queens”

That’s a key quote from the paper. It also probably explains why colonies headed by local queen survive better.

In a follow-up paper to Büchler et al., 2014, the same authors did a more in-depth analysis of a range of colony parameters that correlated with survival 10 which contains an additional piece of the jigsaw explaining why colonies headed by local queens survived better.

“colonies of local origin had significantly higher numbers of bees than colonies placed outside their area of origin”

And, by significantly higher, I mean ~20% higher.

Which finally completes the story and brings us back to the Strong hives = live hives from last week.

Local queens head up colonies that survive better in the local environment to which they (and their workers) are adapted.

The colonies survive significantly longer because the colonies are significantly stronger.

Caveats and conclusions

There are a number of caveats to the ‘count the corpses’ study conducted by Büchler and colleagues.

For example, the local bees might have actually been adapted to the local beekeeping management practices. In future experiments there might be ways to control for this 11.

The absence of Varroa control meant colonies were always weaker in the second year of the study. For the majority of beekeepers this is not a sustainable way to manage colonies. A fourth year would have been impossible as they would have run out of colonies.

Nevertheless, under the conditions tested, this is confirmation that ‘local bees are better adapted to the local environment (and survive better)‘.

But as a scientist there’s always another ‘Why?’ question.

Why are the colonies stronger? Is it increased longevity of worker bees? Perhaps it is better foraging skills, meaning more brood can be reared? Is it an adaptation of the queen to the chemicals in the local pollen that increases her fecundity?

Question, questions, questions …

I can think of at least two additional compelling reasons why local bees and queens are preferable. I’ll cover these at some point in the future.


 

Strong hives = live hives

Science and beekeeping make for interesting contrasts and can be awkward bedfellows 1.

Science is based upon observation of tested single variables. multiple repeats and statistical analysis. It builds on what has gone before but has accepted processes to challenge well-established theories. Some of the greatest advances are made by young researchers willing to test – and subsequently overturn – established dogma.

Over the last three generations science – both how we do it and what we understand – has changed almost beyond recognition.

In contrast, beekeeping is steeped in history, has multiple variables – climate, forage, ability – and very small sample sizes. It tends to be taught by the most experienced, passing down established – though often not rigorously tested 🙁 – methods 2.

As a consequence our beekeeping has barely changed over the last three decades. Established dogma tends to stay established.

Local bees are better adapted to local conditions

So let’s look in a little more detail at one of these established ‘facts’ … that locally reared bees are better adapted to local conditions.

The suggestion here is that locally reared bees, because they’re ‘better adapted’ (whatever that means) are more likely to flourish when the going is good, and more likely to survive when the going gets tough.

Furthermore, the implication is that they’re more likely to do better in that environment than bees reared elsewhere (and that are therefore adapted to a different environment).

This sounds like common sense.

Locally bred queen ...

Locally bred queen …

As Brexit looms and the never-ending supply of early-season Greek or Slovenian queens disappears perhaps it’s also fortunate, rather than just being common sense.

But, as a scientist, I’ve spent a career questioning things.

Every time I read the “locally adapted bees survive better (or perform better, or whatever better)” 3 two questions pop into my head …

  1. What’s local?
  2. How did they prove – or how would I test – this?

Spoiler alert

There is evidence that local bees show adaptive changes to their local environment. There is also evidence that local bees do better in their local environment.

Formally, I don’t think scientists have demonstrated that the former explains the latter. This might seem trivial, but it does mean that our understanding is still incomplete.

However, I’m not going to discuss any of these things today – but I will in the future.

Instead I’m going to deal with those two questions that pop into my head.

If we tackle those I think we’ll be better placed to address that dogmatic statement that local bees are better adapted to local conditions in due course.

But perhaps we’ll first discover that other things are more important?

What’s local?

I live most of the time in central Fife. It’s a reasonably dry, relatively cool, largely arable part of the UK with a beekeeping season that lasts about 5 months (from first to last inspections).

Are my (fabulous 😉 ) locally bred queens adapted for central Fife, or the east of Scotland, or perhaps north-west maritime Europe, or Europe?

Where have all my young girls gone?

What a beauty

Would these locally adapted bees do better here (in Fife) than bees raised in the foothills of the Cairngorms, or the Midlands, or Devon or East Anglia … or Portugal?

If you measure the environment you’ll find there’s significant overlap in terms of the climate, the temperature, the forage, the day length (or a hundred other determinants) with other regions of the UK.

The temperature or rainfall extremes we experience in central Fife aren’t significantly different to those in the Midlands. The season duration is different (because of latitude), but I had lots of short seasons in the Midlands due to cool springs and early autumns.

Local is an ill-defined and subjective term.

But there are differences of course. Are Ardnamurchan bees better able to cope with the rain (and the fantastic scenery) than Fife bees? Are Fife bees better able to exploit arable crops than those foraging on the heather and Atlantic rainforests that cloak the hills in the far west of Scotland?

I don’t know 🙁

And there’s something else I don’t know

I also don’t know how I would meaningfully test this.

Just thinking about these types of experiments makes me nervous. Think of the year to year variation – in weather, forage etc. – compounded by the hive to hive variation.

Then multiply that by the variation between beekeepers.

This last one is a biggy. Two beekeepers of differing abilities will experience very different levels of success – quantified in terms of honey yield or hives that survive for example – in the same season and environment.

Doing a study large enough to be statistically relevant without having such enormous variation that the results are essentially meaningless is tricky.

What a nightmare.

Which, in a roundabout way, brings me to a paper earlier this year by Maryann Frazier and Christina Grozinger from Penn State University.

Ask the question in a different way

The title of the paper tells you most of what you need to know about the study.

Colony size, rather than geographic origin of stocks, predicts overwintering success in honey bees (Hymenoptera: Apidae) in the northeastern United States. 4

But don’t stop reading … let’s look in a bit more detail at what they did.

They approached the question (that local bees are better adapted) from a slightly different angle.

Essentially the question they asked was “Does the geographic origin of the bees influence the overwintering survival of bees in a temperate region?”

This question is easier to answer.

They defined the parameters of the experiment a bit more clearly. For example:

  • Rather than looking at several regions they just studied bees in one area  – Pennsylvania (the temperate region in the title of the paper).
  • The bees came from four sources; two were from a hot geographic region of the USA and two from a cold region.
  • They scored ‘doing better’ only in terms of overwintering survival.

By simplifying the question they could reduce some of the variables. They could therefore increase the quantification of the parameters (colony weight, strength/size etc.) that might influence the ‘doing better’.

And in doing so, they came up with an answer.

