The Mysteries of Colony Collapse

May 15, 2014

COLONY COLLAPSE DISORDER (CCD) of honey bees is one of the lingering mysteries of early 21st Century science in more ways than one: from its microbial, immune system and genetic components to an amorphous almost Orwellian terminology as imprecise and ambiguous as climate change (a slogan wide enough to encompass warming up, cooling down, and even staying the same temperature while the numbers fluctuate around the mean or average). The ambiguous language says both nothing and everything simultaneously, though underlying CCD is a quest for as yet unknown changes in insect rearing circumstances that will produce non-collapsing honey bee colonies. During the 19th century (1800s), a century marked by worldwide famines in the the old colonial empires and phylloxera-ravaged wine-grape vineyards collapsing in France, a revolution in modern medicine was being birthed in the mysteriously collapsing silkworm colonies. Fortunately for lovers of silk fabrics, fashion and textiles, 19th century silkworm farmers had the services of the real-life scientific Sherlock Holmes of the era, the famous French freelance scientist and sometime entomologist, Louis Pasteur.

Pasteur had a knack for solving applied problems like fermentation (beer, wine, vinegar) and silkworm colony collapse, and then using the results to develop broader theories like germ theory, which taught modern doctors to wash their hands and sterilize their instruments so as to stop spreading the germs that commonly killed their patients. How Pasteur almost single-handedly accomplished so much more than whole scientific institutes seemed able to do in the 20th century was the subject of an illuminating mid-20th century book, Louis Pasteur Free Lance of Science, by French-borne microbiologist Rene Dubos. “Toward the middle of the nineteenth century a mysterious disease began to attack the French silkworm nurseries,” wrote Dubos. “In 1853, silkworm eggs could no longer be produced in France, but had to be imported from Lombardy; then the disease spread to Italy, Spain and Austria. Dealers procuring eggs for the silkworm breeders had to go farther and farther east in an attempt to secure healthy products; but the disease followed them, invading in turn Greece, Turkey, the Caucasus–finally China and even Japan. By 1865, the silkworm industry was near ruin in France, and also, to a lesser degree, in the rest of Western Europe.”

“The first triumphs of microbiology in the control of epidemics came out of the genius and labors of two men, Agostino Bassi and Louis Pasteur, both of whom were untrained in medical or veterinary sciences, and both of whom first approached the problems of pathology by studying the diseases of silkworms,” wrote Dubos, who between World Wars I and II worked at the League of Nations’ Bureau of Agricultural Intelligence and Plant Diseases as an editor of the International Review of the Science and Practice of Agriculture. “A disease known as mal del segno was then causing extensive damage to the silkworm industry in Lombardy. Bassi demonstrated that the disease was infectious and could be transmitted by inoculation, by contact, and by infected food. He traced it to a parasitic fungus, called after him Botrytis bassiana (since renamed Beauveria bassiana, a widely used biocontrol agent)…An exact understanding…allowed Bassi to work out methods to prevent its spread through the silkworm nurseries. After twenty years of arduous labor, he published in 1836…Although unable to see the bacterial agents of disease because of blindness, Bassi envisioned from his studies on the mal del segno the bacteriological era which was to revolutionize medicine two decades after his death.”

Chemist Jean Baptiste Dumas, Pasteur’s mentor, prevailed upon the reluctant free lance scientist to head a mission of the French Ministry of Agriculture. “Although Pasteur knew nothing of silkworms or their diseases, he accepted the challenge,” wrote Dubos. “To Pasteur’s remark that he was totally unfamiliar with the subject, Dumas had replied one day: ‘So much the better! For ideas, you will have only those which shall come to you as a result of your observations!’”

A way of life was also at stake. As described in 19th century France by Emile Duclaux, Pasteur’s student and intimate collaborator (in Dubos’ book): “…the cocoons are put into a steam bath, to kill the chrysalids by heat. In this case, scarcely six weeks separate the time of egg-hatching from the time when the cocoons are carried to market, from the time the silk grower sows to the time when he reaps. As, in former times, the harvest was almost certain and quite lucrative, the Time of the Silkworm was a time of festival and of joy, in spite of the fatigues which it imposed, and, in gratitude, the mulberry tree had received the name of arbre d’or, from the populations who derived their livelihood from it.”

