Right: This bee is infested with varroa mites. Varroa mites and microscopic tracheal mites are associated with a die-off of honey bees that has devastated beekeepers in the Northeast.

In the spring of 1993, entomologist Maryann Frazier encountered a mystery. "Beekeepers began calling to report that they had no bees in their colonies," she recalls. "When bees don't have enough food over the winter, beekeepers often will find a big cluster of dead bees in the colony. But these keepers were saying that not one bee was left in their colonies. This was very weird. They had seen bees making flights in February, but by April, there were no bees. What happened to them?" Frazier's investigation into the reasons the bees disappeared continues today. If she and her colleagues can't unravel the mystery of why bee colonies are dying, beekeepers, fruit and vegetable growers, and consumers all are likely to feel the consequences.

Imported from Europe nearly 400 years ago, honey bees spread rapidly throughout the New World. Currently, about 120,000 beekeepers own approximately 3 million colonies of honey bees across the United States. The bees produce a variety of beneficial products, including about 200 million pounds of honey valued at approximately $125 million each year and some 3.9 million pounds of beeswax valued at about $7 million. They are especially valued for the important role they play in pollinating crops. Domesticated honey bee hives can be placed wherever and whenever they are needed, and many beekeepers rent their bees, moving colonies several times during the season to help growers pollinate almonds, apples, blueberries, peaches, strawberries, and other crops. Across the country, more than 2 million honey bee colonies are rented for pollination each year. Nationwide, about $10 billion worth of crops are pollinated by honey bees.

The empty bee colonies raised an alarm about the state of these valuable pollinators. If you're imagining a summer without bee stings, you also should imagine a diet without apples, berries, cherries, melons, pears, plums, pumpkins, and other fresh fruits and vegetables--not to mention honey. "Pennsylvania apples alone are 95 percent dependent on bee pollination," says Frazier.

Millions of honey bees have died in the United States over the past five years, eliminating some beekeeping operations and creating a critical shortage of honey bees for pollination in some areas. In 1981, Pennsylvania had 85,000 commercial honey bee colonies. By 1995, the number had plummeted to less than 27,000 colonies. "That year, some large beekeepers lost 50 to 75 percent of their colonies," Frazier says. "Some beekeepers were wiped out. Because of the shortage of hives available for rent in Pennsylvania and other states, growers face higher pollination fees charged by beekeepers. Many growers--especially smaller orchards--have had trouble finding bees to rent for pollination." To help growers find pollination services, the entomology department maintains a list of beekeepers with hives to rent, which is available through Penn State Cooperative Extension county offices. Frazier and her colleagues also prepared a publication, Hives for Hire, which offers advice on how to contract with beekeepers, place colonies for maximum effect, and assess the strength of colonies.

Once, abundant wild colonies provided a measure of pollination security for fruit and vegetable growers. Today, wild honey bees are nonexistent in many areas. Homeowners have reported a near-absence of honey bees in their gardens. Estimates vary, but the Northeast may have lost 80 percent of its wild honey bees since 1990. Of the 13 wild colonies Penn State researchers located in the fall of 1995, 11 died during the winter of 1995-96.

Researchers have identified suspects in the colony deaths. Two parasites, tracheal mites and varroa mites, have been found in most of the weak and dying colonies. Tracheal mites are microscopic creatures that infest bees' breathing tubes. First found in the United States around 1983, they penetrate the tracheae of honey bees and feed on their blood, which damages the tracheal walls and may block the bees' breathing passages. Varroa mites, which appeared in this country in 1987, are external parasites large enough to be seen with the naked eye. These brownish-red, oval mites feed on the blood of both adult bees and the brood, or developing bees.

The situation has become so critical that the Department of Entomology recently hired a new faculty member to address the mite and disease problems afflicting honey bees. Scott Camazine specializes in honey bee research. In addition to his background as an entomologist, Camazine brings his training as a medical doctor to bear on the maladies affecting honey bees (see At Play in Fields Filled with Bees).

Camazine, Frazier, and others in the College are leading a multistate research and extension program aimed at fighting parasitic mites and educating beekeepers on the latest management practices. The effort, which includes faculty, post-doctoral scientists, graduate students, and technical staff, brings together land-grant universities, state departments of agriculture, and beekeeping organizations in Pennsylvania, New Jersey, Delaware, and Maryland. The College administers the program, conducts research, and provides training and information on honey bee management to extension educators from the four states.

