Know your place - the genetics (and epigenetics) of bee societies

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Back in episode 17 of this series, How to be a queen bee, Sally Le Page took us on a journey to the heart of the hive, exploring how bee societies work and what it takes to become a queen. There’s much more to explore about these fascinating societies, and the genetics that underpin them so I thought I’d take a closer look to see what the buzz is all about…

Bees have long fascinated geneticists. These eusocial insects live in highly ordered societies, with distinct roles, or castes, within them - a place for every bee, and every bee in its place. But how does a hive of genetically near-identical individuals end up diversifying into such different roles? This is most obvious for female bees, which can become either one of many short lived sterile workers or one reigning reproductive queen, along with male drones. And even within this female workforce, there are different jobs to be done, from foraging for food to protecting the hive or raising young.

As Sally explained in her episode, we now know that the journey to becoming a queen bee starts early on in life, and depends on what baby bees are fed. Princesses - those larvae destined to become queens - are fed on royal jelly, while larvae that will become worker bees get bee bread. But rather than royal jelly being the trigger to become a queen, it’s the absence of bee bread that sets her on a regal path, as plant chemicals present in the bee bread suppress the development of reproductive organs - a groundbreaking discovery made in 2015 from May Berenbaum and her team. But how does this actually work?

Now, if you’ve been paying attention to these podcasts over the past few years, you might recognise what’s going on here - where individuals, whether that’s animals or cells, that have the same genome end up specialising into distinct roles through the influence of factors in their environment. Yes folks, it’s epigenetics!

DNA methylation and bee behaviour

Long before Berenbaum’s discovery about bee bread and royal jelly, researchers had been digging into the bee genome in search of clues to their different fates. One of the most obvious telltale signs they were looking for was DNA methylation: small chemical tags that are put on or removed from certain ‘letters’ of DNA and are associated with patterns of gene activity - switching genes on or off.

There’s a long and complicated history of research into DNA methylation and how it might be influenced by diet - not just in bees but in other animals and humans - and whether these marks are controlling gene activity or just reflecting patterns that are established by other means, which we don’t have time to go into here, but there’s more in my book Herding Hemingway’s Cats and episode 15 from series 3 of the podcast - Pimp my genome: the wonderful world of epigenetics if you’re interested.

One of the most famous examples of this phenomenon in mammals is the case of Agouti mice, which come in a range of colours and sizes, from brown and slim to yellow and fat, despite being genetically identical. Changing the amount of the nutrient folate, which alters DNA methylation patterns, in a pregnant Agouti mouse’s diet changes the appearance of her pups, neatly demonstrating how a change in diet might send individuals down different paths, despite having the same genes. So, it would make sense that feeding baby bees a different diet might be altering their DNA methylation, with implications for their gene activity patterns and subsequent development and behaviour.

Back in the noughties, when epigenetics started to become really cool, researchers didn’t have good tools to look at patterns of DNA methylation across the whole genome, let alone get good enough data to compare between queen bees and workers. Instead, researchers led by Ryszard Maleszka at the Australian National University in Canberra came at the problem from the other direction. In 2008 they published a study using a clever technique called RNA interference, or RNAi - using injections of RNA to shut down the activity of an enzyme called DNA methyltransferase 3, or Dnmt3, which is responsible for adding methylation marks to DNA during development. 

Switching off Dnmt3 in female larvae resulting in nearly three quarters of them turning out as queens - far more than would be expected - showing that DNA methylation is definitely playing some kind of role in the switch from worker to queen, mediated by diet. As they say in their paper, “Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioural status.“

Becoming Queen Bee

Over the past decade or more, researchers have been trying to get to the bottom of what makes a queen a queen, epigenetically speaking. By 2010, Maleszka and his team were able to look at DNA methylation across the whole honeybee genome, finding highly distinctive patterns in queens and workers at more than 550 genes, although this has been contradicted by other research showing no difference in methylation between workers and queens. Today, the latest deep sequencing techniques are able to look at methylation patterns in exquisite detail, in ever smaller groups of cells, shedding new light on the connections between epigenetic marks, gene activity and honeybee behaviour. 

In 2018, Paul Hurd at Queen Mary University of London - actually an old buddy from my PhD days - teamed up with Maleszka to go even deeper into the bee genome. They used the latest techniques to map patterns of modifications on histones - these are ball-shaped proteins that DNA wraps around inside the cell, and affect how it is packed and unpacked in order to be read or silenced, and are another key part of epigenetic gene regulation. As might be expected, they found key differences at specific genes between workers and queens, helping to pinpoint specific genetic switches within DNA that control the fate of a developing female bee. 

Curiously, these differences in behaviour between workers and queens may be due to differences in their brains. In 2021, Paul Hurd and his collaborators made an intriguing accidental discovery. They had originally set out to find an antibody that could bind to the bee version of ten-eleven translocation methylcytosine dioxygenase (or TET for short). In mammals, there’s a family of TETs, which are involved in many things in cells including DNA demethylation and controlling networks of gene activity. Seeing as bees only have one version, it should have been a fairly easy task to find an antibody that bound to it. Unfortunately the antibody the team made didn’t bind to TET - curses! Instead, it locked on to an entirely unknown protein, which was produced in bee brains. 

So far, so interesting. But what made this really cool was what they saw when they used fluorescence microscopy to look at where this new protein was located in the brains of bees, comparing male drones, female workers, queens, and larvae at various stages. The antibody lit up a strange rod-like structure inside the nuclei of specific brain cells known as Kenyon cells, but only in drones and workers, with the strongest pattern seen in foraging workers and queens having a much more sporadic pattern. But worker larvae didn’t seem to have any of the protein at all, suggesting that these unusual structures form as their brains develop into adulthood - so perhaps they have something to do with the way in which honeybees brains can grow and change as they mature and learn?

What kind of bee should you be?

Aside from the major differences between queens and workers, epigenetic changes have also been implicated in defining roles within the honeybee working class. Nurse bees get busy in the hive making honeycombs and raising the babies while foragers protect the hive and go out in search of food. Generally, bees are nurses when they’re younger, and then become foragers as they mature. Again, as you might expect, there are differences in DNA methylation between these two groups. But these aren’t fixed. A study in 2012 showed that DNA methylation patterns in foraging bees that are forced to switch roles and go back to stay in the hive as nursemaids also change to look like those of nurses - apparently the “the first evidence in any organism of reversible epigenetic changes associated with behaviour.”

So, while the genes encoding all kinds of bee behaviour and appearance are encoded within the bee genome, it’s the epigenetic state that determines which are active, and therefore what kind of bee a bee can be. As Gro Amdam, one of the researchers, puts it: "It's like one of those pictures that portray two different images depending on your angle of view. The bee genome contains images of both nurses and foragers. The tags on the DNA give the brain its coordinates so that it knows what kind of behaviour to project."

Generational protection

There’s a final intriguing sting in this tale. In 2019, researchers at the University of Cambridge and the Hebrew University of Jerusalem, led by Eyal Maori, uncovered evidence for an even weirder - and cooler - epigenetic phenomenon at work in honeybees. The story starts in 2009, when the scientists were testing a potential way of protecting honeybees from infection with Israeli Acute Paralysis Virus (IAPV), which can cause bee colonies to collapse. The treatment was a form of RNA interference, similar to the experiments I described earlier, and involved getting bees to eat RNA designed from the virus, which would silence the virus by interfering with its gene activity, protecting the bees from infection.

Not only did it work, the hives with bees that were treated with the RNA produced more honey. And, strangely, this effect carried on at least 3 to 4 months after the treatment stopped, when the bees should have died and been replaced. In their 2019 study, the researchers showed that the protective RNA was being transmitted down the generations through royal jelly, passing from one queen to the next. Not only that, but royal jelly also contained a bunch of naturally-occurring RNAs, which look like they should be able to switch off genes in honey bees - potentially contributing to making a queen - as well as infectious agents like viruses, bacteria and fungi, acting as some kind of inherited immune defence for the community. 

There are still plenty of questions about how this works - not least about how a molecule as fragile as RNA can survive in royal jelly and the unfriendly environment of a bee’s belly. But, if this research is to be bee-lieved, it’s a fascinating further layer of complexity in the honeybee’s story. 

All very fascinating, and keeping entomologists busy I’m sure. But it’s important we don’t forget the bigger picture. Not only are bees fascinating for their complex society and intriguing epigenetics, they’re an absolutely essential part of the ecosystem, pollinating the plants that feed us. It’s not quite ‘no bees, no food’, but the impact on global farming and the planet would be devastating. So we need to make sure we keep and eye on the bees and their bee-haviour, because if they all buzz off, we’re going to be in trouble.