Mysterious Missing Bees

Researchers find major new suspect for Colony Collapse Disorder

By Cary Shimek

Lance Sundberg’s visits to that cursed bee yard were like a punch to the gut.

As owner of one of Montana’s largest beekeeping outfits, Sunshine Apiaries of Columbus, Sundberg and his crew were in California near Turlock Reservoir to help pollinate the massive almond crop. It was January 2008, and they had trucked in 18 semi-loads carrying 6,000 hives.

Then his bees started vanishing.

Each day Sundberg went to the yard in his green beekeeping suit to find more of his honeybees had disappeared. The adult insects were simply gone, abandoning hives with plenty of honey, pollen and brood – the eggs and young developing bees.

“When you go into your yard and find that kind of phenomenon, you immediately get sick to your stomach,” he says. “And the (bee) colonies just kept going downhill.”

Sundberg hoped he was dealing with one of many known honeybee maladies, such as varroa mites, because he had heard of a mysterious new “disappearing disease” afflicting beekeepers on the East Coast. At that time he had never heard the term Colony Collapse Disorder.

But it wasn’t mites. CCD stole 38 percent of his bees in California that winter.

It was a huge hit to the Montana businessman, whose bees pollinate everything from avocados and blueberries to cherries and pears across a four-state region. He likes to say his bees help produce one of every 200 apples grown in Washington state.

Sundberg earns about $145 per colony for working the almond harvest. That horrible winter he was forced to purchase $150,000 in Australian bee packages to fulfill his contract.

“Bees are like a canary in a coal mine, and the canaries are tipping over right now,” he says. “And it doesn’t seem like a lot of people are listening.”

Forget horses, cattle, dogs and cats – honeybees are humanity’s most important animal partners. European honeybees went with settlers around the globe and are now used by beekeepers worldwide. In addition to producing honey, bees are required to pollinate the crops that become one-third of all human food. In other words, if CCD wiped out all honeybees in the world tomorrow, a lot of us would follow.

University of Montana researchers now have a major new suspect for CCD: a one-two punch combination caused by an insect virus, previously unknown to North American bees, paired with a fungal pathogen. The research was published Oct. 6 in PLoS ONE, a scientific journal found online at http://www.plosone.org.

When the paper was released, it generated headlines worldwide. The story made the front page of the Missoulian, the front of The New York Times and was featured on “CBS Evening News With Katie Couric” all in the same day – one of the biggest splashes ever for any UM news story.

Research Professor Jerry Bromenshenk is a senior scientist with the UM team that made the breakthrough. An eastern Montana native, Bromenshenk has studied honeybees for decades, learning to use their fuzzy, dust-mop bodies as detectors for pollution, biological hazards, explosives and even buried land mines. He and his partners also are hard at work creating a handheld device that listens to bee buzzing to determine whether the insects are under attack by poisonous chemicals or parasites.

In 2003 the Montana Board of Regents approved creation of Bee Alert Technology Inc. to market UM’s bee breakthroughs, and Bromenshenk and his crew moved off campus in 2004 to a new headquarters at 1620 Rodgers St. in Missoula. The business employs four people in the winter and up to 12 during the busy summer months. The company keeps the lights on by doing bee sampling and contract research, and Bromenshenk hints that some of their major projects are ready to go commercial.

Distressed beekeepers first started complaining about a mysterious new disappearing disease stealing up to 90 percent of their colonies in 2006, and Bee Alert employees attended a March 2007 U.S. Department of Agriculture gathering of national experts trying to unravel the cause. The scientists had different viewpoints, and this original working group soon diverged into factions studying environmental factors, pesticides and pathogens.

Following the pathogen path, the Bee Alert team applied for $4 million in available CCD research funding, but the grant application was rejected. From that point on, support came from “bits and pieces everywhere,” Bromenshenk says. The Almond Board of California and the National Honey Board provided some money, and the U.S. Army offered certain services free of charge. But for the most part, the UM group tackled the CCD mystery with limited means.

The Montana researchers sampled hives struck by CCD in Pennsylvania, California and the Southeast during those early months. Then a group of Penn State researchers published a Sept. 7, 2007, article in Science that claimed Israeli acute paralysis virus of bees “was strongly correlated with CCD.”

It appeared the mystery was solved. But results from this early study didn’t hold up.

“The analysis we did suggested there wasn’t any consistent connection between (the Israeli virus) and the CCD problem,” Bromenshenk says. “In fact, we found more markers for that virus associated with healthy bees than with sick bees.

“That group worked with a preconceived set of viruses – basically the usual suspects,” said Colin Henderson, a Bee Alert researcher and UM College of Technology faculty member. “So they were working from a group of known bee pathogens to try to see if those were causing a completely new disease. And it just didn’t hold up.”

With the race to solve the mystery still on, the Bee Alert crew caught a huge break in 2008 when a small businessman from nearby Florence contacted them. David Wick runs BVS Inc., which detects biological viruses. He called and asked, “How would you like a better way of looking for viruses?”