"During the winter of 1995Ð96, colony losses ranged from 40 percent in Delaware to 53 percent in Pennsylvania to 80 percent in Maine," says Camazine. "We had a long, cold winter followed by a wet spring that year, but weather alone wouldn't cause such severe losses. The typical, average overwintering colony loss in Pennsylvania was just 10 percent before the arrival of tracheal mites."

Left: Jennifer Finley (left) and Maryann Frazier, key members of a regional effort to help beekeepers control parasitic mites, assess the levels of mite infestation in several of Penn State's honey bee colonies.

While researchers can place tracheal and varroa mites at the scene, they're not certain exactly how--or even whether--the mites themselves cause bee colonies to die. For instance, damage by varroa mites to individual bees is difficult to detect. Instead, researchers blame the colony deaths on "parasitic mite syndrome." "We believe tracheal and varroa mites are the major contributing factor in these colony deaths, but viruses and other pathogens, possibly transmitted by the mites, also may be involved," Camazine says. "We need to solve the mystery of exactly why these mite-infested bees are dying. Do infested bees have a lower cold tolerance? Do the mites directly damage bees? Do they spread viral infection or weaken the bees' immune system, allowing other diseases to kill them?"

Bees always have been subject to diseases, parasites, and predators. Some affect the brood, while others affect adult bees. Nosema, a protozoal infection, attacks the cell lining of a bee's digestive tract, giving it diarrhea and decreasing its life span. Chalkbrood is a fungal disease that kills developing larvae and pupae, giving them an appearance like moldy bread. Neither of these are significant threats to the entire colony, but the researchers suspect that mites may put stress on the colony, raising the impact of otherwise minor diseases. The end result is poor brood production, a dwindling colony population, and eventual colony death.

The researchers' first goal is to determine exactly how mite and mite-related diseases affect the health and productivity of honey bees. "With very difficult disease problems, we need to understand the pathology and physiology of the problem step-by-step," says Camazine. "We're interested not just in killing the mites, but in figuring out how the mites are killing the bees."

To identify pathogens possibly associated with the mites when the colonies start to die, the researchers plan to gather bees from mite-infested colonies in late winter and early spring. They will examine these bees for bacterial pathogens and use electron microscopes and laboratory tests to look for viruses. "Then we'll check the colonies in late spring to assess their survival," Camazine says. "This will help us identify pathogens that may be contributing to parasitic mite syndrome. To determine whether pathogens can be transmitted between the mites and the bees, we will attempt to infect varroa mites by letting them feed on diseased bees. Then we will try to transmit pathogens to newly emerged bees by having infected mites feed on them."

The researchers also will study how tracheal mites affect bees. "Tracheal mites may have a direct physical effect on the bees' ability to breathe, but even large numbers of mites within the trachea are unlikely to completely stop air flow," Camazine notes. "We think these mites may have a more subtle pathological effect. In summer, when it's warm, infested bees may be able to fly normally. But in early spring, when temperatures are cool, these bees may be unable to sustain flight and body temperature because they can't breathe efficiently."

To test that theory, Camazine wants to get the bees exercising and measure their activity, just as doctors do when humans with heart disease take exercise tolerance tests. He and Penn State biologist James Marden are conducting flight endurance studies of bees at room temperature and comparing the results with flight performance tests conducted at colder temperatures. They also are investigating whether any measure of flight physiology changes when bees are infested with tracheal mites. "Since mites attack the breathing tubes that supply the bee's flight muscles with oxygen, perhaps infested bees don't get enough oxygen to their flight muscles," Camazine explains. "These muscles may atrophy as a result of mite feeding, and the bee may be unable to fly or maintain its body temperature." To find out, the researchers are measuring the bees' carbon dioxide production. "We put bees in a sealed chamber with oxygen flowing through it," Camazine says. "Then we test the gas emitted for carbon dioxide. The amount of carbon dioxide the bees exhale is a measure of their metabolic rate. When we strike the chamber so the bees fly around, we can compare the amount of carbon dioxide they give off when exercising. This will tell us if there's any difference in the endurance of healthy and infested bees."

