“Men are from Venus, women are from Mars.” Or is it the other way around? No matter. The point is, people communicate, and therefore think, differently. I often think differently than others, probably because I’m a science geek. I also appreciate, and take advantage of, the different thoughts of others. If you read my article “A bee-driven mid-life crisis” in the January issue of Bee Culture, you know I’m struggling with a lot of questions. I’m newly retired and transitioning into a full-time beekeeper. Questions like, how big do I want to be? Where am I going to put all my stuff? What stuff do I really need? What’s my business model? And the list goes on. But the backbone of a bee business is, the bees. The backbone of the bees is their genetics. I’ve been thinking a lot about that lately. How do I want to handle bee genetics?

Every queen rearing class I’ve taken, every book I’ve read and every YouTube video I’ve watched always talks about selecting the best hive for the queen mother. To me, that sounds like a great strategy for creating genetic bottle necks. Think inbreeding, not good. No one ever talks about what it takes to maintain genetic diversity. Last year, I requeened my 150 colonies with queen cells from two queen mothers. Having the source of my entire apiary traced back to two queens could be a genetic nightmare. What saves me is the queens were mated over two counties. The diversity comes in from the drones. But here’s the catch, if you are selecting for traits, the males need to be selected too. By putting the same selection pressure on the drones as you do the queen, you are minimizing diversity.

Why do I care? First, a genetically diverse population is better prepared to handle stressors. As an example, look at COVID. Some people died while others were asymptomatic. A very complex situation, but genetics plays a part. So, my mantra is that genetic diversity is good. I have been contemplating applying to the HILO queen project (www.HiloBees.com) to help them develop their mite tolerant trait. But here’s the thing. It’s a multigenic, recessive trait. In order to be effective, 50% of the drones need to have the traits. If I want to get to a point where I can propagate these queens (getting HILO project permission first, of course), then I must be in an isolated area where the only drones available for mating must be heavily selected for the trait. This situation is a step away from incest. This same argument holds true if I want my bees to be true Caucasians, or true Carniolans. Is this a good thing? In my way of thinking, my mutt bees are better, in the long run, than a highly selected bee stock.

In January this year I got the following text from John Miller of Miller Honey Farms. “I read your piece in BC. It’s time to experience almond bloom. I’d love to host you in the Modesto area in late February. Do a day or two there. Then head north to look at a couple of queen operations.” Perfect. What better way to figure out my future and address my questions than to spend a week hob-knobbing with the experts. For my genetic diversity question, I go back to the “Then head north to look at a couple of queen operations.” This is my chance to get opinions from a couple of the top queen producers in the country. Here’s what I wanted to know. First, are today’s queens better than the queens from 40 years ago? The premise of this question is simple, are current bee breeding practices making better bees? As an example, look at corn. Yields are higher and pest resistance is better so one could argue that corn breeding programs are successful. Not so obvious with bees and I wanted to hear what the experts had to say. Next, I wanted to know how they selected for traits and how did they insure genetic diversity. My hope was to get into some geeky conversations regarding drone:queen mother ratios and population genetic models. Here’s what I learned.

First off, these operations are phenomenal. If you are putting out hundreds of thousands of queens per year, you need a system and you need to know what you are doing. It really put me in my place. I was happy with my 150 queen cells last year. Hah! They are doing a thousand a day and running tens of thousands of mating nucs. This is in the heart of queen production in CA, the so-called golden triangle. Bees are everywhere.

Open mating with multiple drones complicates the propagation of a trait as simple as body color. It makes breeding for complex traits nearly impossible. Instrumental insemination can make breeding doable in small scale. But traits are soon lost with
uncontrolled open mating. How often do you see different colored bees in your hives?