Wick happens to have a brother in a similar line of work, Charles Wick, who is a senior scientist at the Edgewood Chemical Biological Center. The U.S. Army research laboratory in Maryland is tasked with protecting soldiers from biowarfare agents. Researchers there developed a mass spectrometry bioinformatics capability that can quickly identify all the peptides (short lengths of proteins) in any given sample. Every virus, fungus and bacterium has its own unique peptide signature. The Army agreed to use its powerful new capability on samples gathered by the UM researchers even though it had never worked with bees before. This was an opportunity to demonstrate the capability of the new technology on environmental samples.

Henderson says the beauty of the Edgewood method is that it identifies everything in a sample, and these peptide signatures are then cross-referenced with indexes of millions of peptides that scientists worldwide have identified over the years.

“So we basically ground up our bee samples and sent them off to Edgewood,” he says. “They extracted the proteins, digested them into peptide fragments, ran them through the mass spec method that yielded the peptide sequences and compared that to the global library. And we wound up with 30,000 unique proteins from all kinds of critters that showed up in the bees.”

Instead of looking for the usual suspects, UM researchers cast a wider net in their search for a CCD solution. They had no preconceived notions about what they were going to find, but their more inclusive approach eventually paid dividends.

Phillip Welch, a UM grad and Bee Alert senior laboratory analyst, still has that piece of paper with a graph dated April 23, 2008. It represents his “Eureka!” moment.

Welch was one of the people tasked with the unenviable job of sifting through the mountain of data produced by Edgewood from the bee samples.

“I probably had more than 100 hours working on this thing, sitting right here,” Welch says, pointing to the modest conference room table at Bee Alert. “The database is just these massive lines of numbers that we have to break down into grids that we eventually use to produce graphs.”

Henderson says they filtered out all the plant viruses in their voluminous dataset, as well most of the bacteria and fungi, to get to a reasonable virus group that could potentially infect insects. The original samples came from sick, dying and healthy honeybee populations.

Welch says something called insect iridescent virus (IIV) kept showing up in all the infected samples.

“It stuck out for sure,” he says. He printed the graph to show Bromenshenk. “What’s an iridescent virus?” Welch asked.

Bromenshenk didn’t have the foggiest idea. He soon contacted Mexico-based researcher Trevor Williams, a leading expert on insect viral ecology. He says the viruses often hide covertly in their hosts until something triggers them, and then they are almost invariably lethal.

“Virus populations often hide in the host’s genetic code until some trigger activates that segment of DNA,” Henderson says. “Then they start spitting off viral particles and killing cells.”

Williams noted that an iridescent virus was known to infect Asian honeybees, but it was unknown among the European species used by most beekeepers. Maybe the virus had jumped the Pacific.

Henderson says the iridescent virus is a DNA virus, and this is significant because most common honeybee viruses are RNA viruses. DNA viruses use a different infective path.

“Other researchers were focused on RNA viral diseases, and they got real tunnel vision,” he says. “And the only way we found it – I’ll be honest – is that we didn’t know what we were looking for.”

Bromenshenk says iridescents are big compared to other viruses. They get their name because if they become packed together within infected tissue, they can bend light and create a bluish or greenish color. Adult bees with CCD generally disappear, but some beekeepers claim to have spotted green larvae.

When the researchers sifted through the Edgewood data, Henderson was charged with applying advanced statistic programs to the information to discover any unusual associations. He soon learned that CCD-infected bees had different assemblages of viruses and other substances than those in healthy hives.

And the infected colonies with the new iridescent virus had something else every time – a fungus called Nosema ceranae.

“We found a 100 percent occurrence of only two pathogens in every CCD case,” Henderson says, “and it was those two.”

Common Nosema apis is well known to beekeepers and researchers. The fungus attacks bee abdomens, the insects start defecating and it looks like little yellow rain. It’s easy to spot. Nosema apis generally makes bees sick in the spring, but the ceranae variety, which Bromenshenk and fellow researchers suspect might be another Asian transplant, seems to stay active year-round and lacks the apis symptoms.

“At the microscopic level, the fungus looks like something out of an ‘Alien’ movie,” Bromenshenk says. “It has a long filament called a polar tube that it uses to penetrate cell walls of its host so it can use nutrients inside to replicate and eventually kill the host cell.”

Nosema is a common bee disease, and beekeepers have medications available to help knock it down within infected hives. It can negatively impact a beekeeper’s bottom line, but it’s generally more of a nuisance than a major problem. Scientists had noticed that Nosema often was present at CCD-infected hives, but it also is widespread within fairly healthy hives.

But what happens when the iridescent virus and the fungus get together?

“Each of these things alone can make bees sick,” Henderson says. “We don’t know if it’s the two of them together that make bees so sick or if one comes in first to make the bee more susceptible to the secondary infection or co-infection. But the fact is that the two together are found in hives with massive die-offs.”

When the UM researchers realized how important Nosema ceranae was to their CCD theory, they added a new member to their team: Robert Cramer, a fungal pathologist at Montana State University-Bozeman.