“The study of silkworm diseases constituted for Pasteur an initiation into the problem of infectious diseases,” wrote Dubos, who was influenced by the famous Russian soil microbiologist, Serge Winogradsky, who favored studying microbial interactions in natural environments rather than in pure laboratory cultures. “Instead of the accuracy of laboratory procedures he encountered the variability and unpredictability of behavior in animal life, for silkworms differ in their response to disease as do other animals. In the case of flacherie (a disease), for example, the time of death after infection might vary from 12 hours to 3 weeks, and some of the worms invariably escaped death…Time and time again, he discussed the matter of the influence of environmental factors on susceptibility, on the receptivity of the ‘terrain’ for the invading agent of disease. So deep was his concern with the physiological factors that condition infection that he once wrote, ‘If I had to undertake new studies on silkworms, I would investigate conditions for increasing their vigor, a problem of which one knows nothing. This would certainly lead to techniques for protecting them against accidental diseases.’”

“Usually, the public sees only the finished result of the scientific effort, but remains unaware of the atmosphere of confusion, tentative gropings, frustration and heart-breaking discouragement in which the scientist often labors while trying to extract, from the entrails of nature, the products and laws which appear so simple and orderly when they finally reach textbooks and newspapers,” wrote Dubos. “In many circumstances, he developed reproducible and practical techniques that in other hands failed, or gave such erratic results as to be considered worthless. His experimental achievements appear so unusual in their complete success that there has been a tendency to explain them away in the name of luck, but the explanation is in reality quite simple. Pasteur was a master experimenter with an uncanny sense of the details relevant to the success of his tests. It was the exacting conscience with which he respected the most minute details of his operations, and his intense concentration while at work, that gave him an apparently intuitive awareness of all the facts significant for the test, and permitted him always to duplicate his experimental conditions. In many cases, he lacked complete understanding of the reasons for the success of the procedures that he used, but always he knew how to make them work again, if they had once worked in his hands.”

Though famed for disproving the spontaneous generation of life, immunization via attenuated living vaccines and the germ theory of infectious disease: “Pasteur often emphasized the great importance of the environment, of nutrition, and of the physiological and even psychological state of the patient, in deciding the outcome of the infectious process,” wrote Dubos. “Had the opportunity come for him to undertake again the study of silkworm diseases, he once said, he would have liked to investigate the factors which favor the general robustness of the worms, and thereby increase their resistance to infectious disease…A logic of Pasteur’s life centered on physiological problems is just as plausible as that which resulted from the exclusive emphasis on the germ theory of contagious disease.”

The 21st century is riddled with insect colony conundrums and mysteries. For example, why among the social insects are honey bees plagued by Colony Collapse Disorder, while “Colony Expansion Disorder” prevails for other social insects in the USA. Rather than collapsing, USA colonies of Argentine ants are forming “super-colonies,” and red imported fire ant colonies are growing stronger by the day and annually expanding their North American geographic range; this despite being deliberately dosed with pesticides and attacked by biocontrol organisms (perhaps even more so than the beleaguered honey bees). And quite independently of mortgage rates and housing sales, Formosan subterranean termite colonies damaging billions of dollars of USA housing stock are happily munching away at both live trees and “dead-tree” wooden housing assets with little collective danger of colony collapse, though individual colonies come and go.

Perhaps beekeeping and crop pollination would be easier if Colony Collapse Disorder were an actual “disorder” as defined by the Diagnostic and Statistical Manual of Mental Disorders (DSM), and honey bees were endowed with sufficient consciousness and behaviors amenable to bee psychology or psychiatry.

