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Colony Collapse Disorder is comprised of a wide variety of problems with some problems making others more serious due to weakening the hive. Of these problems the infamous Varroa Mite which feed on the blood of bees weakens the colony and leaves the remaining workers at the mercy of other problems such as hive beetles. Large numbers of these beetles can lay an overwhelming number of eggs in unprotected combs at a time which builds their numbers so high that even strong colonies can be overwhelmed. Pesticides can serve as another death sentence to any hive, especially as the usage increases in areas around them.

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CCD Why Should the Public Care About What Happens to Honey Bees?
About one mouthful in three in our diet directly or indirectly benefits from honey bee pollination. Commercial production of many high-value and specialty crops like almonds and other tree nuts, berries, fruits and vegetables depend on pollination by honey bees. These are the foods that give our diet diversity, color, and flavor.

Honey bees are not native to the New World; they were brought here from Europe in the 1500s and 1600s by colonists. But many of our crops also came from the Old World and evolved in the same places as honey bees. There are native pollinators in the United States, but honey bees are more prolific and easier to manage, especially on a commercial level for pollination of a wide variety of crops. Almonds, for example, are almost completely dependent on honey bees for pollination. In California, the almond industry makes use of almost three-quarter of all managed honey bee colonies in the United States, brought from all over the country during one short window of time in January and February each year.
Honey Bee Health Problems
Parasites and pests: Varroa mites ( Varroa destructor) are essentially a modern honey bee plague. The Varroa mite has been responsible for the deaths of massive numbers of honey bee colonies since its arrival in the United States in 1987. A native of Asia, Varroa normally parasitizes the Asian honey bee, Apis cerana, which is a different species from the European or western honey bee, Apis mellifera, on which this country primarily depends for crop pollination.

Varroa mites directly damage honey bees by attaching and sucking the bees' equivalent of blood (hemolymph fluid) somewhat like ticks. They also indirectly damage honey bees because, similarly to mosquitos, Varroa mites also transmit an array of pathogenic viruses to honey bees such as deformed wing virus.*

Beekeepers have identified Varroa mites as their single most serious problem causing colony losses today.

Small hive beetles, native to sub-Saharan Africa, were first found in the United States in 1996 and had spread to 30 States by 2014. Large beetle populations are able to lay enormous numbers of eggs. These eggs develop quickly and result in rapid destruction of unprotected combs in a short time. If large populations of beetles are allowed to build up, even strong colonies can be overwhelmed in a short time.

Wax moths arrived in the United States in 1998 in Florida. This can be a very destructive insect pest, damaging beeswax comb, comb honey, and bee-collected pollen. Wax moths are rarely the initial cause of colony failure but can overcome weak colonies.

Pathogens: Since the 1980s, many new exotic pathogens that infect honey bees have been found in this country. These include deformed wing virus, paralytic viruses such as Israeli acute paralysis virus, which was first found in 2004, European foulbrood bacteria, and Nosema ceranae fungi, which arrived in 2005. They have all become major problems for U.S. honey bees and beekeepers.

Poor nutrition: Honey bees' natural diet comes primarily from nectar and pollen gathered from a wide variety of flowers. Insufficient or incomplete nutrition has come to be recognized as an essential factor that weakens the honey bee's immune systems and is likely to make bees more susceptible to all of the other problems troubling them today.

As demand for pollination services grows, bee colonies often are kept for more time on sites in a mono-crop environment before being moved directly to the next mono-crop area. As more and more land is lost to urbanization and suburbanization, it also means a loss of habitat with a diverse mix of nutritious bee forage plants. In addition, when it comes to helping bee colonies survive the winter and droughts, both times when nectar supplies can be scarce for bees, beekeepers often provide an artificial diet. Scientists are still trying to perfectly duplicate a bee's natural pollen/nectar diet for those times of the year when good forage is not available.