“When Nosema gets inside a cell, it keeps growing until there is no more room for the fungus, and then the cell bursts open,” Cramer says. “The fungus can then go and infect other cells in the gut. Then you add in the virus. We theorize that the bees get one infection or the other, and this causes the bees to become stressed, which then allows the second infection to come in and more effectively cause disease.”

Using the connection with Williams, the iridescent virus specialist at the Instituto de Ecologia in Mexico, the Bee Alert team was introduced to insect virus specialist Shan Bilmoria at Texas Tech University. Bilmoria had studied iridescents for use as a possible biopesticide. The Texans provided an iridescent virus similar to the one that turned up in the CCD hive samples. This virus, however, infects wax moths, which often are found in beehives. It wasn’t a perfect match, but it was close.

Cramer did some inoculation experiments with Nosema and the wax moth iridescent.

“Dr. Cramer figured out how to infect bees with both pathogens,” Henderson says. “He did some laboratory inoculations with each pathogen alone and then both together. There was a significant increase in mortality when both were present.”

“And wouldn’t you know it, he ended up with the occasional wax moth or bee that turned green,” Bromenshenk says.

Bromenshenk has seen a microcosm of CCD play out right in his office.

Bee Alert maintains several bee colonies for its research, and – somewhat ironically – one winter most of them were wiped out by CCD. Bromenshenk kept a fancy glass hive in his office with a tube that gave access to the outdoors. That winter he decided to populate the hive with the queen and a few stoic honeybee survivors from a colony that nearly had been wiped out by the disappearing disease.

“It was amazing to watch,” he says. “The first thing they did was produce another queen, which is rare, and both those queens just laid their hearts out. Soon they were pretty recovered. But then I came back from a business trip, and it was like a hammer had been dropped on them. We watched as CCD swept through them, and their population dropped down to only one queen and six bees.”

The researchers monitored the oscillations of the fungus and virus loads. They repeatedly would see the populations drop as the pathogens peaked. Bromenshenk says the bees would start recovering a bit, the diseases would rebound, and “Boom! The bees would drop back down again.”

Since that time UM has protected its research colonies by working aggressively to control Nosema with fungicide. Bromenshenk also recommends beekeepers keep their hives away from damp, foggy areas, as there have been more reports of CCD outbreaks in such places or after dramatic rain events. Improving nutrition also seems to help, but occasional CCD attacks on the Bee Alert hives and others nationwide continue.

“Our research is a work in progress,” Bromenshenk says, “but it may be the most important advance for the cause of CCD in the previous three years.”

“I would say we are probably 85 percent certain that we are on the right track for the cause,” Henderson says. “We found the initial correlation between the two pathogens, and it’s not a casual correlation. We also have done inoculation experiments with closely related species and found higher mortality in bees infected with both pathogens. We want to isolate and identify the Nosema virus we see with CCD and then infect some whole colonies. If that produces symptoms of CCD, that would seal the deal for us.

“That’s the next step,” he says. “We need to re-infect with the exact virus and fungus, observe for disease development and then pull the agents back out. That’s standard scientific procedure.”

“We aren’t claiming cause,” Bromenshenk says, “but we have multiple lines of evidence. We’ve got bees from all parts of the country that have a commonality with these two types of pathogens.”

Even if the pairing of the virus and fungus doesn’t turn out to be the cause of CCD, he says, the presence of a new iridescent virus preying upon European honeybees alone is an exciting discovery.

The UM team strongly suspects the iridescent bee virus came to America from Asia. Bromenshenk says that 20 years ago India faced major collapses and a “clustering disease” among Asian honeybees. British virologists noted that infected colonies often had an iridescent virus associated with the Nosema fungus. Those scientists suggested against intermixing European honeybees with their Asian counterparts.

“But guess what?” Bromenshenk says. “They intermixed.”

One question still begs to be asked: Why do CCD-afflicted bees disappear?

“There are a variety of ideas on that,” Bromenshenk says. “I think it’s that they are sick with something affecting their digestive systems, and they leave the hives and just don’t have the energy to make it back. It would be like if you or I hiked into the wilderness and got an incredibly bad stomach flu. We might not make it back either.”

Sundberg, the Montana beekeeper, hasn’t experienced extremely heavy CCD losses since that horrible 2008 winter. He updated the nutrition for his honeybees – going to a straight sucrose feed predominantly – and this seems to have bolstered the ability of his hives to resist CCD. But the disappearing disease continues to steal some of his charges every year, and he still hears of Big Sky beekeepers losing 50 percent or more of their hives to the mysterious illness.

CCD is to beekeepers what hailstorms are to farmers: an unlucky bolt from the blue.

“But I think (this UM work) will help in the long run,” Sundberg says. “If you are dealing with an unknown and you don’t know how to deal with it, it’s extremely disheartening. Now if it’s an iridescent virus and Nosema ceranae, as least you know what got you there.

“Our research community has really stepped up to the plate on trying to figure out different aspects of beekeeping,” he says. “I think the American public needs to know we need to keep fighting for beekeepers, and we need to keep the research finances coming.

“Maybe then we can finally figure this whole phenomenon out.”

For more information, email Bromenshenk at beeresearch@aol.com or Henderson at colin.henderson@umontana.edu.