Is the Key to Saving Pollinators … Honey Bee Semen?    By Simran Sethi   July 19, 2018

In the hopes of preserving their genetic diversity, entomologists are collecting and freezing this valuable fluid

A male bee releasing its seminal fluid at the USDA bee lab in Baton Rouge, Louisiana. The male does not survive the process. (Anand Varma) The first question everyone wants to know is: how?

“I’m surprised it took you so long to ask,” Brandon Hopkins says with a laugh. The 35-year-old entomologist is preparing samples to be sent to the USDA Agricultural Research Service National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado, a facility dedicated to securing our food supply by collecting genetic material from agricultural species. “You pretty much just squeeze them, and the stuff pops out,” he says.

Hopkins is the apiary and lab manager of Washington State University’s Apiary Program, and the “stuff” he’s referring to is honey bee semen.

Yes, semen. Hopkins spends a lot of his time visiting beekeepers and collecting seminal fluid from drones, the male honey bees that exist primarily to impregnate queen bees. Or, as Hopkins puts it: “They’re flying genitalia. They don’t collect nectar; they don’t collect pollen. The only thing they do is mate.”

He prefers to capture drones during flight, when they are on their way back from their daily attempts to mate with a queen. Between 1 and 5 p.m.—their flight time—he sets mesh screens in front of the entrances to hives. Worker bees are small enough to get through the screens and back into their dwelling, but drones can’t. As they cling to the dividers, Hopkins springs into action, gathering the stinger-less bees in cages and placing them, one by one, under the microscope.

He explains his process: “When you squeeze a male, if he’s mature, his genitalia pops out. And then, floating on a bit of mucus, is about one microliter of semen.” Sadly, in nature, drones put so much blood and energy into reproduction that they die after successful mating. And this is what Hopkins mimics in the lab: “We squeeze them to the point where they die,” he says. It takes Hopkins about an hour to process 300-500 drones and fill a single 100-microliter tube with their reproductive fluid.

The follow-up question, of course, is: why? That is: why in the world are scientists collecting bee semen?


In short, as a hedge for the future. “There could be unique and valuable [variants of a gene] that may not be noticeably valuable at this point,” but could become incredibly important in the face of a yet-unknown future threat, Hopkins says of the genetic material he collects. Most of the semen is frozen, catalogued and stored in Fort Collins, where the hope is that it will stay viable for years, perhaps decades, just waiting to be thawed out so it can impregnate a honey bee far in the future.

Or not so far in the future. Honey bees already face plenty of threats: pests and diseases, pesticides and fungicides, nutrition and the way colonies are managed, both in terms of beekeeping and breeding and genetics. Topping the list is a parasitic mite called Varroa destructor, which reproduces in honey bee colonies and lives up to its sinister name by sucking the blood from adults and developing larvae. It has been devastating bee populations since it was first detected in the United States in 1987.

By the numbers, the situation is dire. According to the USDA National Agricultural Statistics Service, in the late 1940s, we had nearly 6 million managed beehives in the United States. By 2008, that number dropped to just over 2 million—and has stayed there ever since. The semen Hopkins collects, then, could help protect, or even expand, future generations of honey bees—which means safeguarding billions of dollars in agricultural crops and an inestimable wealth of biodiversity for the planet.

Brandon Hopkins, hard at work collecting bee semen. (Steve Sheppard)

While the United States is home to around 4,000 native bees, our agricultural pollinator of choice is the non-native honey bee, which hails from South and Southeast Asia. That’s because honey bees are prolific and multipurpose pollinators, says Bob Danka, the research leader of the USDA Honey Bee Lab in Baton Rouge, Louisiana. While some bees pollinate a single species of plant, honey bees forage on over 100 commercial cropsdelivering nearly $3,000 worth of pollination services per hectare per crop.

In the U.S., honey bees handle “something like 90 percent of pollination,” Danka explains, and one colony averages a peak summer population of upwards of 60,000 bees. “Other bees can’t exist in large enough numbers to pollinate vast acreages of crops,” he says. The bees can also be moved in and out of various locations with relative ease, which is essential for crops like almonds, which require cross-pollination.