Right: Maryann Frazier prepares to treat an infested colony for tracheal and varroa mites. She says that although fewer colonies were lost in 1996 and 1997, beekeepers can't be complacent about mites.

If there is a difference, it could explain the absence of bees in some dead colonies. "During winter, bees eat honey, but they won't defecate inside the hive," Frazier explains. "Instead, they wait for a relatively warm day to go outside. Infested bees in cold weather might go out for cleansing flights, but if they can't get their metabolic rate and flight capacity up to what it should be, they can't return from the flight and die in the field."

Tracheal mites also may have an indirect effect on bees by impairing their immune systems and making them more susceptible to pathogens. Working with entomologist Diana Cox-Foster, the researchers are looking at enzyme systems that bees use to fight disease. "While sucking the bee's blood, tracheal mites also inject saliva and other substances that the bee's body reacts to," says Camazine. "The tracheae tend to get very dark and blackened, a reaction that may be caused by the enzyme system we are studying. Perhaps a response to the tracheal mite saliva prevents air exchange across the tracheae, or perhaps the bees are mounting an immune response against the tracheal mite injections that causes some inadvertent damage to their own bodies. We have to find out more about the honey bee immune system in order to use what we discover to deal with the problem."

Since mites, especially tracheal mites, may adversely affect honey bee queens, the researchers also embarked on a project to look at the health and productivity of honey bee queens. "Many beekeepers reported a loss of queens," says Camazine. "We wondered whether the mites were affecting queens directly. There are about 40,000 workers in a colony, and one queen, who lays all the eggs. If she doesn't lay eggs well, the colony will be weak and there won't be enough bees to sustain the colony. We need to know whether tracheal mite infestation of the queen is a significant factor in poor colony performance."

To find out, the researchers obtained new queens from commercial breeders and collected data on tracheal mite infestation, queen ovary development, and other factors. In the course of this effort, the researchers examined queens from commercial breeders across the country. "We ordered 15 queens from each breeder, then performed autopsies on them," says Camazine. "We analyzed the pheromones they give off, which control colony behavior. We looked at the thorax for tracheal mite infestation and the intestine for nosema disease. We took out the ovaries to assess their quality and measured levels of sperm stored in the spermatheca, the little sac that holds the sperm after the queen mates. We found remarkably variable results." Almost 75 percent of one breeder's queens were infested with tracheal mites, while another had no infested queens. About 40 percent of some breeders' queens were infected with nosema. "We were surprised to find that much disease among the queens supplied by breeders," Camazine notes. "These findings should alert beekeepers that not all queens are of equal quality. We're also sending individual letters to the breeders explaining the results of our study, which should suggest that they need to keep their queens healthier."

Since queens play such a major role in the health of an entire hive, having beekeepers requeen their colonies more frequently might be a quick fix for some mite problems. "The current recommendation is to requeen every two to three years," Camazine says. "But it costs only $10 to requeen a colony, while a new colony can cost up to $50. Requeening wouldn't solve the mite problem, but it could be an important component of an integrated approach to mite management for beekeepers."

Any new mite management strategies are likely to be eagerly embraced by beekeepers. "Beekeeping is no longer possible without close attention to mite control, but there are few proven options available to protect colonies," says Frazier. "Unless new control methods are developed and beekeepers are educated on proper management practices, we expect continued losses of colonies in the years to come. An effective program for controlling parasitic and mite-related diseases will incorporate a number of different control methods and practices. You can't just rely on chemical control, because the mites will develop resistance over time. Our goal is to develop methods that are safe, effective, sound, economical, and long term."

Currently, only one material--Apistan--is available for treating varroa mites. "Mites in Italy develop resistance to it within five years," notes Frazier. "If we lose this material, we have nothing to replace it with." Desperate beekeepers are trying everything they can to keep their hives alive. "They are using the materials that are registered for control, like Apistan strips for varroa mites," Frazier says. "But because that's the only thing that's available, the price for Apistan keeps rising, and the beekeepers are very discouraged. In addition, beekeeping is such a small industry that chemical companies may be reluctant to invest in registering new materials."