The study

Sixty colonies were established in three apiaries in Pennsylvania. Two of the apiaries (A & B) were within 1 mile of each other, with the third (C) about 15 miles away. Colonies were generally established from packages 5, to which a queen was introduced from one of four different queen breeders.

Two of the queen breeders were from southern USA (Texas or Florida) and two from northern USA (Vermont and West Virginia 6.

The authors used microsatellite analysis to confirm that the queens – after introduction – headed genetically distinct colonies by midsummer 7.

So far, so good …

They then used standard beekeeping methods to manage the colonies – regular inspections, Varroa treatments as appropriate, feeding them up for winter etc.

They scored colonies for a variety of ‘parameters’; net weight, frames of brood, adult bees and stores.

Four queens failed before winter.

And then they overwintered the remaining 56 colonies …

The results

… of which only 39 survived until April 🙁

39/56 sounds a pretty catastrophic loss to me but it’s actually about the same (~30%) as the average winter losses reported each year in the USA.

So, did the ‘cold-adapted’ 8 Vermont queens survive and prosper? Did the ‘Southern Belles’ 9 from Texas all perish in the cold Pennsylvanian winter?

No.

That’s no to both questions.

There was no significant difference in survival of colonies headed by queens from the north or the south.

The geographic ‘origin’ of the bees did not determine colony survival.

They may have been ‘locally adapted’ (to Vermont, or Texas or wherever) and they were certainly genetically distinct, but it made no difference to whether the colony perished or not in Pennsylvania.

So if the source of the queen didn’t influence things, what did?

Weighty matters

This is the key figure from the paper.

Overwintering success is significantly associated with colony weight.

The heavier a colony was in October, the more likely that the colony survived until April.

The left hand panel shows the probability of a colony surviving (vertical axis, solid line) plotted against the net weight of the colony.

Below about 30 kg colony survival dropped significantly.

The right hand panel shows that net weight alone was not the only determinant. This plots colonies ranked by weight (vertical axis) and indicates whether they survived or not. An underweight (i.e. under 30 kg) colony in apiary C was much more likely to survive than a similar weight colony from the other two apiaries.

Allee, Allee 10

The heavier the colony, the greater the chance it survived. Furthermore, it wasn’t simply the amount of stores available.

Heavier colonies were also larger colonies.

This indicates a so-called Allee effect 11 which is a positive correlation between population density and individual fitness.

This has been shown before for honey bees (and other social insects). For bees we know that the larger the winter cluster the better they are able to maintain the correct overwintering temperature. These large clusters show lower per capita honey consumption to maintain the same temperature when compared to small clusters.

However, in addition to not running out of stores (due to more frugal usage) 12, large colonies will also be better able to rear brood in early spring … ‘it takes bees to make bees’.

Taken together these results demonstrate that colony size and weight, rather than geographic adaptation, is probably the most important determinant of overwintering colony survival.

Disease interlude

These studies were conducted in 2013 (and published in 2019 … a feature of some of my science 🙁 ). In the previous year the authors set up a similar study but did not manage Varroa levels.

Under these conditions only 12% of the colonies survived.

There’s a lesson there I think 😉

This disastrous 2012 study used the same queen breeders to source their queens (from Texas, Florida, West Virgina and Vermont). Some of these queens were described and sold as ‘Varroa-resistant’.

There was no difference in survival (or, more accurately, death) rates between colonies headed by queens described as ‘Varroa-resistant’ or not.

Another lesson perhaps?

Is there a geographic component to Varroa-resistance? Are Varroa-resistant Vermont colonies only actually resistant to mites from Vermont?

Or their viruses? 13

OK, we’re getting distracted … let’s return to apiary C.

Forage diversity and abundance is also important

Colonies in apiary C survived better at lower overall net weights than colonies from other apiaries. In addition, average colony weights were higher in apiary C than in the other two apiaries.

Apiary location significantly affected colony weight and survival.

And the abundance and range of nectar sources was significantly different between the three apiaries used in this study, with colonies from apiary C – located in a less forested and more agricultural area – surviving better.

The proportion of land cover/land use types surrounding apiaries.

The authors suggest that the forage diversity and abundance around apiary C increased the size of the colonies (by boosting brood rearing, adult longevity and colony growth) and that it was this larger adult population, rather than colony weight per se, that was important.

Are we getting the message?

This is the second time in a month that I’ve discussed the importance of strong colonies.

A few weeks ago I discussed how strong colonies are more profitable because they generate a surplus of honey or bees, both of which are valuable.

In this post I show that the primary determinant of overwintering success is the strength and weight of the colony. The source of the queen – whether from the balmy south or the frosty north – had no significant influence on colony survival.

This doesn’t mean local bees aren’t better adapted to local conditions. That wasn’t what was being tested.

However, it does suggest that other things that may be as important, or perhaps more important.

The take home message from this study is keep strong colonies in a forage-rich environment.

In a future post I’ll discuss the evidence that local bees are better adapted … and I’ll make the suggestion that some of these adaptations might be explained because the local genotype actually produces stronger colonies 😉


Note

This was originally published with the title Correlates of winter survival on 8/11/2019 but a hamster running amok in the server meant that the email to those registered to receive announcements of new posts was never sent. Rather than let the post disappear into digital oblivion – as the take home message is an important one – I’m re-posting it again.

With apologies to those who read the original …

Spotty brood ≠ failing queen

I thought I’d discuss real beekeeping this week, rather than struggle with the high finance of honey sales or grapple with the monetary or health consequences of leaving supers on the hive.

After all, the autumn equinox has been and gone and most of us won’t see bees for several months 🙁

We need a reminder of what we’re missing.

Beekeeping provides lots of sensory pleasures – the smell of propolis on your fingers, the taste of honey when extracting, the sound of a full hive ‘humming’ as it dries stored nectar … and the sight of a frame packed, wall-to-wall, with sealed brood.

Brood frame with a good laying pattern

This is a sight welcomed by all beekeepers.

Nearly every cell within the laid up part of the frame is capped. All must therefore have been laid within ~12 days of each other (because that’s the length of time a worker cell is capped for).

However, the queen usually lays in concentric rings from the middle of the frame. Therefore, if you gently uncap a cell every inch or so from the centre of the frame outwards, you’ll see the oldest brood is in the centre and the most recently capped is at the periphery.

It’s even more reassuring if the age difference between the oldest and the youngest pupae is significantly less than 12 days. Hint … look at the eye development and colouration.

This shows that the queen was sufficiently fecund to lay up the entire frame in just a few days.

What are these lines of empty cells?

But sometimes, particularly on newly drawn comb, you’ll see lines of cells which the queen has studiously avoided laying up.

That'll do nicely

That’ll do nicely …

It’s pretty obvious that these are the supporting wires for the sheet of foundation. Until the frame has been used for a few brood cycles these cells are often avoided.

I don’t know why.

It doesn’t seem to be that the wire is exposed at the closed end of the cell. I suspect that either the workers don’t ‘prepare’ the cell properly for the queen – because they can detect something odd about the cell – or the queen can tell that there’s something awry.

However, after a few brood cycles it’s business as usual and the entire frame is used.

Good laying pattern ...

Good laying pattern …

All of these laid up frames contain a few apparently empty cells. There are perhaps four reasons why these exist:

  • Workers failed to prepare the cell properly for the queen to lay in
  • The queen simply failed to lay an egg in the cell
  • An egg was laid but it failed to hatch
  • The egg hatched but the larvae perished

Actually, there’s a fifth … the cell may have been missed (for whatever reason) but the queen laid in it later and so it now contains a developing larva, yet to be capped.

What are all these empty cells?

But sometimes a brood frame looks very different.

Worker brood 1 is present across the entire frame but there are a very large number of missed cells.

Patchy brood pattern

Patchy brood & QC’s …

Note: Ignore the queen cells on this frame! It was the only one I could find with a poor brood pattern.

This type of patchy or spotty brood pattern is often taken as a sign of a failing queen.

Perhaps she’s poorly mated and many of the eggs are unfertilised (but they should develop into drone brood)?

Maybe she or the brood are diseased, either reducing her fecundity or the survival and development of the larvae?

Sometimes spotty brood is taken as a sign of inbreeding or poor queen mating.

Whatever the cause, colonies producing frames like that shown above are clearly going to be less strong than those towards the top of the page 2.

So, if the queen is failing, it’s time to requeen the colony …

Right?

Perhaps, perhaps not …

Which brings me to an interesting paper published by Marla Spivak and colleagues published in Insects earlier this year 3.

This was a very simple and straightfoward study. There were three objectives, which were to:

  • Determine if brood pattern was a reliable indicator of queen quality
  • Identify colony-level measures associated with poor brood pattern colonies
  • Examine the change in brood pattern after queens were exchanged into a colony with the opposite brood pattern (e.g. move a ‘failing queen’ into a colony with a good brood pattern)

If you are squeamish look away now.

Inevitably, measuring some of the variables relating to queen quality and mating success involve sacrificing the queen, dissecting her and counting ‘stuff’ … like viable sperm in the spermathecae.

Unpleasant, particularly for the queen(s) in question, but a necessary part of the study.

However, in the long run it might save some queens, so it may have been a worthwhile sacrifice … so, on with the story.

Queen-level variables in ‘good’ and ‘poor’ queens

By queen level variables I mean things about the queen that could be measured – and that differ – between queens with a good laying pattern or a poor laying pattern.

Surprisingly, good and poor queens were essentially indistinguishable in terms of sperm counts, sperm viability, body size or weight.

Poor queens i.e. those generating a spotty brood pattern, weren’t small queens, or poorly mated queens. They were also not more likely to have fewer than 3 million sperm in the spermathecae (a threshold for poorly mated queens in earlier studies).

Furthermore, the queens had no statistical differences in pathogen presence or load (i.e. amount), including viruses (DWV, Lake Sinai Virus, IAPV or BQCV), Nosema or trypanosomes (Crithidia). 

Hmmm … puzzling.

Colony-level variables

So if the queens did not differ, perhaps colonies with spotty brood patterns had other characteristics that distinguished them from colonies with good brood patterns?

Spivak and colleagues measured pathogen presence and amount in both the good-brood and poor-brood colonies.

Again, no statistical differences.

So what happens when queens laying poor-brood patterns are put into a good-brood pattern hive?

And vice versa …

Queen exchange studies

This was the most striking part of the study. The scientists exchanged queens between colonies with poor-brood and good-brood and then monitored the change in quality of the brood pattern 4.

Importantly, they monitored brood quality 21 days after queen exchange. I’ll return to this shortly.

Changes in sealed brood pattern after queen exchange

Queen from good-brood colonies showed a slight decrease in brood pattern quality (but not so much that they’d be considered to now generate poor brood patterns).

However, surprisingly, queens from poor-brood colonies exhibited a greater improvement in brood quality (+11.6% ± 9.9% more sealed cells) than the loss observed in the reverse exchange (-8.0% ± 10.9% fewer sealed cells).

These results indicate that the colony environment has a statistically significant impact on the sealed brood pattern.

Admittedly, a 10-20% increase (improvement) in the sealed brood pattern on the last frame photograph (above) might still not qualify as a ‘good brood pattern’ queen, but it would certainly be an improvement.

Matched and mismatched workers

Since exchanged queens were monitored just 21 days after moving them all the workers in the receiving hive were laid – and so genetically related to – the previous queen.

The authors acknowledge this and comment that it would be interesting to extend the period until surveying the hive to see if ‘matched’ workers reverted to the poor brood pattern (assuming that was what the queen originally laid).

This and a host of other questions remain unanswered and will undoubtedly form the basis of future studies.

The authors conclude that “Brood pattern alone was an insufficient proxy of queen quality. In future studies, it is important to define the specific symptoms of queen failure being studied in order to address issues in queen health.”

Notwithstanding the improvements seen in some brood patterns I suspect they would be insufficient to justify not replacing an underperforming queen … when considering the issue as a practical beekeeper i.e. there may be improvements but they were much less than could be achieved by replacing the queen from a known and reliable source.

But it might be worth thinking twice about this …

Insufficient storage space

In closing it’s worth noting that I’ve seen spotty or incomplete brood patterns when there’s a very strong nectar flow on and the colony is short of super storage space.

Under these conditions the bees start to backfill the brood box, taking up cells that the queen would lay in.

Usually this is resolved just by adding another super or two.

If there remains any doubt (about the queen) and you’ve provided more supers you can determine the quality of the laying pattern by putting a new frame of drawn comb into the brood nest.

The queen should lay this up in a day or two if she’s “firing on all cylinders”.

In which case, definitely keep her 🙂


 

Crime doesn’t pay

At least, sometimes it doesn’t.

In particular, the crime of robbery can have unintended and catastrophic consequences.

The Varroa mite was introduced to England in 1992. Since then it has spread throughout most of the UK.

Inevitably some of this spread has been through the activities of beekeepers physically relocating colonies from one site to another.

However, it is also very clear that mites can move from colony to colony through one or more routes.

Last week I described the indirect transmission of a mite ‘left’ by one bee on something in the environment – like a flower – and how it could climb onto the back of another passing bee from a different colony.

Mite transmission routes

As a consequence colony to colony transmission could occur. Remember that a single mite (assuming she is a mated female, which are the only type of phoretic mites) is sufficient to infest a mite-free hive.

However, this indirect route is unlikely to be very efficient. It depends upon a range of rather infrequent or inefficient events 1. In fact, I’m unaware of any formal proof that this mechanism is of any real relevance in inter-hive transmission.

Just because it could happened does not mean it does happen … and just because it does happen doesn’t mean it’s a significant route for mite transmission.

This week we’ll look at the direct transmission routes of drifting and robbing. This is timely as:

  • The early autumn (i.e. now) is the most important time of year for direct transmission.
  • Thomas Seeley has recently published a comparative study of the two processes 2. As usual it is a simple and rather elegant set of experiments based upon clear hypotheses.

Studying phoretic mite transmission routes

There have been several previous studies of mite transmission.

Usually these involve a ‘bait’ or ‘acceptor’ hive that is continuously treated with miticides. Once the initial mite infestation is cleared any new dead mites appearing on the tray underneath the open mesh floor must have been introduced from outside the hive.

All perfectly logical and a satisfactory way of studying mite acquisition.

However, this is not a practical way of distinguishing between mites acquired passively through drifting, with those acquired actively by robbing.

  • Drifting being the process by which bees originating from other (donor) hives arrive at and enter the acceptor hive.
  • Robbing being the process by which bees from the acceptor hive force entry into a donor hive to steal stores.

To achieve this Peck and Seeley established a donor apiary containing three heavily mite-infested hives of yellow bees (headed by Italian queens). These are labelled MDC (mite donor ccolony) A, B and C in the figure below. This apiary was situated in a largely bee-free area.

They then introduced six mite-free receptor colonies (MRC) to the area. Three were located to the east of the donor hives, at 0.5m, 50m and 300m distance. Three more were located – at the same distances – to the west of the donor apiary. These hives contained dark-coloured bees headed by Carniolan queens.

Apiary setup containing mite donor colonies (MDR) and location of mite receptor colonies (MRC).

Peck and Seeley monitored mite acquisition by the acceptor hives over time, fighting and robbing dynamics, drifting workers (and drones) and colony survival.

Test a simple hypothesis

The underlying hypothesis on the relative importance of robbing or drifting for mite acquisition was this:

If drifting is the primary mechanism of mite transmission you would expect to see a gradual increase of mites in acceptor colonies. Since it is mainly bees on orientation flights that drift (and assuming the egg laying rate of the queen is constant) this gradual acquisition of motes would be expected to occur at a constant rate.

Conversely, if robbing is the primary mechanism of mite transmission from mite-infested to mite-free colonies you would expect to see a sudden increase in mite number in the acceptor hives. This would coincide with the onset of robbing.

Graphically this could (at enormous personal expense and sacrifice) be represented like this.

Mite acquisition by drifting (dashed line) or robbing (solid line) over time (t) – hypothesis.

X indicates the time at which the mite-free acceptor colonies are introduced to the environment containing the mite-riddled donor hives.

These studies were conducted in late summer/early autumn at Ithaca in New York State (latitude 42° N). The MDC’s were established with high mite loads (1-3 mites/300 bees in mid-May) and moved to the donor apiary in mid-August. At the same time the MRC’s were moved to their experimental locations. Colonies were then monitored throughout the autumn (fall) and into the winter.

So what happened?

Simplistically, the three mite donor colonies (MDC … remember?) all collapsed and died between early October and early November. In addition, by mid-February the following year four of the six MRC’s had also died.

In every case, colony death was attributed to mites and mite-transmitted viruses. For example, there was no evidence for starvation, queen failure or moisture damage.

But ‘counting the corpses‘ doesn’t tell us anything about how the mites were acquired by the acceptor colonies, or whether worker drifting and/or robbing was implicated. For this we need to look in more detail at the results.

Mite counts

Mite counts in donor (A) and receptor (B, C) colonies.

There’s a lot of detail in this figure. In donor colonies (A, top panel) phoretic mite counts increased through August and September, dropping precipitously from mid/late September.

This drop neatly coincided with the onset of fighting at colony entrances (black dotted and dashed vertical lines). The fact that yellow and black bees were fighting is clear evidence that these donor colonies were being robbed, with the robbing intensity peaking at the end of September (black dashed line). I’ll return to robbing below.

In the receptor colonies the significant increase in mite numbers (B and C) coincided with a) the onset of robbing and b) the drop in mite numbers in the donor colonies.

Phoretic mite numbers in receptor colonies then dropped to intermediate levels in October before rising again towards the end of the year.

The authors do loads of statistical analysis – one-way ANOVA’s, post-hoc Wilcoxon Signed-Rank tests and all the rest 3 and the data, despite involving relatively small numbers of colonies and observations, is pretty compelling.

Robbery

So this looks like robbing is the route by which mites are transmitted.

A policeman would still want to demonstrate the criminal was at the scene of the crime.

Just because the robbing bees were dark doesn’t ‘prove’ they were the Carniolans from the MRC’s 4. Peck and Seeley used a 400+ year old ‘trick’ to investigate this.

To identify the source of the robbers the authors dusted all the bees at the hive entrance with powdered sugar. They did this on a day of intense robbing and then monitored the hive entrances of the MRC’s. When tested, 1-2% of the returning bees had evidence of sugar dusting.

Returning robbers were identified at all the MRC’s. Numbers (percentages) were small, but there appeared to be no significant differences between nearby and distant MRC’s..

Drifting workers and drones

The evidence above suggests that robbing is a major cause of mite acquisition during the autumn.

However, it does not exclude drifting from also contributing to the process. Since the bees in the MDC and MRC were different colours this could also be monitored.

Yellow bees recorded at the entrances of the dark bee mite receptor colonies.

Before the onset of significant robbing (mid-September) relatively few yellow bees had drifted to the mite receptor colonies (~1-2% of bees at the entrances of the MRC’s). The intense robbing in late September coincided with with a significant increase in yellow bees drifting to the MRC’s.

Drifting over at least 50 metres was observed, with ~6% of workers entering the MRC’s being derived from the MDC’s.

If you refer back to the phoretic mite load in the donor colonies by late September (15-25%, see above) it suggests that perhaps 1% of all 5 the bees entering the mite receptor colonies may have been carrying mites.

And this is in addition to the returning robbers carrying an extra payload.

Since the drones were also distinctively coloured, their drifting could also be recorded.

Drones drifted bi-directionally. Between 12 and 22% of drones at hive entrances were of a different colour morph to the workers in the colony. Over 90% of this drone drifting was over short distances, with fewer than 1% of drones at the receptor colonies 50 or 300 m away from the donor apiary being yellow.