Between February and March of each year, 80 to 90 percent of the country’s available commercial bees—about 1.8 million colonies—are trucked to California to pollinate almond blossoms. But the work doesn’t end there. These bees are used year-round for their labor, writes Ferris Jabr writes in Scientific American:

“After the almond bloom, some beekeepers take their honeybees to cherry, plum and avocado orchards in California and apple and cherry orchards in Washington State. Come summer time, many beekeepers head east to fields of alfalfa, sunflowers and clover in North and South Dakota, where the bees produce the bulk of their honey for the year. Other beekeepers visit squashes in Texas, clementines and tangerines in Florida, cranberries in Wisconsin and blueberries in Michigan and Maine. All along the east coast migratory beekeepers pollinate apples, cherries, pumpkins, cranberries and various vegetables. By November, beekeepers begin moving their colonies to warm locales to wait out the winter: California, Texas, Florida and even temperature-controlled potato cellars in Idaho.”

This overreliance on honey bee labor, however, has its dangers. “We, in North America, have painted ourselves into this corner using honey bees because of modern agricultural practices and our need to produce large amounts of crops efficiently,” Danka says. And the work is starting to stress the bees out: “The pressure on them is very real, and it seems to be getting worse.”

Today, you might think of these bees as fully dependent on humans. “When Varroa mites came to the U.S., it eliminated 99 percent of the feral population of honey bees,” Hopkins says. “Some are saying there are no wild honey bees now because they can’t survive without human intervention. They’re like a domestic species.”

This codependent relationship with humans is revealed in changes in bee nutrition. Bees are just like us: They need a varied diet in order to thrive. As our diets have become less diverse, so have theirs. The expansion of industrialized agriculture and increase in monocrops grown in monoculture means there is little diversity in the plants from which bees source pollen and nectar. The habitats where they forage have turned into what Marla Spivak, a professor of entomology at the University of Minnesota, describes as “food deserts.”

The challenge is exacerbated, Spivak explains in a 2012 TED talk, by the convergence of supply and demand. At the same time we’re experiencing a decline in bee populations, we’re also growing an increasing number of crops that rely on them. In the last half-century, she says in her talk, “there has been a 300 percent increase in crop production that requires bee pollination.” Just last year, American beekeepers lost approximately 40 percent of their honey bee colonies.

Cryopreserved tubes of honey bee semen stored at the USDA’s genetic preservation center in Fort Collins, Colorado. (Simran Sethi)

That’s why, in 2016, the USDA’s Agricultural Research Service decided to add honey bee semen to its Fort Collins collection, which also stores a range of other materials—from seeds and stems to animal blood and embryos—that are essential for sustaining our domestic food supply. “It is part of [our] response to the ongoing crisis that the country’s beekeepers are facing,” the institution wrote in its online post announcing the launch.

The man tasked with the glamorous job of collecting the semen? Brandon Hopkins.

In 2008, the modern-day honey bee sperm collector was wrapping up a master’s degree in biology at Eastern Washington University focusing on the reproductive biology of frogs and mice. When Hopkins learned about the challenges bee populations were facing, however, he decided to explore a method that has been used to preserve the semen of cows and other animals: cryogenic freezing. Traditionally, bee semen specimens were extracted, stored at room temperature and stayed viable for about two weeks.

“I had never even really seen a honey bee hive,” Hopkins says. “But, fortunately, my master’s advisor had been [working] long enough in the mammalian world—with cattle and sheep and goats and all that stuff—and he said, ‘It doesn’t have to be perfect, it just has to work.’ Rather than waiting to get a perfect system, we went ahead and did it.”

In fact, Hopkins explains, they set about freezing honey bee semen despite the fact that one of the last papers written about cryopreservation from the 1980s stated the results weren’t good enough and that researchers should stop pursuing that method of storage. Nevertheless, Hopkins extracted a single capillary tube of semen (100 microliters), froze it and had “pretty good success.”

This was happening at the same time that Washington State University researcher Steve Sheppard, head of the WSU Apis Molecular Systematics Laboratory, was out in the field, collecting fresh material of the same variety. That year, he had been awarded the only permit given by USDA to import semen from global bee populations into the United States. Those samples became the foundation of what has become the largest collection of bee germplasm in the world, stored at WSU and containing subspecies native to Europe, Western Asia and Central Europe.

Sheppard subsequently became Hopkins’ PhD advisor, and the two of them started traveling together, collecting bee semen and freezing it on-site. The work came with unique challenges. “The problem with fresh semen is that you only get that one shot,” Hopkins explains. “It’s very expensive and time-consuming to collect overseas. Then you use it and may have a queen that doesn’t even produce any progeny.”

But it also paid off: Hopkins says the material collected and frozen five years ago is “the same as if it had been frozen for five days.”

