Breeders toughen up bees to resist deadly mites

The death of his favorite queens in 2013 was the final straw for BartJan Fernhout, an amateur beekeeper in Boxmeer, the Netherlands. Fernhout's queens, which he had purchased from a specialty breeder, produced workers with prized traits: They were calm and made plenty of honey. Then, Fernhout's hives became infested with a parasitic mite named Varroa destructor, which has become a major contributor to the demise of bee colonies worldwide.

Chemicals and other methods can control the parasites. But the mites are developing resistance, and the treatments sometimes don't work—or even backfire. The chemical Fernhout used to fight his mites, for example, stopped his queens from laying eggs. That caused the workers to kill the barren queens and begin to raise new royalty, a ruthless reaction the bees evolved long ago to ensure the future of their hives.

Frustrated, Fernhout decided there had to be a better way to combat the mites. The next year, he took a buyout from his research job at a veterinary firm to found Arista Bee Research, a nonprofit that has joined a growing global quest to breed honey bees able to resist Varroa mites on their own.

It's been slow, laborious work. Since the mite jumped from Asian honey bees (Apis cerana) to the common domesticated European honey bee (A. mellifera) more than a half-century ago, researchers have discovered some bees can keep the mite in check through behaviors such as fastidious grooming and removing mite-infested larvae. But identifying bees able to mount these responses is tedious. A standard way to evaluate grooming, for example, is to count how many mite legs have been chewed off by vigilant bees. And the complexities of bee reproduction make it cumbersome to combine mite-resistance traits with others valued by apiarists. Although researchers and breeders have created bees that require fewer pesticides, even these colonies can be overrun by mites—and very few lines can yet survive without any treatment. "There is progress, but not very significant," says Benjamin Dainat, a bee researcher and breeder at the Swiss Bee Research Centre in Bern.

New molecular tools promise to accelerate those efforts. A new protein-based test, for example, would allow beekeepers to simply send a laboratory a few dozen antennae, plucked from their bees, to learn whether the insects have mite-detecting powers. Other scientists are sequencing the genomes of huge numbers of bees, hoping to create a relatively cheap and easy way to identify bees that carry genes for the protective behaviors. Such a test "is almost the Holy Grail" of anti-Varroa research, Fernhout says.

A success would help secure the future of the multibillion-dollar honey bee industry, which supplies honey and enables the largescale pollination of high-value crops, such as almonds. If breeders can spread resistant bees, then "the long term is looking good" for controlling the mite and stemming the bee die-offs, says John Harbo, a retired biologist and bee breeder in Baton Rouge.

Like other ruinous pests, Varroa started to cause trouble after it moved to a new host. One species, V. jacobsoni, is a long-standing parasite of Asian honey bees throughout their home range of southern Asia. It reproduces in the bees' brood cells, where it feeds on the larvae, but it typically doesn't destroy colonies. One reason is that the mite lays its eggs only on larvae that will become drones—the males that mate with queens—and hives produce only a few drones. If the mite does target the more numerous larvae of worker bees, they commit suicide (a process called social apoptosis), preventing the mite from reproducing. The natural process of starting a new colony, called swarming, also gives colonies a fresh start; when a queen and a swarm of workers abandon their old hive, they leave behind the reproducing mites as well.

In the mid–20th century, after apiarists brought European colonies to Asia, the mite found its new host. The European bee, which beekeepers prefer for its large colonies and docile workers, generally lacked the Asian variety's defenses. Breeders had selected against swarming behavior, for example, because keepers don't like queens to abandon their hives. The mite quickly adapted to its new host, and it routinely infests the larvae of European worker bees. The result was a new strain of Varroa—defined as the new destructor species in 2000—which ran amok. It now afflicts European bees everywhere except Australia and a few islands.

The role of pesticides such as neonicotinoids in honey bee die-offs is debated. But there is no question that the mites have been a major factor. V. destructor weakens both adult and larval bees by consuming their fat stores. The mite also spreads viruses, including a lethal one that deforms wings, preventing bees from flying. Parasitized colonies lose workers, make less honey, and often fail within a year if not treated.

Modern beekeeping, which involves keeping hives in close proximity, appears to have accelerated the mite's spread. When one colony is collapsing, bees from others come to rob honey and also pick up mites and viruses. "If you are commercial beekeeper and you stop treating, basically you lose your operation," says Fanny Mondet, a bee researcher at the French National Institute for Agricultural Research in Avignon. The rise of V. destructor in the late 1980s and '90s made other beekeeping problems look "like child's play," says Robert Danka, a bee biologist with the U.S. Department of Agriculture's (USDA's) Agricultural Research Service in Baton Rouge.

In theory, there is a simple solution: Don't do anything and let natural selection eliminate bees that can't resist the mite. It's brutal, but it works. Populations of feral honey bees crashed after Varroa arrived, for example. Then, in a few places they began to recover, suggesting some colonies had defenses. Beekeepers who let their hives fend for themselves also saw results. In the early 1990s, Daniel Weaver, a bee breeder in Navasota, Texas, let the mite run wild in 1000 of his colonies. Just nine survived the first year, and from these he bred mite-resistant bees. "It was a painfully expensive experience," he recalls, and costly enough to put many beekeepers out of business. Such natural culling could also mean losing valuable bee strains produced by decades of breeding.

As a result, many beekeepers treat their hives with pesticides. Others add in or substitute nonchemical methods, although they are more work and can pose trade-offs. For example, mimicking swarming by transferring a queen and some workers to a new hive means a smaller colony and less honey for a while.

Breeding mite-resistant bees is an increasingly appealing alternative. "You can reduce the use of treatments, increase your survival, and reduce the number of colonies that you need to replace every year," says Greg Hunt, a bee biologist who recently retired from Purdue University in West Lafayette, Indiana.

People were saying that breeding bees for resistance to Varroa was like breeding sheep for resistance to wolves. John Harbo, a retired biologist and bee breeder in Baton Rouge

One source of inspiration for breeders is Karl Kehrle, a Benedictine monk known as Brother Adam, who worked at Buckfast Abbey in the United Kingdom. In the early 1900s, U.K. honey bees were dying from a different mite, which infested their airways. Brother Adam traveled the globe, collecting resistant bees. Over 70 years, he bred a robust strain known as the Buckfast bee.

To replicate Brother Adam's success with the new threat, bee breeders hope to enhance mite-fighting behaviors such as grooming. Some bees vigorously shake off mites, or bite the parasites until they drop off—a behavior whose effect can be measured. Three times a year, researchers at Purdue University enlist students to visit 100 to 150 bee colonies and collect mites that have dropped to the bottom of the hive. Then, they put the parasites under a microscope and count how many legs are missing or partially chewed off. By breeding queens from colonies where mites showed high levels of damage, they have developed relatively resistant colonies. But not everyone has the time and patience for laborious limb counts.

In a lab in a suburb of Berlin, technician Karla Rausch stares intently at a video of bees scurrying inside a hive. She replays one short clip several times, tracking a single bee that has a tiny number glued to its back. A hexagonal brood cell holding a larva has caught its attention—and for good reason. Researchers at the Institute for Bee Research (LIB) here have placed a Varroa mite inside this cell and many others. Rausch is documenting how bees respond as part of an effort to identify genetic markers for mite resistance, which could be a shortcut to breeding better bees.

The worker taps its antennae on several cells, checking for chemical cues. Then it nibbles a hole in the mite-infested cell. Other bees, responsible for cleaning, will remove the larva from the hive, preventing the mite from reproducing. This behavior, called Varroa sensitive hygiene (VSH), is heritable and a key target for breeders.

In the 1990s, however, the idea of breeding Varroa-resistant bees was considered a long shot, recalls Harbo, who worked at USDA's bee lab in Baton Rouge, where VSH was first observed. "People were saying [that would be] like breeding sheep for resistance to wolves," he says.

Harbo and his colleagues proved them wrong. By breeding bees from colonies with a lower proportion of reproducing mites, they were able to create colonies that had improved Varroa resistance. But the new strains never caught on with beekeepers. One weakness was that they were inbred, lacking the genetic diversity that helps create vigorous hives.

Since then, Danka and other breeders have continued to improve these bees. The newest group to follow in Harbo's footsteps is Arista Bee Research. One of its principal efforts has been to create a network of more than 100 bee breeders in Europe, mostly amateurs. (Some teams wear T-shirts boasting they are "Varroabusters!") Together, they have experimentally infested more than 1500 colonies with mites, then selected queens from the colonies that were good at VSH. In the best hives, the bees were able to detect and remove every reproducing mite. "The first time I saw this, I didn't believe my own eyes," Fernhout says.

At LIB, researchers hope that identifying genetic markers for VSH will help in breeding resistant bees without sacrificing other valuable traits. They are looking for individual bees that are hygiene superstars, with the help of videos like those Rausch is studying. Then, the researchers sequence these bees to identify markers. That will help refine a prototype DNA chip, recently developed at LIB, that could quickly tell breeders whether a bee carries those genes. Its queen could then be bred with bee strains that already perform well in their regions. The use of genetic markers for breeding has "been very successful in cattle and chicken, and I think it will be even more successful in the honey bee," says Kaspar Bienefeld, director of LIB. He hopes a genetic test will be available to breeders starting next year, at a cost of about $60 per test.