Discussion and conclusions

This was a simple and elegant experiment. It provides compelling evidence that robbing of weak, collapsing colonies is likely to be the primary source of mite acquisition in late summer/early autumn.

It also demonstrates that drifting, particularly over short distances, is likely to contribute significant levels of mite transmission before robbing in earnest starts. However, once collapsing colonies are subjected to intense robbing this become the predominant route of mite transmission.

There were a few surprises in the paper (in my view).

One of the characteristics of colonies being intensely robbed is the maelstrom of bees fighting at the hive entrance. This is not a few bees having a stramash 6 on the landing board. Instead it involves hundreds of bees fighting until the robbed colony is depleted of guards and the robbers move in mob handed.

As a beekeeper it’s a rather distressing sight (and must be much worse for the overwhelmed guards … ).

I was therefore surprised that only 1-2% of the bees returning to the mite receptor colonies carried evidence (dusted sugar) that they’d been involved in robbing. Of course, this could still be very many bees if the robbing colonies were very strong. Nevertheless, it still seemed like a small proportion to me.

It’s long been known that mites and viruses kill colonies. However, notice how quickly they kill the mite receptor colonies in these studies.

The MRC’s were established in May with very low mite numbers. By the start of the experiment (mid-August) they had <1% phoretic mites. By the following spring two thirds of them were dead after they had acquired mites by robbing (and drifting) from nearby collapsing colonies 7.

It doesn’t take long

The science and practical beekeeping

This paper confirms and reinforces several previous studies, and provides additional evidence of the importance of robbing in mite transmission.

What does this mean for practical beekeeping?

It suggests that the late-season colonies bulging with hungry bees that are likely to initiate robbing are perhaps most at risk of acquiring mites from nearby collapsing colonies.

This is ironic as most beekeepers put emphasis on having strong colonies going into the winter for good overwintering success. Two-thirds of the colonies that did the robbing died overwinter.

The paper emphasises the impact of hive separation. Drifting of drones and workers was predominantly over short distances, at least until the robbing frenzy started.

This suggests that colonies closely situated within an apiary are ‘at risk’ should one of them have high mite levels (irrespective of the level of robbing).

If you treat with a miticide, treat all co-located colonies.

However, drifting over 300 m was also observed. This implies that apiaries need to be well separated. If your neighbour has bees in the next field they are at risk if you don’t minimise your mite levels … or vice versa of course.

And this robbing occurred over at least 300 m and has been reported to occur over longer distances 8. This again emphasises both the need to separate apiaries and to treat all colonies in a geographic area coordinately.

Most beekeepers are aware of strategies to reduce robbing i.e. to stop colonies being robbed. This includes keeping strong colonies, reduced entrances or entrance screens.

But how do you stop a strong colony from robbing nearby weak colonies?

Does feeding early help?

I don’t know, but it’s perhaps worth considering. I don’t see how it could be harmful.

I feed within a few days of the summer honey supers coming off. I don’t bother waiting for the bees to exploit local late season forage. They might anyway, but I give them a huge lump of fondant to keep them occupied.

Do my colonies benefit, not only from the fondant, but also from a reduced need to rob nearby weak colonies?

Who knows?

But it’s an interesting thought …

Note there’s an additional route of mite transmission not covered in this or the last post. If you transfer frames of brood from a mite-infested to a low mite colony – for example, to strengthen a colony in preparation for winter – you also transfer the mites. Be careful.


Colophon

The idiom “Crime doesn’t pay” was, at one time, the motto of the FBI and was popularised by the cartoon character Dick Tracy.

Woody Allen in Take the Money and Run used the quote “I think crime pays. The hours are good, you travel a lot.”

Flower mites

Where do all those pesky mites come from that transmit pathogenic viruses in and between colonies?

Unless you are fortunate enough to live in the remote north west of Scotland 1 or the Isle of Man then bees, whether managed or feral, in your area have the parasitic mite Varroa destructor.

And if you take a mite-free colony from, say, north west Scotland and stick it in a field in Shropshire 2 it will, sooner or later, become mite-infested.

Sooner rather than later.

In our studies we see mite infestation (capped drone pupae with associated mites) within a few days of moving mite-free colonies to out apiaries.

Where did these mites originate and how did they get there?

Direct or indirect? Active or passive?

They don’t walk there.

Mites are blind and have no directional abilities over long distances.

Essentially therefore there are just two routes, both involving the host honey bee 3.

Direct, in which phoretic mites are transferred on honey bees between colonies, or indirect, in which they are transferred via something that isn’t a bee in the environment.

Like a flower.

Mite transmission routes

With an infested hive (the Donor) and a mite-free hive (the Acceptor 4) the direct routes involve the well-established processes of drifting and robbing.

As far as the acceptor hive is concerned, drifting is a passive process. The bees just arrive at the entrance and are allowed access.

In contrast, robbing is an active process by the acceptor hive. The foragers that rampage around pillaging weak colonies bring the phoretic mites back with them.

There have been two recent papers that have considered the relative importance of these routes and, in the case of indirect transmission, whether there is evidence that it can occur.

Both papers are from Thomas Seeley and colleagues at Cornell University. Seeley conducts simple and elegant experiments and, apart perhaps for the statistics, both papers are pretty readable, even without a scientific background.

I’ll deal with indirect transmission here and return to drifting and robbing in the future.

Say it with flowers … send her a mite

There is quite a bit of circumstantial evidence that horizontal transmission via flowers may occur. This includes evidence that mites can survive on flowers for several days (in the absence of bees). If ‘presented’ with live or dead bees these mites could then climb onto the bee.

But clambering aboard a dead bee held in a pair of tweezers is very different from boarding a live bee making a transient visit to a flower.

Like this.

This short video is by David Peck, the lead author on a 2016 manuscript on acquisition of mites by bees visiting flowers 5. The paper is open access and freely available so I’ll cut to the chase and just present the key details.

The mites and bees came from the same colony. Mites were harvested by sugar roll and placed on flower petals. Different flower species were baited with the same anise-flavoured sugar solution to make them equally attractive to foraging bees.

Video recording of bee visits enabled the scientists to determine whether the mite attached to a bee, if it was subsequently groomed off (in the vicinity of the flower) and how long any interaction took. The latter was measured in bee seconds i.e. the cumulative number of seconds a bee was present before the mite attached.

Mite transmission to bees from flowers

In 43 independent tests, using a total of three different flower species, every mite successfully managed to clamber onto a visiting bee. Of these, 41 left the flower with the bee (the two that didn’t fell off or were groomed by the bee).