When asked if he ever envisioned this as his life’s work, Hopkins was clear: “No. For sure not.” But he sees the incredible value in the work he’s doing. “The cool thing about the incorporation of cryopreservation in bee breeding is that it will allow us to breed across space and time,” Sheppard said in an email. “We can retrieve genetics years after it’s been placed in storage. So, you can envision that, in 2030, we could cross the bees back to material from 2015 that we have [stored] in the liquid nitrogen tank.”

And that’s why it’s important to preserve material that’s both commercially viable and diverse. “While I don’t really think that we’re going to suddenly lose all our honey bees and need to tap into this frozen stock to repopulate the planet with bees, it is too bad that we weren’t doing this before, say, Varroa mites came,” Hopkins says. “We lost a huge amount of genetic diversity in the U.S. population that we can’t really get back because we didn’t have any frozen material.”

To get back to that level of diversity, he says, there is more work to be done. “Honey bees are an agricultural domestic species now,” says Hopkins. “They need the same research and attention that cattle, for example, get. It would be great if they were better recognized—in conservation, breeding techniques, selection, all [it takes] to improve them.”

Refrigerating Honey Bees to Fight Mites, Colony Collapse

Washington State University     By Scott Weybright, College of Agricultural, Human and Natural Resouce Sciences     April 23, 2018

PULLMAN, Wash. – Saving Honey Bees Is Easier When Varroa Mite Infestation Is Reduced. WSU Researchers Are Hoping Mid-Season Hibernation Can Help In The Fight Against The Mighty Mites.

The black bump on this honey bee's back is a varroa mite. Mites weaken bees’ immune systems, transmit viruses, and siphon off nutrients. Photo by Scott Bauer, USDA Agricultural Research Service.

Varroa Mites Are Pests That Weaken Bees’ Immune Systems, Transmit Viruses And Siphon Off Nutrients. They’re A Huge Factor In Colony Collapse Around The Country.

“Most Treatments Only Kill Varroa On Adult Bees, And Are Generally Only Effective For Three Days,” Said Brandon Hopkins, Assistant Professor Of Entomology And Manager Of The WSU Bee Program. “But A Lot Of Mites Live In The Brood, Which Are Under A Wax Cap That Treatments Can’t Touch. Those Bees Hatch Out And Are Already Afflicted.”

Currently, Treating For Mites Requires Three Treatments Over A 21-Day Period To Make Sure You Treat All The New Bees That Come Out Infested With Mites.

These Treatments Are Difficult And Expensive Because Beekeepers Must Treat All Their Colonies On A Specific Schedule. It’s Very Labor Intensive To Treat Thousands Of Colonies By Hand Three Times At Precise Timing Cycles, Hopkins Said.

Cold Storage

Bees Don’t Truly Hibernate, But They Do Change Their Behavior In Winter. Queens Stop Laying Eggs, So No New ‘Brood’ Is Created At That Time.

Last August, WSU Researchers Put 200 Honey Bee Colonies Into Refrigerated Storage. This Is A Time When Bees Are Still Active, But Have Finished Making Honey For The Season, And There Are No Crops That Require Pollination. It’s Also When Beekeepers Normally Do A Round Of Mite Treatments.

By Placing Colonies In Refrigerators, The Queen Stops Laying New Eggs, Which Stops The Production Of Brood. When The Bees Come Out Of Refrigeration, There Is No ‘Capped Brood’.

At That Point, Hopkins And His Team Apply A Varroa Treatment On The Adult Bees.

The Initial Results Were Overwhelmingly Positive. Researchers Found An Average Of Five Mites Per 100 Bees On The Control Colonies (Not Refrigerated) One Month After The Normal Three-Cycle Mite Treatment.

The Refrigerated Colonies Had An Average Of 0.2 Mites Per 100 Bees One Month After The Single Mite Treatment.

“That’s A Significant Decrease,” Hopkins Said. “Refrigeration Is Expensive, So We Need To Do More Work To Prove The Cost Is Worth It For Beekeepers, But We’re Really Excited So Far.”

Additionally, The Infestation Levels Varied Tremendously From Colony To Colony In The Control Samples. That’s Because Of The Difficulty In Treating Colonies Consistently Over Three Cycles. The Colonies That Had The Refrigeration Treatment Had Consistent Mite Numbers With Little Variation.

Doubling Down

Brandon Hopkins in his bee lab.