A variety of alternative synthetic and botanical compounds, especially essential oils, are being marketed as mite control agents, including thymol and wintergreen oil. "But more work needs to be done to determine whether any of these agents are effective for mite control," Frazier says. "There's no published data that proves they are effective mite treatments."

Other materials that do show promise aren't yet legal in this country. Formic acid, legal in Canada, is naturally present in honey. Some insects produce it as a defense mechanism. Because it's extremely caustic, mites are unlikely to develop resistance to it. "Some beekeepers feel that the approval system is not acting fast enough," Frazier says. "A lot of work has been done to show that formic acid is effective and can be used safely. Beekeepers read these things, and even though it's not approved, they see no reason not to use it."

But formic acid needs to be applied over a long period of time. Most beekeepers are using it in liquid form, which evaporates in a day or two, so they have to reapply it six to eight times. Research associate Jennifer Finley is collaborating with U.S. Department of Agriculture scientists to study a slow-release formic acid gel. "New, safer formulations, such as the incorporation of formic acid into a gel, would let beekeepers put it on once or at most twice. It will last in this gel, which evaporates slowly over a period of several weeks," she says.

The researchers also are looking for resistant strains of honey bees. "We have evaluated strains of honey bees reported to have some resistance to mites," says Camazine. "So far, these strains have not showed significant resistance, but we are still evaluating one strain of bees."

Until the researchers can pinpoint exactly why the colonies are dying and develop specific management techniques, a general guideline is that more seems to be better when it comes to controlling mites. Finley and her colleagues asked beekeepers to provide information on their colony losses in the 1995Ð96 season, including the time of year colonies died, the mite and disease incidence in their colonies, and the treatments they applied after harvesting honey. Of the 6,054 colonies included in the survey, 3,030 died during the winter. "Fifty-three of the 252 beekeepers who responded did not apply any kind of treatment for mites or disease," Finley says. "On average, these beekeepers lost 71.6 percent of their colonies, which was significantly higher than the loss rates of beekeepers who applied at least one treatment." Apistan was the most popular treatment, with 70 percent of beekeepers using it. Other beekeepers applied menthol for tracheal mites, Fumidil-B for nosema, or grease patties infused with an antibiotic. Each individual treatment reduced average colony losses at least marginally, and the antibiotic patties, Apistan, and Fumidil-B significantly decreased losses. Twelve beekeepers who treated with a combination of antibiotic patties, menthol, Apistan, and Fumidil-B had the lowest colony loss rate--about 25 percent. "These beekeepers really reduced their losses compared to those who didn't treat at all," says Finley. "Those who are treating aggressively are having the best luck keeping overwintering colonies alive. Our recommendation to beekeepers is to hit mites with everything you've got. Many beekeepers tend to skip antibiotic and Fumidil-B treatments. These don't treat mites, but they really help mite- infested colonies."

To spread the word about managing the mites, Frazier, Finley, and Camazine talk to beekeepers at meetings and maintain a World Wide Web site that includes information on current research and recommendations (http://www.psu.edu/dept/beehive/index.html). They publish a regional newsletter called Bee Aware. They also developed the Bee Aware expert system, a computer-based tool that includes an informational module, as well as a module that beekeepers can use to diagnose diseases and get information about how to treat them.

The honey bee outlook for the near future is mixed. "Colony losses weren't as bad in 1996 and 1997," says Frazier. "We don't know precisely why. The winter wasn't as harsh, which might have helped. The bees may be developing some resistance, the disease may be going through cycles, or the beekeepers simply may be treating more aggressively. Also, in the previous season, so many infested colonies died that we decreased our load of diseased colonies. There wasn't as much transmission of disease from one colony to another, because a large proportion of mites died with the infested colonies. The 1995-96 winter was particularly bad, and it may be another five years until it's that bad again. But beekeepers can't afford to be complacent."

Faculty and staff referenced in this article are Scott Camazine, assistant professor of entomology; Diana Cox-Foster, associate professor of entomology; Jennifer Finley, research associate in entomology; and Maryann Frazier, senior extension associate in entomology. James Marden is an assistant professor of biology in the Eberly College of Science. Research discussed in this article has been funded by the Pennsylvania Department of Agriculture and the Fund for Rural America of the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture.