Tag Archives: virus resistance

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.


 

Leave and let die

If you follow some of the online discussions on Varroa you’ll see numerous examples of amateur beekeepers choosing not to treat so as to ‘select for mite-resistant bees’.

For starters it’s worth looking at the ‘treatment-free’ forums on Beesource.

DWV symptoms

DWV symptoms

The principle is straightforward. It goes something like this:

  • Varroa is a relatively new 1 pathogen of honey bees who therefore naturally have no resistance to it (or the viruses it transmits).
  • Miticide treatment kills mites, so favouring the survival of bees.
  • Consequently, traits that confer partial or complete resistance to Varroa are not actively selected for (which would otherwise happen if an untreated colony died out).
  • Treatment is therefore detrimental, at the population level if not the individual level, to the development of Varroa-resistant bees.
  • Therefore, don’t treat and – with a bit of luck – a resistant strain of bees will appear.

A crude oversimplification?

Yes, I don’t deny it.

There are all sorts of subtleties here. These range from the open mating of queens, isolation of apiaries, desirable traits (with regards to both disease resistance and honey production 2), livestock management ethics, our responsibilities to other beekeepers and other pollinators. I could go on.

But won’t.

Instead I’ll discuss a short paper published in the Journal of Apicultural Research. It’s not particularly novel and the results are very much in the “No sh*t Sherlock” category. However, it neatly emphasises the futility of the ‘do nothing and expect evolution to find a solution’ approach.

But I’ll start with a simple question …

How many colonies have you got?

One? (in which case, get another)

Two?

Ten?

One hundred?

Eight-two thousand? 3

Numbers matters because evolution is a numbers game. The evolutionary processes that result in alteration of genes (the genotype of an organism) that confer different traits or characteristics (the phenotype of an organism) are rare.

For example, viruses are some of the fastest evolving organisms and, during their replication, mutations (errors) occur at a rate of about 1 in 104 at the genetic level 4.

This is why we treat ...

This is why we treat …

But so-called higher organisms (like humans or bees) have much more efficient replication machinery and make very many fewer errors. A conservative figure for bees might be about 10,000 times less than in these viruses (i.e. 1 in 108), though it could be as much as a million times less error-prone 5

There are lots of other evolutionary mechanisms in addition to mutation but the principle remains broadly the same. The chance changes that are acquired by copying or mixing up genetic material are very, very infrequent.

If they weren’t, most replication would result – literally – in a dead end.

OK, OK, enough numbers … what about my two colonies?

So, since the evolutionary mechanisms make small, infrequent changes, the chance of a beneficial change occurring is very small. If you start with small numbers of colonies and expect success you’re likely to be disappointed.

Where ‘likely to be’ means will be.

The chances of picking the Lotto jackpot is about 1 in 45 million for each ticket purchased. If you expect to win you will be disappointed.

It could be you … but it’s unlikely

If you buy two tickets (with different numbers!) your chances are doubled. But realistically, they’re still not great 6.

And so on.

Likewise, the more colonies you have, the more likely you’ll get one that might – by chance – acquire a beneficial mutation that confers some level of resistance to Varroa.

Of course, we don’t really know much about the genetic basis for resistance (or tolerance?) to Varroa in honey bees. We know that there are behavioural changes that increase survival. We also know that Apis cerana can cope with Varroa because it has a shorter duration replication cycle and exhibits social apoptosis.

There are certainly ‘hygienic’ and other traits in bees that may be beneficial, but at a genetic level I don’t think we know the number of genes that are altered to confer these, or how much each might contribute.

So we don’t know how many mutations will be needed … One? One hundred? One thousand?

If the benefit of an individual mutation is very subtle it might offer relatively little selective advantage, which brings us back to the numbers again.

Apologies. Let’s not go there.

Let’s cut to the chase …

Comparison of treated vs untreated colonies over 3 years

Miticides – whether hard chemicals like Amitraz or Apistan or organic acids like formic or oxalic acid – work by exhibiting differential toxicity to mites than to their host, the bee. They are not so specific that they only kill mites. They can harm other things as well … e.g. if you ingest enough oxalic acid (5 – 15g) it can kill you.

Amitraz

Amitraz …

Jerzy Wilde and colleagues published their study 7 comparing colonies treated or untreated over a three year period. The underlying question addressed in the paper is “What’s more damaging, treating with potentially toxic miticides or not treating at all?”

The study was straightforward. They started with 100 colonies, requeened them and divided them randomly into 4 groups of 25 colonies each. Three received treatment and one was a control.

The ‘condition’ of the colonies was measured in a variety of ways, including:

  • Colony size in Spring (number of combs occupied)
  • Nosema levels (quantified by numbers of spores)
  • Mite drop over the winter (dead mites per 100g of ‘hive debris’)
  • Colony size in autumn (post-treatment) and egg laying rate by the queen
  • Winter losses

The last one needs some explanation because in one group (guess which?) there were more winter losses than they started the experiment with.