Other groups have launched similar campaigns. Leonard Foster, a molecular biologist at the University of British Columbia in Vancouver, Canada; genomicist Amro Zayed of York University in Toronto, Canada; and their colleagues are now completing a sequencing study of bees from about 1400 colonies across Canada. And the French National Institute for Agricultural Research and Arista Bee Research are sequencing other colonies. Geneticist Brock Harpur, who now leads the Purdue effort, is using his startup funding to sequence the genomes of mite-biting bees. The rising activity, Zayed says, suggests "we're ready for the breakthrough."

In Canada, one research team has been pursuing a different approach to quickly identifying bees with good mite hygiene. It is led by Foster, who grew up around bees; his parents were amateur beekeepers.

Foster notes that past efforts to identify genetic markers for mite resistance have tended to fail because honey bees have one of the highest rates of genetic recombination in the animal kingdom. The constant gene shuffling means that a DNA sequence used as a marker of a desired trait, such as VSH, can drift away from the gene or genes that actually control that trait, rendering useless any test that looks for the marker. (Whole genome sequencing promises to minimize this problem by finding markers close to the genes and thus less at risk of drift.)

To address the problem, 12 years ago Foster decided to search instead for proteins associated with desired traits, such as the ability to detect diseased larvae. The massive effort involved snipping and analyzing antennae from tens of thousands of bees taken from some 600 colonies across western Canada. Then, the researchers compared the proteins expressed in antennae from bees with good hive hygiene with those in the antennae of less finicky bees. (Researchers assess hygiene with what is called the freeze kill test. They place a bottomless tin can on the wax comb and pour in liquid nitrogen, killing the brood underneath. After 24 hours, the researchers count how many dead larvae the worker bees have removed.)

Foster's team found that certain levels of 13 antennae proteins correlate with good hygiene. When they used those markers to guide breeding, they found it could identify bees with promising traits just as well as the freeze kill test, the group reported in Scientific Reports in 2017. "They're demonstrating that it's feasible to use marker-assisted selection," says bee biologist and breeder Marla Spivak of the University of Minnesota in St. Paul. "It's superexciting."

Foster hopes to eventually commercialize the test, which requires antennae from about 25 bees and costs roughly $100. His group is now wrapping up the project, which also has identified protein markers for a dozen other desirable traits, such as honey production and disease resistance. "My hope is to see this used in a wide fraction of the industry," Foster says. "The dream is a gene pool so strong we won't have to worry about disease."

Even if new approaches make it faster and easier to create new, mite-resistant bee strains, getting beekeepers to use the insects could be a challenge. Creating a well-rounded bee that satisfies all the demands of beekeepers remains "really a tricky task," says biologist and bee breeder Ralph Büchler of the Kirchhain Bee Institute in Germany.

And getting a resistance trait into a queen is just half the battle. Keeping it in her offspring is also a challenge. That's because queens mate with multiple drones while flying up to 10 kilometers from their hive. The behavior provides beneficial genetic diversity to the colony but can undo a breeder's efforts if a resistant queen mates with drones that lack mite-fighting genetics.

Breeders can overcome this problem by artificially inseminating queens. It's a tricky technique that requires collecting semen from drones, anaesthetizing the queen with carbon dioxide, and then inseminating her under a microscope. Research institutes and amateur breeding groups help train beekeepers in the necessary skills, and there is increasing interest. LIB, for instance, has a 2-year waiting list for its course.

Another approach is to send queens to isolated mating stations, where the only drones available for mating are ones brought there by beekeepers. Many islands in the Netherlands, Germany, and Denmark host such stations, run by amateur beekeeping associations. One of these stations, operated by the Association of Tolerance Breeding, a group of about 150 German breeders, tries to provide mite-resistant drones by not treating their colonies with pesticides for at least one season—relying on natural selection to weaken or eliminate drones vulnerable to the parasite.

Economics may also slow the adoption of resistant strains. The cost of treating and living with mites is low enough that many commercial beekeepers don't see an advantage to buying improved, resistant queens. And many breeders—who can already sell every queen they produce—don't have an incentive to invest in selecting for Varroa resistance. Researchers predict that will change if the mite continues to develop resistance to amitraz, now the pesticide of choice in many countries. "If amitraz fails," Danka says, "the landscape changes overnight."

Fernhout and other breeders want to be ready for the eventuality. They are close, they believe, to creating a world in which mite-resistance genes are widespread in honey bee populations, and beekeepers can set aside their failing pesticides. Fernhout, now 55, has a timeline in mind: "I just want to have resistant bees when I retire."