After Hearing About This Research, A Few Beekeepers Approached The WSU Scientists About Doing A Similar Round Of Refrigeration In The Early Spring. Most Commercial Beekeepers In The U.S. Take Their Colonies To California For Almond Pollination In February And March. But There’s A Time Gap Between The End Of The Almond Pollination Season And The Start Of Pollination Season In The Northwest.

“Beekeepers Generally Have Two Periods Of Time For Mite Treatments, Before The Bees Make Honey And After,” Hopkins Said.

Once Bees Have Mites, The Infestation Increases During The Pollination And Honey Production Months.

“But If They Can Start With Low Mite Numbers, The Bees Are Healthier During The Honey Production Period,” Hopkins Said. “A Lot Of Varroa Damage Comes While The Bees Are Making Honey.”

Calculated Risk With 100 Colonies

This Spring, Belliston Bros., A Commercial Idaho Beekeeper, Donated 100 Honey Bee Colonies To Do A Refrigeration Study Just Like The One Done In August Last Year.

“It’s A Big Risk For Them,” Hopkins Said. “But If It Works, Beekeepers Would Have Significantly Better Varroa Control While Using Fewer Chemicals. And They’ll Have Better Colony Survival During The Following Pollinating Season. It’s A Win All-Around.”

Nobody Really Knows How Bees Will React To Being Put Back Into Their Winter Mode In What Is Normally The Middle Of Their Active Season, He Said. But That’s What Science Is All About. And If This Works, It Could Be A Major And Environmentally Sound Victory In The Great Varroa Mite Battle That Beekeepers Have Been Waging For Decades.

“We’re Hopeful,” Hopkins Said. “We Won’t Have Results Back For Several Months, But We’re Excited We May Have A Way To Help Beekeepers Keep Their Colonies Strong And Stable.”

No Offense, American Bees, But Your Sperm Isn't Cutting It

NPR The Sale    By Ryan Bell    July 13, 2017

With an American honeybee queen for a mother and a European honeybee drone for a father, this worker bee has a level of genetic diversity unseen in the U.S. for decades. Researchers at Washington State University hope a deeper gene pool will give a new generation of honeybees much-needed genetic traits, like resistance to varroa mites. The parasite kills a third of American honeybees each year. Megan Asche/Courtesy of Washington State University

Editor's note: This story is for mature bees only.

Seducing a honeybee drone – one of the males in a colony whose only job is to mate with the queen – is not too difficult. They don't have stingers, so you just pick one up. Apply a little pressure to the abdomen and the drone gets randy, blood rushing to his endophallus, bringing him to climax.

"They're really accommodating," says Susan Cobey, a honeybee breeder on Whidbey Island, Wash. "One ejaculate is about 1 microliter, and it takes 10 microliters to artificially inseminate a queen."

Since 2008, Cobey has done her share of bee abdomen rubbing as part of a research team from Washington State University traveling through Europe and Asia. They've collected sperm from native honeybees in Italy, Slovenia, Germany, Kazakhstan and the Republic of Georgia – countries where honeybees have favorable genetic traits, like resistance to the varroa mite. The deadly parasite has been cited as a major factor in bee deaths, along with genetics, poor nutrition and pesticide exposure, according to a major report from the USDA and EPA in 2013.

Varroa mites are an invasive parasite from Asia that sucks hemolymph (bee blood) from adult and larval honeybees, weakening their immune systems and transmitting deadly pathogens, like bent wing virus. If left untreated, a varroa infestation can kill a colony in one year. First detected on U.S. soil in 1987, varroa has spread quickly, infesting upwards of 50 percent of American hives. Last year, 33 percent of U.S. honeybee hives died. That's troubling for the plight of honeybees and U.S. agriculture, which relies on pollinators to produce one-third of the food we eat.

The buzz on American bees: too much inbreeding

According to the WSU research team, the root cause of the U.S. honeybees' vulnerability to varroa is a dwindling gene pool that has left them short on genetic traits that help honeybees resist varroa elsewhere in the world.

"Honeybees aren't native to America," Cobey says. "We brought them here. But the U.S. closed its borders to live honeybee imports in 1922, and our honeybee population has been interbreeding ever since."

WSU has monitored the genetic diversity of honeybee queens in Washington and California since 1994, showing a steady decline. Dr. Brandon Hopkins, the team's expert in freezing and thawing bee sperm, likens honeybee breeding to a poker game played with an incomplete deck of cards. "There's no way to get a four-of-a-kind if there aren't four aces in the deck," Hopkins says.