Overwintering colony losses were made up from splits of colonies in the same group the following year, so that each year 25 colonies went into the winter i.e. surviving colonies were used to generate additional colonies for the same treatment group.

Treatment and seasonal variation

To add a little complexity to the study the authors compared three treatment regimes:

  1. Hard chemicals only – active ingredients amitraz or the pyrethroid flumethrin (the research group are Polish, so the particular formulations are those licensed in Poland – Apiwarol, Bayvarol and Biowar).
  2. Integrated Pest Management (IPM) – a range of treatments including Api Life Var (primarily a thymol-based treatment) in spring, drone brood removal early/mid season, hard chemical or formic acid in late summer/autumn and oxalic acid in midwinter.
  3. Organic (natural) treatments only – Api Life Var in spring, the same or formic acid in late summer and a midwinter oxalic acid treatment.

The fourth group were the untreated controls.

To avoid season-specific variation they conducted the experiment over three complete seasons (2010-2012).

The apiary in winter ...

The apiary in winter …

The results of the study are shown in a series of rather dense tables with standard deviation and statistic significance … so I’ll give a narrative account of the important ones.

Results …

The strength of surviving colonies in Spring was unaffected by prior treatment (or absence of treatment) but varied significantly between seasons. In contrast, late summer colony strength was significantly worse in the untreated control colonies. In addition, the number of post-treatment eggs laid by the queen was significantly lower (by ~30%) in untreated control colonies 8.

Remember that early autumn treatment is needed to reduce Varroa infestation and so protect the winter bees that are being reared at this time from the mite-transmitted viruses.

Out, damn'd mite ...

Out, damn’d mite …

The most dramatic effects were seen in winter losses and (unsurprisingly) mite counts.

Mites were counted in the hive debris falling through the open mesh floor during the winter. In the first year the treated and untreated controls had similar numbers of mites per 100g of debris (~12). In all treated colonies this remained about the same in each subsequent season. Conversely, untreated controls showed mite drop increasing to ~43 in the second year and ~114 in the final year of the study.

During the three years of the study 30 untreated colonies died. In contrast, a total of 37 colonies from the three treatment groups died.

The summary sentence of the abstract to the paper neatly sums up these results: 

Failing to apply varroa treatment results in the gradual and systematic decrease in the number of combs inhabited by bees and condition of bee colonies and consequently, in their death.

… and some additional observations

Other than oxalic acid, none of the treatments used significantly affected the late season egg laying by the queen. Api Life Var contains thymol and many beekeepers are aware that the thymol in Apiguard quite often stops the queen from laying. Interesting …

I commented last week on queen losses with MAQS. In this Polish study, 8 of 50 colonies treated with formic acid suffered queen losses.

In the third season (2012) 45% of the 100 colonies died. More than half of these lost colonies were in the untreated controls. In contrast, overall colony losses in the first two years were only 9% and 13%. Survival of untreated colonies for a year or two is expected, but once the Varroa levels increase significantly the colony is doomed.

Overall, colonies receiving integrated pest management or hard chemical treatment survived best.

Evolution …

March of Progress

Evolution …

Remind yourself where the colonies came from that were used to make up the losses in the treatment (or control) groups … they were splits from colonies within the same group. So, colonies that survived without treatment were used to produce more colonies to not be treated the following season.

Does this start to sound familiar?

Jerzy Wilde and colleagues started with 25 colonies in the untreated group. They lost 30 colonies over a 3 year period and ended up with just two colonies. Had they wanted to continue the study they would have been unable to recover their losses from these two remaining colonies.

If you don’t treat you must expect to lose colonies.

Lots of colonies.

Actually, almost all of them.

… takes time

This study lasted only three years. That’s not very long in evolutionary terms (unless you are a bacterium with a 20 minute replication cycle). 

It would be unrealistic to expect Varroa resistance to almost spontaneously appear. After all, there are about 91 million colonies worldwide, the majority of which are in countries with Varroa. Lots of these colonies will not be treated. If it was that easy it would have happened many times already.

What happens when you start with more colonies and allow more time to elapse?

Well, this ‘experiment’ has been done. There are a number of regions that have well-documented populations of feral honey bees that are living with, if not actually resistant to, Varroa.

One well known population are the bees in the Arnot Forest studied by Thomas Seeley. These bees have behavioural adaptations – small, swarmy colonies – that lessen the impact of Varroa on the colony 9.

Finally, returning to the title of this post, there is the so-called “Bond experiment” conducted on the island of Gotland in the Baltic Sea. Scientists established 150 colonies of mite-infested bees and let them get on with it with no intervention at all. Over the subsequent six years they followed the co-evolution of the mite and the bee 10.

It’s called the “Bond experiment” or the Live and Let Die study for very obvious reasons.

Almost all the colonies died.

Which is why the title of this post is more appropriate for those of us with only small numbers of colonies.