In 2004 a group of researchers in Japan published a study in Journal of Virology (1, here) suggesting that they had discovered a new virus that was only found in specific areas of the brains of aggressive honeybees, but not more docile ones.  When I came across it I was certain that it would make a great episode for the virus podcast,  this wouldn’t be the first report of a virus affecting the behavior, but as far as I know it was the first about a virus affecting bee behavior.  Combine the general misperception that viruses are always a bad thing with the (correct) perception that aggressive stinging insects are scary – you would think that would make a great episode.  But alas, it has led me on an years-long deep dive/rabbit hole that started with a simple question: is this really a new virus?  I have posted a few times before about the deeper depths of this rabbit hole, but not about the paper that started me on this dig.

I have the perfect sound effect ready for when I do make an episode on this because in order to separate the aggressive bees from the more docile ones the researchers tied a live hornet  and dangled it in front of a honeybee hive entrance.  Some of the bees aggressively attacked this lure (there’s the sound effect) while others fled.  Actually, the scientists write that “Some of the bees (attackers) scrambled and grappled with the hornet obstinately, shaking their wings and beating their abdomens.” and on a side note, part of me wants to start a whole nother podcast on wonderful adverbs and adjectives in scientific papers.  Scientists are frequently taught to boil their prose to the point where the flavor is gone, and its refreshing when an unnecessary yet juicy adverb like “obstinately” is left in.

After dissecting and making a goo out of a specific part (mushroom bodies) of the brains of each group (aggressive and docile), the researchers used a technique called “differential display” to identify RNAs that are present in one of these groups and not the other.  One RNA they find specifically in the brains of aggressive bees and so they name it “Kakugo RNA” for the Japanese “ready to attack”.  This wouldn’t mean much if it only came from one hive, so they repeat the entire procedure with another hive and get the same results.

The sequence of this RNA turns out to have a high degree of similarity to various picorna-like viruses, specifically a group called the deformed wing viruses. Other picorna virus that you might have heard of include polio virus, hepatitis A virus and the first animal virus ever discovered: foot and mouth disease virus (great episode).  One trivial explanation for these results might be that this RNA is coded by DNA in the honeybee genome, and perhaps their results are explained by differential transcription of this hypothetical gene – but by southern blot they can find no such gene.   They also perform RT-PCR for Kakugo in nurse bees, attacker bees and forager bees and only find it in the attackers, specifically in their brains and not the head, thorax or abdomen.  The lack of detection in the head is odd, perhaps these were the heads minus the brains?

If Kakugo is indeed a virus you would expect that at least some of these RNAs would be found inside virions, so they layer bee brain lysates onto a sucrose gradient (essential placing it on top of a solution of sucrose that gets more and more concentrated towards its bottom) and centrifuge.  Virions are more dense than free nucleotides and will penetrate lower into that tube during centrifugation; to facilitate the detection of the virus they dope the lysates with polio virus (which should have the same physical characteristics).  They do find Kakugo RNA lower in the tube at the same location as the polio virus.

If Kakugo virus is indeed a virus you would expect it to be capable of infecting other bees, so somehow they identified uninfected worker bees and injected one microliter of goo from infected attacker bee heads.  They do see an increase in Kakugo virus RNA after three days by RT-PCR but oddly they don’t measure beyond that.  The average level of increase on day three is just below two fold, with a pretty wide variation (almost zero to six fold).  No mention is made of any behavioral changes by the presumably infected bees, and unfortunately the researchers hint that these presumably infected bees died 3 days after the injection by writing “The workers inoculated with attacker head lysate or PBS were viable for at least three days”.  “At least three days” suggests to me that even the injection of buffer (phosphate buffered saline “PBS) results in death after 3 days.  Certainly these results are suggestive that maybe these RNAs are replicating, but the result doesn’t look reproducible/consistent bee to bee

This paper feels a lot like most of my results in the lab with adenovirus: I had lots of suggestive evidence that would support various hypothesis to super-interesting questions, but none felt like a slam dunk.  That’s how science frequently feels – slam dunks are rare.

A later paper in 2006 (2) would go on to show that the presence of deformed wing viruses isolated from honeybees in Cyprus did not correlate with aggressiveness and would point out that Kakugo virus is so closely related to DWV (they cite 98% identical at the nucleotide level) –> so close that they note that it was impossible to design RT-PCR primers that would allow they to distinguish the two.   I just did a BLAST in NCBI and Kakugo virus is 99% identical to a sequence isolated from the whole body of bees in Sweden in 2018 that the depositers named “deformed wing virus”, not “Kakugo virus” – so it appears, at the very least, that Kakugo is perhaps a variant of DWV and that other researchers haven’t started using the Japanese name.

Is this a new virus?  It would be really if there was a virus that was different from deformed wing virus that made honeybees aggressive, but looking at all the information suggests that probably its just a variant of deformed wing virus.

References

(1) Fujiyuki et al 2004 Journal of Virology “Novel insect picorna-like virus identified in the brains of aggressive worker honeybees”

(2) Rortais et al 2006 Virology Journal “Deformed wing virus is not related to honey bee’s aggressiveness”