Brandon Hopkins, a cryopreservation specialist, works in Washington State University's fruit tree orchard in Pullman, Wash. As a doctoral student at WSU, Hopkins perfected a system of freezing and thawing bee semen for use in artificial insemination. Shelly Hanks/Courtesy of Washington State University

The imported semen has restacked the deck. WSU's crossbred honeybees already test positive at a higher level of genetic diversity than the first queens tested in 1994. "This doesn't mean they are superior in performance to the other bees," Hopkins says. "It means we have a better chance of finding rare and unique traits."

It used to be that honeybee breeders selected for bees that produced more honey, grew more populous hives, and were gentler to handle. Now, they want honeybees that can resist varroa. Without it, beekeepers must rely on costly "miticide" treatments to control varroa.

However, studies suggest the mites are developing resistance to pesticides and the chemicals may be harming honeybees, compounding the problem of widespread bee deaths known as Colony Collapse Disorder.

"I lost 40 percent of my colonies to varroa last fall," says Matthew Shakespear, whose company, Olson's Honeybees, raises 16,000 hives in central Washington. "I'm not taking any more chances. We've already done five treatments, compared with the two treatments we applied this time last year."

A problem that blooms in almond orchards

Pollination services like Olson's Honeybees are the cornerstone of a $15 billion segment of U.S. agriculture. A hefty share of that is the almond industry, whose trees are completely reliant on honeybees for pollination. It's also the industry most susceptible to fallout from the varroa epidemic in bees: California's almond groves serve as an incubator for the growth and spread of varroa mites across the United States.

"There are 800,000 acres of almonds in California," says Patrick Heitkam, owner of Heitkam's Honey Bees in Orland, Calif. "It takes two hives to pollinate one acre, so that's a need for 1.6 million hives. There are only 500,000 hives in the state, so the rest are trucked in from around the country."

Almond trees bloom in January, a time of the year when most honeybee varieties are dormant in their hives. But an Italian species of honeybee, Apis mellifera linguistica, which evolved in the warm Mediterranean climate, is active when the first almond blossom pops in late-January, making them the most popular variety in the U.S.

The trouble is, Italian honeybees are extremely susceptible to varroa mites, because their hives grow so big, so fast and so early.

"Italian honeybees rear their babies and varroa mites nearly one-for-one," says Dr. Robert Danka, a research entomologist at the USDA's Honey Bee Breeding, Genetics and Physiology Research Unit in Baton Rouge, La.

Lessons from mighty Russian stingers

Like the WSU team, Danka has also looked to the Old World for an answer to varroa mites. Between 1994 and 2000, he traveled to the Russian far east, where a local honeybee, Apis mellifera, has developed resistance to varroa. They are descended from European honeybees brought by Russian settlers traveling the Trans-Siberian Railway at the turn of the 20th century. The journey inadvertently transplanted the honeybees into the native range of varroa mites in east Asia, where they evolved resistance to the pest.

These Russian bees groom themselves, biting and crushing the mites. They also have a prevalent genetic trait called varroa sensitive hygiene (VSH), aborting larval honeybees infested with mites and removing them from the hive before the parasite can spread.

Danka brought back 360 queens, the basis for what is now a robust Russian honeybee population in the United States. While their prowess as mite biters continues, Russian honeybees haven't proven up to task as commercial pollinators. The queens are used to long Russian winters, so they are slow at building up their hives, meaning only a small number are ready to fly in the almond groves come January.

Survival of the fittest bees

Still, Danka says the Russian honeybee offers proof that a European subspecies can develop varroa mite resistance through natural selection. That evolutionary process is interrupted in commercial beehives, because of the "prophylactic use of miticides," Danka says. "We're maintaining varroa-susceptible bees through chemistry. If we took away all those pesticide treatments – and to be clear, I'm not advocating for this – the results would be horrific. But in a rather short period of time, only varroa-resistant bees would be left." And those bees could be the basis of a new population.

Matthew Shakespear, the commercial honeybee keeper in Yakima, Wash., would rather not spend money treating his hives for varroa mites. Starting last year, he diversified his business to include hives of Carniolan honeybees, Apis mellifera carnica, a subspecies native to Eastern Europe.

The queens he bought were the great-great-great granddaughters of a honeybee drone that Susan Cobey found in the mountains of Slovenia.

"Maybe these new genetics can deal with the varroa mites naturally, rather than having to rely on chemicals," Shakespear says. "It's time to start widening our gene pool.