Speed and efficiency

It took on average just two minutes of bee visits for the mite to climb aboard. In one test the mite successfully attached in just 2 seconds.

About 50% of the mites attached after the first contact with a bee. The average number of contacts needed was just over two (usually to the same bee).

We’ve all watched bees visiting flowers. They approach, orientate, land, take off again, reorientate, land again. Sometimes they walk across the inflorescence.

That’s all it takes.

The mites didn’t move about the flower much. They didn’t chase the bee around the flower. None moved more than 1 cm.

They simply waited for the bee to come close enough.

Mites haven’t got eyes but they have exquisitely sensitive chemosensory receptors on their forelegs (not four legs, they have eight 😉 ). They use these to detect the approaching bee and are then nimble enough to embark, as the video above shows.

Mites on daisy (Bellis sp.) or speedwell (Veronica sp.) relocated to a bee much more rapidly than those placed on an Echinacea flower. It’s not clear why – the flowers are larger on Echinacea so perhaps it’s something to do with the way a bee interacts with these when foraging?

Case proven m’lud?

Mites are transferred between colonies via flowers … it’s a fact.

Not quite.

What this study shows was that mites on flowers can readily attach to a visiting bee.

Specifically to a visiting bee from the same hive that the mite was ‘harvested’ from for the experiments.

Mites absorb the cuticular hydrocarbon profile of their host hive i.e. they smell like the bees do. Perhaps they were less readily detected by the visiting mite-free bee? Would they transfer to bees from a foreign colony less efficiently?

Conversely, host-parasite theory would suggest that the mite would have evolved mechanisms to preferentially infest ‘foreign’ visiting bees 6. At least they should if this route provided a suitable selective pressure, which would involve it providing an advantage to the mite (over other routes like robbing or drifting, for example). This remains to be tested.

But there’s something else missing until we can be certain that mites are transferred indirectly between colonies via flowers.

Have you ever seen a flower with a mite on it?

I haven’t either.

Which of course doesn’t help support or refute a role for flowers in mite transmission.

Absence of evidence is not evidence of absence.

A limited survey of flowers around apiaries also failed to detect Varroa 7 which is as little help as our own observations (see above).

So we’re left with half a story. Mites can transfer (quite efficiently) from flowers to bees. What we don’t know is whether – or how – they get from infested bees to the flower in the first place.

And if they do, whether it happens frequently enough to be of any real relevance as a mite transmission route between hives.

Next week I’ll revisit robbing and drifting as mite transmission routes to discuss some recent studies looking at their relative importance.

One last thing … one of the co-authors of the 2016 study described above is Michael L. Smith. In 2014 he published the honey bee sting pain index. I’m pleased to see he’s moved on to less painful scientific studies 🙂


Colophon

Flour mite (c) Joel Mills

The flour mite (Acarus siro), a distant relative of Varroa destructor, is a contaminant of grain and – unsurprisingly – flour which “acquires a sickly sweet smell and becomes unpalatable”.

Which isn’t a huge recommendation for Mimolette cheese. This cheese originates from Lille in France. It has a grey crust and an orange(ish) flesh, looking a bit like a cantaloupe. The crust hardens over time.

The appearance, the hardening (?) and certainly the flavour of the crust is due to the addition of flour mites (aka cheese mites) which are intentionally introduced during production of the cheese. Yummy.

Virus resistant bees?

In the early/mid noughties there was a lot of excitement about a newly discovered pathogen of honey bees, Israeli Acute Paralysis Virus (IAPV). This virus was identified and initially characterised in 2004 and, a couple of years later, was implicated as the (or at least a) potential cause of Colony Collapse Disorder (CCD) 1..

CCD is, and remains (if it still exists at all), enigmatic 2. It is an oft-misused term to describe the dramatic and terminal reduction in worker bee numbers in a colony in the absence of queen failures, starvation or obvious disease. It primarily occurred in the USA in 2006-07 and was reported from other countries in subsequent years 3.

Comparisons of healthy and CCD-affected colonies showed a correlation between the presence of IAPV and colony collapse, triggering a number of additional studies. In this and a future post I’m going to discuss two of these studies.

I’ll note here that correlation is not the same as causation. Perhaps IAPV was detected because the colony was collapsing due to something else? IAPV wasn’t the only thing that correlated with CCD. It’s likely that CCD was a synergistic consequence of some or all of multiple pathogens, pesticides, poor diet, environmental stress, migratory beekeeping, low genetic diversity and the phase of the moon 4.

IAPV

Israeli Acute Paralysis Virus is an RNA virus. That means the genome is made of ribonucleic acid, a different sort of chemical to the deoxyribonucleic acid (DNA) that comprises the genetic material of the host honey bee, or the beekeeper. The relevance of this will hopefully become clear later.

RNA viruses are not unusual. Deformed wing virus (DWV) is also an RNA virus as is Sacbrood virus and Black Queen Cell Virus. In fact, many of the most problematic viruses (for bees or beekeepers [measles, the common cold, influenza, yellow fever, dengue, ebola]) are RNA viruses.

RNA viruses evolve rapidly. They exhibit a number of features that mean they can evade or subvert the immune responses of the host, they can acquire mutations that help them switch from one host to another and they rapidly evolve resistance to antiviral drugs.

To a virologist they are a fascinating group of viruses.

IAPV isn’t a particularly unusual RNA virus. It is a so-called dicistrovirus 5 meaning that there are two (di) regions of the genetic material that are expressed (cistrons) as proteins. One region makes the structural proteins that form the virus particle, the other makes the proteins that allow the virus to replicate.

Schematic of the RNA genome of Israeli Acute Paralysis Virus

There are many insect dicistroviruses. These include very close relatives of IAPV that infect bees such as Acute Bee Paralysis Virus (ABPV) and Kashmir Bee Virus (KBV). They are very distant relatives of DWV and, in humans, poliovirus; all belong to the picorna-like viruses (pico meaning small, rna meaning, er, RNA i.e. small RNA containing viruses … I warned you about the Latin).

Phylogenetic relationships between picorna-like viruses

Like DWV, IAPV-infected bees can exhibit symptoms (shivering, paralysis … characteristic of nerve function or neurological impairment in the case of IAPV) or may be asymptomatic. The virus probably usually causes a persistent infection in the honey bee and is transmitted both horizontally and vertically:

  • horizontal transmission – between bees via feeding, direct contact or vector mediated by Varroa (not all of these routes have necessarily been confirmed).
  • vertical transmission – via eggs or sperm to progeny.

IAPV resistance

An interesting feature of IAPV is that some colonies are reported to be resistant to the virus. This is stated in an interesting paper by Eyal Maori 6 but, disappointingly, is not cited.