The very real plight of honey bee colonies or hives is still in what Dubos would call the “atmosphere of confusion, tentative gropings, frustration.” At the most recent Entomological Society of America annual meeting, roughly a century and a half after silkworm colony collapse was eliminated by better more sanitary rearing practices, honey bee health was still puzzling. Honey bee colony loss in Virginia increased to 30% from 5-10% in recent years, possibly due to disease pathogens, pesticides and immune system suppression, say Virginia Tech researchers (e.g. Brenna Traver) studying glucose oxidase (GOX), an indicator of immunity in social insects. Honey bee social immunity is complex, involving factors as diverse as pheromones and grooming, and honey bee production of hydrogen peroxide (H2O2), which sterilizes food for the colony.

Nosema ceranae, a global gut pathogen, was seen all around the USA in 2007 at the same time as Colony Collapse Disorder. Black queen cell virus is another culprit, along with deformed wing virus, which is spread among honey bees by varroa mites. Then it is hard to overlook that over 120 different pesticides and their metabolites have been found in honey; including common beekeeper-applied pesticides such as coumaphos, fluvalinate, chlorothalonil and the antibiotic fumagillin. At the University of Puerto Rico, Gloria Dominguez-Bello is testing oxytetracycline and other commonly used antibiotics for their effects on honey bee microbes similar to those known to affect everything from obesity and brain function to organ transplants.

Those familiar with Pasteur’s entomological research on silkworm colony collapse in the 1800s would have experienced a sense of deja vu at the most recent Entomological Society of America meetings listening to Gloria DeGrandi-Hoffman, a research leader at the USDA-ARS Carl Hayden Bee Research Center in Tucson, Arizona. Nutrition, stress and pesticides may indeed be involved, but more focus is warranted for honey bee microbial health and gut microbes. Honey bee nutrition and microbiology is complicated by seasonal variations with changing food sources. According to DeGrandi-Hoffman, a lack of beneficial microbes may set honey bees up for infectious diseases like chalkbrood.

For example, pesticides used for Varroa mite control and potent beekeeping antibiotics like thymol and formic acid can affect the Lactobacillus microbes bees need for digestion and preservation of pollen as beebread, said DeGrandi-Hoffman. When bacterial plasmids found in high numbers in beebread are plated with the pathogen Aspergillus flavus, the pathogen rapidly loses virulence.

It is likely honey bees rely on beneficial microbes to protect from harmful pathogens, as honey bees have among the fewest immune system genes of any insect. Thus, when California almond growers spray fungicides, insecticides and miticides, a side effect could be fewer beneficial microbes in honey bee guts and in beebread. Thus, the honey bees would be less healthy and more susceptible to diseases like chalkbrood. Probiotic supplements designed to add beneficial microbes to honey bee diets are being tested in some California orchards. No doubt a familiar concept to those shopping for probiotic yogurts.

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Honey Bees, 24-Hour Surveillance Cameras & Pesticides

February 22, 2011

HONEY BEE HEALTH had the entomologists buzzing and the grad students searching for answers at the Entomological Society of America (ESA) annual meeting in San Diego. For several years now specialists have been spinning speculative theories as to why the pollinating honey bees of commerce, mostly the species known as Apis mellifera, have been in such sad shape. Isaac Newton had the proverbial apple bonk him awake to gravity. Bee entomologists have not yet had that magical bee sting in the butt “Aha” moment.

But there seems no getting away from the problem, as keepers of bee hives an ocean away from the USA are also getting stung with big losses, from what is dubbed colony collapse disorder (CCD). What has entomologists scurrying to their Petri dishes and bee hives and firing up surveillance cameras, chromatographs and mass spectrometers is a study titled “High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health.” Christopher Mullin of The Pennsylvania State University, a self-described connoisseur of how poisons work, and several colleagues “found 121 different pesticides and metabolites within 887 wax, pollen, bee and associated hive samples” from 23 states and one Canadian province. Enough to induce sleep-loss and second thoughts about the health and sleep-inducing effects of commercial honey products.