I was a little nervous about asking my first question as it could be construed as an “attack” on their livelihood. But the conversation that ensued was fabulous. “Are queens better now than when you first started or, are we just maintaining quality?” First, it’s a difficult question because environmental stressors have changed over time. So, the question resembles the “apples to oranges” comparison. In general, they have been maintaining queen quality. There aren’t huge differences. However, improvements have been made. Chalkbrood and foulbrood are much rarer than they used to be. That is likely due to the continued selection for disease-free lines to use as queen mothers. In addition, they’ve also been selecting for hygienic behavior. Hygienic behavior is a recessive trait, requiring it to come in from both the drones and the queen. So, how do they select for hygienic behavior yet maintain genetic diversity? The key is that there was a “group” decision decades ago to select for hygienic behavior among many, if not most of the queen producers in the region. The result of that decision is that drones from many different genetic sources were selected for the trait. Over time, a large number of genetically diverse drones containing the hygienic traits became available for mating. With mating nucs set up throughout the county, genetic diversity was “maintained” by the drones. So, like my situation where the diversity came in from the drones in different areas, so too in Northern CA, just times a million.

I have not spoken with the breeders of the HILO project, so I don’t know their breeding strategy or their plans for genetic diversity. The one thing I do know is that the tools to properly bring this recessive trait quickly and efficiently into the hands of beekeepers are not available. What’s needed is a good genetic map of the honey bee and marker assisted breeding. We need to get to a point where the trait no longer defines the line. Instead, the trait is introduced into several lines to increase the quality of those lines. These are the tools used in almost every other agricultural industry, including both plants and animals.

In my opinion, the lag in honey bee breeding has two causes. First, bee genetics are hard. Why? The promiscuous mating behavior of the queen makes it impossible to control which drones mate with her. The drones from a region gather in drone congregation areas (DCAs). This is where the queen goes to mate with 10-20 drones. It has been reported that drones can travel seven km (over four miles) to reach a congregation area. Now, take a map and draw a circle with a four mile radius around your apiary. That’s a large area, over 12 square miles. How many colonies other than your own are in that area? Any queen you produce, whether by grafting, supercedure, or on the spot queen rearing will have access to drones from that entire area. Plus, she is mating with multiple drones, each carrying different genetics. This is great for genetic diversity, because it results in several different genetic populations of bees in the colony (have you ever seen different colored bees in a colony?). But, it also means that the likelihood that your queen mated with the appropriate drones is tiny. The inability to control mating in large scale makes true queen breeding extremely difficult. Now, think of the “golden triangle” which is full of queen breeders. Think how difficult it is for them to maintain even the simplest traits because of the diversity of drone population.

For the small-time beekeeper like me, unless truly isolated, breeding for improvement is probably a waste of time. You can, however, maintain good, healthy queen stock. Selecting your best colonies for queen mothers will work because all the queen producers are using similar selection criteria. If you are selecting for chalkbrood-free colonies and those virgin queens go out to mate, it’s likely the drones came from colonies where the queens were also selected from chalkbrood-free colonies. However, if you are trying to select for a complex trait like hygienic behavior, you could have the best queen in the world and you won’t be able to propagate it. The moment those daughter queens reach the DCA, packed with non-hygienic drones, the trait is lost. This is why it was so important for the queen producers to work together to select for hygienic behavior. Having everyone in the region doing the same selection meant that, over time, the complex hygienic trait was carried by a good proportion of drones. Therefore, the odds of a queen mating with at least a few drones carrying the trait are good.

I believe the second reason why honey bee breeding lags behind the rest of agriculture is because the bee industry is relatively small. There is neither the money nor the people-power to develop the tools. There are groups working on it but developing the appropriate lines to evaluate will take a large, concerted effort. I hope the beekeeping community, its benefactors, and its researchers can come together to develop a large, collaborative effort to reach this long-term goal in as short a time as possible.

Click here to go directly to Part 1 – Bee Driven Mid-Life Crisis

Click here to go directly to Part 3 – Where Do I Put It?

It’s Not The Bees

“With the introduction of China’s Home Responsibility System in the 1980s, the farmers of Hanyuan County in Sichuan Province found it economically beneficial to replace their rice paddies with fruit orchards.

The mountainous slopes of the region lent themselves well to fruit production, particularly pears, for which Hanyuan County is now renowned.

Any crops grown beyond the quotas of China’s collectivized farming program could now be sold on the open market and, in order to maximize their yield, the farmers began to increase their use of pesticides.

This, in turn, had a negative effect on the population of the natural pollinators, and the local beekeepers were driven to relocate their colonies out of the cultivation areas.