At the same time these studies were being conducted there was a lot of interest in genetic exchange between pathogens and hosts (e.g. where genetic material from the pathogen gets incorporated into the host) and an increasing awareness of the importance of a process called RNA interference (RNAi) in host resistance to pathogens 7.

Maori and colleagues screened the honey bee genome for the presence of IAPV sequences (i.e. a host-acquired pathogen sequence) using the polymerase chain reaction 8. About 30% of the bees tested contained IAPV sequences derived from the region of the genome that makes the structural proteins of the virus. Other regions of the virus were not detected.

Two additional important observations were made. Firstly, the IAPV sequences appeared to be integrated into a number of location of the DNA of the honey bee (remember IAPV is an RNA virus, so this requires some chemical modifications to be described shortly). Secondly, the IAPV sequences were expressed as RNA. This is significant because RNA is an intermediate in the production of RNAi (with apologies to the biologists who are reading this for the oversimplification and to the non-scientists for some of this gobbledegook. Bear with me.).

And now for the crunch experiment …

Virus challenge

Maori and team injected 300 white eyed honey bee pupae that lacked the integrated IAPV sequence with virus.

Only 2% survived.

They went on to inoculate a further 80 pupae selected at random. Thirteen of these survived (16%) and emerged as healthy-looking adults. The 67 corpses all showed evidence of virus replication and lacked the integrated IAPV sequence in the bee genome.

In contrast, the 13 survivors all contained integrated IAPV sequences but showed no evidence for replication of the virus.

This is of profound importance to our understanding of the resistance of honey bees to pathogens … and in the longer term for the selection or generation of virus-resistant bees.

If it is correct.

Subsequent studies

It’s of such profound importance that it’s extraordinary that there have been no subsequent follow-up papers (at least to my knowledge).

What there have been are number of outstanding but indirectly related studies that have demonstrated a potential mechanism for the integration of RNA sequences into a DNA genome.  We also now have a much improved understanding of how such integrated sequences could confer resistance to the host of the pathogen.

Perhaps the best of these follow-up studies is one by Carla Saleh 9 on the molecular mechanisms that underlie the integration of viral RNA sequences into the host DNA genome. This study also demonstrates how an acute virus infection of insects is converted to a persistent infection.

One of the big problems with the Maori study is explaining how RNA gets integrated. RNA and DNA are chemically similar but different. You can’t just join one to the other.

Saleh showed the an enzyme called an endogenous reverse transcriptase (an enzyme that converts RNA to DNA) was required. In the fruit fly virus model system she worked with she showed that this enzyme was made by a genetic element within the fruit fly genome (hence endogenous) called a retrotransposon.

Importantly, Saleh also showed that the integrated virus sequences acted as the source for interfering RNAs (RNAi) which then suppressed the replication of the virus.

The study by Saleh and colleagues is extremely elegant and explains much of the earlier work on integration of RNA pathogen sequences into the host genome.

However, it leaves a number of questions unanswered about the bits of IAPV that Maori claim are associated with virus resistance in honey bees.

Unfinished business

The Saleh study is really compelling science. Perhaps the same process operates in honey bees?

This is where issues start to appear. The honey bee genome has now been sequenced. Perplexingly (if the Maori study is correct) it contains few transposons and no active retrotransposons.

Without a source of the reverse transcriptase enzyme there’s no way for the RNA to be converted to DNA and integrated into the host genome.

The second major issue is that there are conflicting reports of the presence of viral sequences integrated in the honey bee genome. The assembled sequence 10 appears to contain no virus sequences but there are conference reports of sequences for IAPV, DWV and KBV using a PCR-based method similar to that used by Maori.

Where next?

There’s a lot to like about the Maori study on naturally transgenic bees (a phrase they used in the conclusion to their paper).

It explains the reported IAPV resistance of some bees/colonies (though this needs better documentation). It implicates a molecular mechanism which has subsequently been demonstrated to operate in a number of different insects and host/pathogen systems.

It’s also a result that as a beekeeper and a virologist I’d also like to think offers hope for the future in terms honey bee resistance to the pathogens that can blight our colonies.

Monoculture ... beelicious ...

Monoculture … beelicious …

However, the absence of some key controls in the Maori study, the lack of any real follow-up papers on their really striking observation and the contradictions with some of the genomic studies on honey bees is a problem.

What’s new?

Eyal Maori has a very recent paper (PDF) on RNAi transmission in honey bees. It was in part prompted by the second of the IAPV studies I want to discuss that arose after IAPV was implicated as a possible cause of CCD. That study, to be covered in a future post, demonstrates field-scale analysis of RNAi-based suppression of IAPV.

It is important for two reasons. It shows a potential route to combat virus infections and, indirectly, it emphasises the importance of continuing to properly control Varroa (and hence virus) levels for the foreseeable future.


 

Magic mushrooms not magic bullets

Bees are very newsworthy. Barely a week goes by without the BBC and other news outlets discussing the catastrophic global decline in bee numbers and the impending Beemaggedon.

These articles are usually accompanied by reference to Colony Collapse Disorder (CCD) and the apocryphal quote attributed to Albert Einstein “If the bee disappears from the surface of the earth, man would have no more than four years to live” 1.

They also generally illustrate news about honey bees with pictures of bumble bees … and conveniently overlook the global increase in honey bee colonies over the last 50 years.

Never let the truth get in the way of a good story 2

‘shrooms

And the story is particularly newsworthy if it includes the opportunity for a series of entirely predictable (but nevertheless amusing) puns involving mushrooms or fungi 3.

And for me, it is even better if it involves viruses.

It was inevitable I’d therefore finally get round to reading a recent collaborative paper 4 from Paul Stamets, Walter Sheppard, Jay Evans and colleagues. Evans is from the USDA-ARS Beltsville bee labs, Sheppard is an entomologist from Washington State University and Stamets is a really fun guy 5, an acknowledged mushroom expert and enthusiast, award-winning author 6 and advocate of mushrooms as a cure for … just about anything. Stamets is the founder and owner of Fungi Perfecti, a company promoting the cultivation of high-quality gourmet and medicinal mushrooms.

An an aside, you can get a good idea of Stamets’ views and all-encompassing passion for ‘shrooms by watching his YouTube video on the Stoned Ape [hypothesis] and Fungal Intelligence.

Fungi and viruses

It has been shown that extracts of fungi can have antiviral activity 7, though the underlying molecular mechanism largely remains a mystery (for a good overview have a look at this recent review in Frontiers in Microbiology by Varpu Marjomäki and colleagues). I’m not aware of any commercial antivirals derived from fungi 8 and none that I’m aware of are in clinical trials for human use.