Surveillance cameras, 24-hours a day, are the best way to monitor and gather numerical data on how pesticides affect honeybees, Cornhusker grad student Bethany Teeters told the ESA in her prize-winning poster, “Bees under surveillance.” Being more video than even an insomniac can sanely watch, the University of Nebraska-Lincoln entomology lab delegates the task to “state-of-the-art detection” software: namely EthoVision XT, which Noldus Information Technology calls “the most widely applied video tracking software that tracks and analyses the behavior, movement, and activity of any animal” from “lab animals in mazes to farm animals in stables.” No doubt what Geoge Orwell would have used in his Animal Farm novel, had he written it in 2011 rarther than 1946.

“Honey bees are exposed to sublethal doses of pesticides on a regular, often chronic, basis,” Teeters told the ESA. “For instance, the pyrethroid tau-fluvalinate (Apistan(R)) is one of many pesticides applied directly into the hive to control the parasitic mite Varroa destructor. Although tau-fluvalinate is considered safe for honey bees, potential effects of sublethal intoxication remain unexplored.” Same goes for coumaphos, also used to treat for Varroa mites.

“Honey bees may also encounter sublethal doses of pesticides while foraging,” said Teeters. “Systemic pesticides, including the neonicotinoid imidacloprid, have become prominent in U.S. crop pest management. This raises concerns about the consequences of sublethal exposure to systemic pesticides in nectar and pollen that honey bees visit in addition to chronic exposure to residues in the hive. Decline in colony health has been associated with ppm (parts per million) pesticide residues in hive products, and the neonicotinoid can impair honey bee health at ppb (parts per billion) levels.”

Teeters surveillance videos of bees exposed to sublethal pesticide doses in Petri dishes revealed that bees exposed to tiny traces of tau-fluvalinate spend more time socially interacting. Bees exposed to imidacloprid spend less time socially interacting and more time eating. Next step is studies to see if this is true in actual honey bee hives, and whether colony health is impacted.

Natalie Boyle, a graduate student at Washington State University in Pullman, studied the effects of Varroa mite pesticides on honey bee hives in Moscow, Idaho. Honey bee adults stressed by miticide residues died sooner and did less reproductive swarming. But they compensated with increased brood production. “While our results are preliminary, if we find that pesticide residues in brood comb adversely affect colony health, it would suggest that regular brood comb replacement in beekeeping operations might be a suitable management strategy,” said Boyle. “Similarly, approaches to reduce miticide applications in beehives and pesticide exposure in agricultural field settings would be highly beneficial.”

Back at The Pennsylvania State University, graduate student Daniel Schmehl noted that the Varroa mite-killing chemicals coumaphos and tau-fluvalinate were found in almost every honey bee hive sampled in North America. Furthermore, these two chemicals were “associated with reduced queen weight and reduced ovary development.” After six days chronic exposure to tau-fluvalinate in cage studies, worker bees were less attracted to queen bees. This was possibly “due to changes in pheromone production from the queen or pheromone recognition by the workers.”

On the West Coast, at the University of California, San Diego, graduate student Daren Eiri explored how sublethal doses of the pesticide imidacloprid can subtly alter foraging habits in ways that weaken honey bee colonies. A common lab assay used to assess foraging is stimulating the honey bee antenna with sucrose, which elicits the proboscis (tongue) extension reflex (PER). PER is the lab equivalent of natural honey bee behavior in the field when foragers are stimulated by nectar. The pesticide seemed to make the bees “become pickier when foraging for nectar sources, possibly limiting the colony intake and storage of their only carbohydrate.” Pollen foraging may also be reduced, and “the colony would therefore suffer a protein deficit, resulting in lessened brood production and a dwindling population.”

Though the mystery of colony collapse disorder (CCD) is far from solved, current agriculture practices do not seem to be making honey bee colonies healthier, to say the least. But the collapse of the imported honey bee may have a silver lining: It is spurring agriculture to turn to previously neglected native pollinators. But the rise of the native pollinators is another story, for another time.