With the disappearance of the bees, along with the desire to control the quality and purity of the pear varieties, the farmers began the labor-intensive task of pollinating their crops by hand.

Every member of the family is involved in the hand-pollination process in some way. Old woman selecting the stamens from the Yali (main pollinizer) flowers. This is a preparation process before the farmers go to hand pollinate. Farmers need to dry the stamens on 20-23°C temperature.

An aged farmer works on his orchard. In Juixiang even the oldest are capable to complete physically challenging tasks such as pollinating each blossom one by one, even in the most diffi cult and dangerous environment.

Chen Tao pollinating the family orchard in Dalian. He uses a duster made out of chicken feathers. The duster touches pollen mixture, and then shaken over the target pear trees for one to three times a day in order to ensure adequate pollination of the pears.”

A farmer prepares a small pollination stick. The feathers are degreased in alcohol before tying on the bamboo stick.

Flowering pear trees in Hangduan mountains. The average number of pear trees owned by each household is around 80-110. To pollinate these trees three to six polliniser trees are suffi cient. A 10 year old polliniser tree can produce fl owers enough to pollinate approximately 50 trees.

Shiqin Tan stands amongst his pear orchard. He planted the trees 20 years ago. His orchard consists of 45 trees. A person can pollinate 30-40 trees a day.

The transport of masses: motorcycles are the most common form of transport in the mountainous area of Jiuxiang.Young couple on its way to start pollinating their pear trees.

In preparation for hand pollinating. Woman brushing off the stamens from the Yali variety. She collects them into a pot before they start the drying process.

With simple tools such as a bamboo stick and chicken feathers they embarked on a journey of learning, not just how and when to pollinate, but when to collect the stamens, how to dry them, and which varieties respond to which pollinizer.

Additionally, not all the pear varieties are self-compatible, so cross-pollination is needed in order to achieve a desirable crop.

With skill and patience, the farmers can produce high quality, high yield product, albeit with increased labor costs than if they relied on nature alone.

As industrialization continues to push up the cost of hiring a workforce, the farmers must find an alternative way of cultivating their crops in order for them to remain viable. With pear production accounting for 40 to 50 percent of the household income, the stakes are high and adaptability will be key to their success.

The return of the natural pollinators is possible, but this is unlikely without a coordinated approach to limiting the use of agrochemicals.
What the future holds is uncertain and further work is needed to find a successful solution that balances the economy with ecology.”

The process of science involves new discoveries and continuous revision. The culture of science has placed a premium on novelty, but the revision process is often slow, and hampered by context that is difficult to interpret. Stack this atop the highfalutin’ superorganism (AKA: honey bees) and yep, information can become a sticky swarming mess. Reasonable context in science comes from experimental design, statistical application, and the ability of the scientist/author to construct a reality sandwich from the meat of new results and the bread of present knowledge. Scientists learn how to present their findings clearly, and must eventually sell their ideas forward to maintain future funding. When a scientific manuscript is sent for peer-review, the author must target a general science audience by placing the work in context. However, when that same piece of work becomes a news release or popular article, it is typically painted with a more colorful brush. Like an invasion of “Murder Hornets”, it must buzz the public ear.

My personal favorite: “Bees Need Meat”, explains that microbes in flowers are a crucial part of the bee diet. It goes on to say that microbes are basically “meat”, and microbiome changes caused by various environmental and agricultural factors could be starving the insects. While it seems bizarre to consider bees eating meat, this news release aspired to awaken the world to the vital connection between ALL pollinators, agriculture and microbes. As a devout microbial ecologist, I wholeheartedly embrace a future wherein agriculture accounts for microbial factors across the landscape including flowers, water sources, soil, natural systems, and all pollinators, including honey bees. After all, there are thousands of pollinating bee species, and microbes are essential to nearly every life process. However, I recommend that you stop short of replacing your pollen substitute with probiotic hamburger. Although a novel and highly relevant discovery, “Bees Need Meat” was NOT about honey bees. In fact, honey bees were conspicuously absent from these studies, and for good reason. The fact is, honey bees store and process their collected pollen very differently from the bees that eat microbial meat. Moreover, the way in which honey bees control microbial growth and exposure is a hallmark of advanced social life and honey bee success.

First, let’s examine the bees that eat microbial meat. These bees are native to the Americas, and mostly solitary or socially primitive. Every female solitary bee must function as a forager, nest builder and queen. They mate, then forage for their own pollen and nectar, and lay individual eggs on nectar-laden pollen balls containing some microbes from flowers. As the larvae develop, it is sometimes a race between microbe and larvae to consume the pollen and nectar provisions. As a result, many native bees end up eating a big load of microbes along with their pollen. These microbes were called “meat” because the measured nutritional value was more similar to a meat eater than a cow grazing grass. Earlier research on native bees detailed how mold growth could devour a large proportion of solitary larvae along with its pollen ball. In response, some bee species return to the nest site to police fungal growth, walling off infected from non-infected larvae. Other solitary bee species developed ways to combat fungal growth, including the application of plant materials and salivary secretions that protect developing larvae. As happens in every microbial ecosystem, various microbes compete for access to the sugary pollen ball including opportunistic pathogens (mostly molds) and potentially beneficial microbes that suppress mold growth (Lactobacillus, Acetobacteraceae and sugar tolerant yeasts). Generally, the microbial function of these sticky pollen balls resembles that of silage production, wherein acid produced by fermentative microbes inhibits mold growth, preserving, and in some cases, altering the nutritional quality.

As a pinnacle of bee evolution, honey bees possess more and better strategies to combat unwanted microbial growth, and advanced social organization has provided some unique modifications. First off, honey bee larvae consume very little if any pollen, so by definition, cannot eat the microbial meat growing on pollen. Honey bees possess a functional subgroup called nurse bees that control multiple aspects of colony nutrition. This group of adult bees eat and digest the pollen, bulk up their fat bodies, then feed larvae with highly nutritious jelly using special head glands. Secondly, honey bee stored pollen (or beebread) has virtually no microbial meat. Stored pollen is 50% honey by weight and a highly preservative environment that suppresses microbial growth. In fact, both honey and jelly are notorious microbe assassins each containing a devastating variety of antimicrobials. Together, these two substances buffer the effects of environmental change and allow the honey bee to survive extended times with no forage. The perennial honey bee lifestyle would be impossible without honey and jelly. Not only do these substances buffer nutritional dearth but have also evolved to confront the microbial challenges associated with periods of limited nutrition. One might even hypothesize that the evolution of honey bee sociality was continually refined by relentless microbial challenge during the nutritional shift to a perennial lifestyle. But I digress…

Honey bees do not eat microbial meat. The scientific process relies on thoughtful revision, and revision relies on context. As a social insect ecologist who peer reviews 75-100 scientific papers yearly, I’m constantly asking myself three context reliant questions; 1) Am I capable of reviewing this material? Many manuscripts about bees I cannot agree to review because I lack the context (education or experience) to judge them. 2) Are the methods and results valid?, This is difficult to judge and requires an expert versed in the methods and statistics used by the authors. 3) How were the results placed in context? In other words, does the author’s tasty sandwich square with hypothesized reality, and if not, do their evidence and arguments convincingly address the inconsistencies? This judgement typically requires a deep knowledge of present and past literature surrounding the subject matter. In short, context is everywhere, it is both theory and hypothesis, hardware and software, tool box and blueprint. Automatically created but rarely considered, the mind quickly imposes context on every situation. At the most fundamental level, it is how you distinguish important things from the background. The space devoted to context can dominate an article, or be largely ignored. Unfortunately, context is complicated, longwinded and often boring. To drive my point home, context is defined as: “the circumstances that form the setting for an experiment, statement, or discussion, and in terms of which, it can be fully understood and assessed”. The devil is in the details. Only the science-minded could love context.

In contrast, novelty is fast and fun, and like a new puppy… has sharp little teeth. Context and novelty always compete for space, especially when reducing a scientific article to a twitter blurb, or when sampling select bits of science to support a new product on the market. Presentation of a novel finding requires a dramatic distillation of context, typically producing a short catch phrase that rings the brain bell “wait, what?”…microbial meat? murder hornets? In this age of fast information, novelty is linked inexorably with sensationalism. It’s the silver bullet of science. The breakthrough of the century. It’s the brush used to paint all advertisement, snake oils, and conspiracy theories. Almost by default, novelty is misinterpreted due to limited or missing context. “Based on science” only works if the context is correct and complete. Returning to our example, “bees need meat”, the press release was crafted to highlight the importance of landscape; the flowers and associated microbes sustained by the landscape. The reader was first “hooked” by the scent of sensationalism, satiated by meaty novelty, then for dessert, served a small scoop of context.

But like you, when someone says bees, I see a box with honey supers, a colony on a landscape, an integrated system, a factory in a fortress. I think of beekeeping, honey bee research, whether it happened in a cup, a cage, a nuc, a super, what bees did you sample and why, what time of year, treated for mites, antibiotics, viral load, landscape history…All these factors add variation to the results, some predictable, some stochastic. How do these parts contribute to the collective whole? For the majority of bee publications, much of this variation is either unknown, ignored completely, difficult to attach to the experimental design, or poorly accounted for in the article. Even when you’ve done your due diligence accounting for these factors, honey bee colonies are famous for behaving in ways you’ve never seen before. While I’m sure you’ve experienced this as a seasoned beekeeper, I relearn it repeatedly trying to get bee hives to “beehave” for bee research.

Reducing colonies to cages or nukes can yield statistically relevant results, but often does not predict what will happen to full size hive A or B, on landscape A or B, in season A or B, and so on. To understand any complex system, you must approximate many things. Data is piecemeal, and must be stitched together to assemble a gestalt, a platform from which to build novel understanding.

It is in this context that we interpret the biological and statistical reality associated with honey bee microbial ecology. Not easily reduced to individual terms, a colony is composed of physiologically interrelated groups of activities. Colonies can stay in place or move quickly across a highly variable landscape. The colony is a robust and dynamic social entity, both nurturing and deadly. Colony function relies on the interdependence between groups of individuals performing a variety of different processes in parallel. At the active fringes of the factory, the hive itself is an antibiotic, a prebiotic and a probiotic, populated by a special set of microbes evolved to endure the hive environment. The lion’s share of these microbes are not recently transmitted from flowers, but are present throughout the year, and may be considered “native” to the hive environment. Although not convincingly demonstrated in its entirety, this statement about native hive microbiota reflects an educated opinion based on one scientist’s microbial understanding of honey bees, native bees, and the pollination environment. More context available on request.

Knowing the complete genome of an animal, whether human, bee, or worm, can go a long way toward predicting how that animal looks, grows, and behaves. But to really know the source of interesting traits, one must study which parts of that genome are active at key times. Genes that provide the blueprints for individual proteins (a big part of the ‘active’ genome) are silent much of the time. When triggered, they spin off messenger RNAs, whose message is then translated into strings of amino acids that make up unique proteins. This triggering, or ‘transcription’, into messenger RNAs can be precise to individual genes or can link tens or hundreds of genes that turn on and off in a coordinated way. Coordinating the arrival of many proteins at once is essential for complex events such as building a limb, enacting an immune response, building a bank for memories, and numerous other needs of even the simplest organisms.

Humans have about 21,000 protein-coding genes and bees have maybe 16,000. These are depressingly average counts for mammals and insects, respectively, and far less than, say, earthworms and potatoes. Our exceptionalism, then, must reflect how well we use the genes we have. This relates directly to how well our cells turn particular genes on and off. When I first studied genetics, we were not given the whole story. Genes were turned on by promoters that hit a cartoonish landing pad on the chromosome just in front of the gene – and protein-building ensued. This view was nuanced a bit by the study of transcription factors that matched to varying degrees sites ‘near’ key genes, turning them all on at once. Science fans will know that anything with ‘factor’ in its name still has a bit of mystique (e.g., ‘virulence’, ‘queen’, and ‘fudge’ factors). Transcription factors are now well studied, however, and are validated as good predictors of which proteins need to work together during particular events in the lives of cells.

Many decades ago, discussion began on still more mysterious epigenetic (‘above the genome’) factors that regulated the activity of larger chromosome regions. Generally, epigenetic controls squash gene activity. One prime example for organisms with sex chromosomes (humans, yes, bees, no) involves the regulation of genes found on the chromosome shared by both sexes (the ‘X’ chromosome in our case). For many genes, it is healthier if their protein levels are roughly equal in males and females. Since females have two X chromosomes in their cells, there has to be some sort of dosage compensation for the proteins not related to sex differences, so they play well with proteins from the rest of the genome. To do this, genes encoding proteins on the X chromosome in males might be hyperactive to mimic those from XX females – but in reality, it is mostly the female X-encoded genes that slow down to match the boys. In fact, a good fraction of X chromosomes in female mammals is silenced by a process called methylation, an enzyme-driven swap of DNA components that makes these components less likely to be turned on. For you science buffs who remember the four bases of DNA, it is generally the cytosines that get dinged up in this way, neat by itself but also a perfect landmark for scientists to predict which parts of the genome are silenced.

Other than hearing these stories of dosage compensation by a somewhat sloppy brush, I admit to not really thinking much about methylation – UNTIL some fascinating work by my friend and hero, Professor Ryszard Maleszka from the Australian National University. Prof. Maleszka toiled for years as a leader in the sequencing of the honey bee genome. When it all came together, he was almost giddy to find that the bee genome had a full set of enzymes needed to tag specific chromosome segments with methylation and, importantly, maintain the faithfulness of that methylation tag over time. Soon, he and his team showed that these epigenetic forces were important in one of the most beautiful processes in bee biology, the generation of queen bees.

Queens and workers are indistinguishable at the gene level, a fact known by queen breeders who can graft without discrimination from female larvae to get their future queens. What sets them apart is the coordinated production of caste-biased proteins that, in the case of queen-destined larvae, speed metabolism and development and set the seeds for prodigious ovaries. Worker-destined larvae grow more slowly, and have different brain structures and barbed stingers, among many other differences. While the environmental causes of this split involve larval diet (royal jelly and its queen ‘factors’), inside each bee the queen path is set by the epic and apparently epigenetic regulation of hundreds of genes tuned to either a queen or worker fate. In a highly cited paper from 2008, Dr. Maleszka, his scientist wife Joanna, and their team produced clear evidence that silencing by methylation was key for this process. The paper, “Nutritional control of reproductive status in honeybees via DNA methylation” is freely available from the journal Science (https://science.sciencemag.org/content/319/5871/1827). Methylation is an imprecise paintbrush, more Monet than Michelangelo, but a critical gene showed shifts in methylation that reflected the queen or worker pathway. Interestingly, worker-destined larvae apparently had a higher rate of methylation for caste-related genes and the proof for the whole story came when the ANU team silenced a key enzyme that drives methylation and successfully pushed more larvae down the queen road, irrespective of diet!

Science moves on and additional epigenetic processes have been discovered since this work, but the message that whole cohorts of caste-biased genes can be tagged in a lasting way to impact their activity has held up beautifully. For a really recent take on it all, Professor Maleszka’s group recently teamed up with Marek Wojciechowski, Paul Hurd, and colleagues from England in a fascinating and freely available paper titled “Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development” (https://genome.cshlp.org/content/28/10/1532). These epigenetic changes are undeniable and the signals they leave behind have helped to identify numerous genes products tied to key traits like queen production, disease responses and even stinging tendencies. I will follow up with reviews of those traits in the future, and show how this discovery has been franchised over the past decade to help explain traits like queen production, or perhaps reproduction generally, in species far beyond honey bees. As a teaser, I mentioned that these methylation-related enzymes can repaint the same site even after cells divide. It turns out they ensure that some methylation patterns are faithful from drones or queens to their offspring, presenting great possibilities for a parent’s environment to shape the ways their offspring preps for life. Think ‘you are what your parents ate’, or suffered through, etc. There are also indications that some offspring traits more strongly reflect their dad’s versus mom’s contribution, a tendency that might either strengthen or challenge colony life. Stay tuned, it will be epigenetic.