Stamets cites his own observations of honey bees foraging on mycelia (the above-ground fruiting body we call ‘mushrooms’) and speculates that this may be to gain nutritional or medicinal benefit.

Shrooms

Mushroom

This seems entirely reasonable. After all, bees collect tree resins to make propolis, the antimicrobial activity of which may contribute to maintaining the health of the hive.

I’ve not seen bees foraging on fungi, but that certainly doesn’t mean they don’t.

Have you?

Whatever … these observations prompted the authors to investigate whether mushroom extracts had any activity against honey bee viruses.

Not just any viruses

Specifically they tested mushroom extracts against deformed wing virus (DWV) and Lake Sinai Virus (LSV).

DWV is transmitted by Varroa and is globally the most important viral pathogen of honey bees. It probably accounts for the majority of overwintering colony losses due to a reduction in longevity of the fat bodied overwintering bees.

LSV was first identified in 2010 and appears to be widespread, at least in the USA. It has also been detected in Europe and is a distant relative of chronic bee paralysis virus. It has yet to be unequivocally associated with disease in honey bees.

Not just any ‘shrooms

Mycelial extracts were prepared from four species of fungi. As a lapsed fly fisherman I was interested to see that one of those chosen was Fomes fomentarius, the hoof fungus which grows on dead and dying birch trees. This fungus, sliced thinly, is the primary ingredient of Amadou which is used for drying artificial flies 9.

Hoof fungus … and not a honey bee in sight.

Mycelial extract preparation took many weeks and generated a solution of ethanol, aqueous and solvent soluble mycelial compounds together with potentially contaminating unused constituents from the growth substrate. This was administered in thin (i.e. 1:1 w/v) sugar syrup.

Don’t just try hacking a lump off the tree and placing it under the crownboard 😉

Results

In laboratory trials all the fungal extracts reduced the level of DWV or LSV in caged honey bees by statistically significant amounts.

Unfortunately (at least for the layman trying to comprehend the paper) the reductions quoted are n-fold lower, based upon an assay called a quantitative reverse transcription polymerase chain reaction. Phew! It might have been preferable – other than it being appreciably more work – to present absolute reductions in the virus levels.

Nevertheless, reductions there were.

Encouragingly they were generally dose-dependent i.e. the more “treatment” added the greater the reduction. A 1% extract of hoof fungus in thin syrup reduced DWV levels by over 800-fold. Against LSV the greatest reductions (~500-fold) were seen with a different extract. In many cases the fold change observed were much more conservative i.e. less activity (though still statistically significant).

A) Normalised DWV and LSV levels in individual bees. B) Activity of mushroom extracts against LSV.

These lab studies encouraged the authors to conduct field trials. Five frame nucleus colonies were fed 3 litres of a 1% solution of one of the two most active extracts. Virus levels were quantified 12 days later. Control colonies were fed thin syrup only.

These field trials were a bit less convincing. Firstly, colonies fed syrup alone exhibited 2- to 80-fold reduction in DWV and LSV levels respectively. Against DWV the fungal mycelial extracts reduced the level of the virus ~40-fold and ~80-fold better than syrup alone. LSV levels were more dramatically reduced by any of the treatments tested; ~80-fold by syrup alone and ~90-fold or ~45,000-fold better than the syrup control by the two mycelial extracts.

Or is it any ‘shrooms … or ‘shrooms at all?

It’s worth emphasising that syrup-alone is not the correct control for use in these studies. As stated earlier the mycelial extract likely also contained constituents from the fungal growth media (sterilised birch sawdust).

The authors were aware of this and also tested extracts prepared from uninoculated birch sawdust. This definitely contained endogenous fungal contamination as they identified nucleic acid from ‘multiple species’ of fungi in the sterilised sawdust, the majority from three commonly birch-associated fungi (none of which were the original four species tested).

The authors are a little coy about the effect this birch sawdust extract had on virus levels other than to say “extracts from non-inoculated fungal growth substrate also showed some activity against DWV and LSV”. In lab studies it appears as though ‘some activity’ is between 8- and 120-fold reduction.

Without some additional controls I don’t think we can be certain that the compound(s) responsible for reducing the viral levels is even derived from the mushroom mycelium, whether the endogenous ones present in the sawdust, or those grown on the sawdust.

For example, perhaps the active compound is a constituent of birch sawdust that leaches out at low levels (e.g. during the extraction process) but that is a released in large amounts when fungi grow on the substrate?

Hope or hype?

Readers with good memories may recollect articles from fifteen years ago about fungi with activity against Varroa. In that case the fungus was Metarhizium anisopliae. There are still groups working on this type of biological control for mites but it’s probably fair to say that Metarhizium has not lived up to its early promise 10.

A lot more work is needed before we’ll know whether mushroom extracts have any specific activity against honey bee viruses. There are lots of unanswered questions and it will take years to have a commercial product for use by beekeepers.

Don’t get rid of your stocks of oxalic acid or Apivar yet!

Questions

What are the active ingredient(s) and mode of action?

Do the extracts actually have any activity against the viruses per se, or do they instead boost the immune response of the bee and make it better to resist infection or clear established infections?

How specific are the extracts? Do they have activity against other RNA viruses of honey bees? What about Nosema? Or the foulbroods? If they boost immune responses you’d expect a broad range of activities against bee pathogens.

You’d also expect that bees would have evolved to actively forage on mushroom fruiting bodies and so be a common sight in late summer/early autumn.

Are they toxic to bees in the longer term? Are they toxic for humans? Fomes formentarius is considered “inedible, with a slightly fruity smell and acrid taste”. Delicious!

Finally, is the reduction in virus levels observed in field studies sufficient to have a measurable positive influence on colony health? It’s worth remembering that Apivar treatment reduces mite levels by 95% and virus levels by about 99.9999%.


Colophon

Magic!

Magic mushroom is a generic term used to refer to a polyphyletic group of fungi that contain any of various psychedelic compounds, including psilocybin, psilocin, and baeocystin. Talk to Frank to find out more about the effects and dangers of magic mushrooms. The de facto standard guide for the identification of magic mushrooms is Psilocybin Mushrooms of the World by … you guessed it, Paul Stamets.

The term magic mushroom was first used in Life magazine in 1957.

A magic bullet is a highly specific drug or compound which kills a microbial pathogen without harming the host organism. The term (in German, Zauberkugel) was first used by Nobel laureate Paul Ehrlich in 1900. Ehrlich discovered/developed the first magic bullet, Salvarsan or Arsphenamine, an organoarsenic compound that is effective in the treatment of syphilis.

Mycelial extracts of fungi are not (yet at least) a magic bullet for use in the control of honey bee viruses.