Pesticides: The U.S. Environmental Protection Agency (EPA) has strict regulations to protect managed honey bee colonies form incidents of pesticide misuse in formulation or application. Tips and complaints alleging pesticide-related bee incidents may be reported to State or tribal authorities or directly to the EPA Office of Pesticide Programs, beekill@epa.gov, National Pesticide Information Center: http://npic.orst.edu/ or https://www.epa.gov/compliance/guidance-inspecting-alleged-cases-pesticide-related-bee-incidents.

Sublethal pesticide effects: A survey of honey bee colonies conducted in 2010 by ARS researchers looked at 170 pesticides or their residues in honey bees, beeswax, and pollen. The data showed no consistent pattern of pesticide that differed between healthy and CCD-affected colonies. The most commonly found pesticide in the study was coumaphos, which is used by beekeepers to treat honey bees for Varroa mites.

The pesticide class neonicotinoids (for example, clothianidin, thiamethoxam, and imidacloprid) has been accused of damaging or killing honey bees or being the cause of CCD even when the exposure is below the level expected to be toxic. The nicotine-based neonicotinoids were developed in the mid-1990s in large part because they showed reduced toxicity to wildlife compared with previously used organophosphate and carbamate insecticides.

The scientific data about the impact of pesticides and neonicotinoids in particular at environmentally and agriculturally realistic levels is mixed. Some findings have shown that neonicotinoids have sublethal effects on honey bees at or below approved doses and exposures. Documenting such sublethal effects is very difficult due to the many factors that can influence individual situations in field studies and during grower use including timing of use, health and nutritional state of the bees, total mix of pesticides, pathogens and parasites present, crop type, weather during the growing season, and accumulation of pesticides from year to year. Other studies have indicated that healthy colonies appear not to be impacted.*

While these four areas are easy to categorize on paper, in reality these factors often may overlap or interact with one another. Honey bees might be able to survive many of these problems if the problems occurred one at a time. But when they hit in any of a wide variety of combinations, the result can weaken and overcome the honey bee colony's ability to survive.
ARS Research Directions
ARS is focused on directly improving the health of managed honey bees by finding ways to mitigate the impacts of pathogens, pests, and pesticides and enhancing bee nutrition and management. Agency scientists are also working on projects that take a bigger-picture view toward helping honey bees. This includes developing better knowledge about areas such as gut microbes and their interactions with honey bee immune systems, preservation and expansion of honey bee genetic diversity, and evaluating the effect of land management practices on bees to assure better productivity of pollinators.

For more information about ARS honey bee research programs, see ARS
National Program #305
Protecting Pollinators and Crops from Pests
ARS News about Honey Bees Parasite Hijacks Iron in Honey Bees Pollens to Fit A Honey Bee's Every Season Four Nutrients in Flower Pollens Improve Honey Bee Gut Health
Microalgae Food for Honey Bees

Deformed Wing Virus Genetic Diversity in U.S. Honey Bees Complicates Search For Remedies

New Tool Improves Beekeepers' Overwintering Odds and Bottom Line

Microbes on the Menu for Bee Larvae Weak Honey Bee Colonies May Fail From Cold Exposure During Shipping Honey Bee Colonies More Successful By Foraging on Non-Crop Fields ARS Microscopy Research Helps Unravel the Workings of a Major Honey Bee Pest Bacterial Imbalances Can Mean Bad News for Honey Bees
ARS Research Leads to Better Understanding of Bee Health August 2, 2016

USDA Scientists and Beekeepers Swap Colonies to Better Bees June 21, 2016
Back to Contents Best Recommendation for Beekeepers
Since little is known for sure about the cause(s) of CCD, mitigation must be based on improving general honey bee health and habitat and countering known mortality factors by using best management practices. This includes supplemental feeding in times of nectar/pollen scarcity.
Best Recommendations for the Public
The best action the public can take to improve honey bee survival is not to use pesticides indiscriminately. In particular, the public should avoid applying pesticides during mid-day hours, when honey bees are most likely to be out foraging for nectar and pollen on flowering plants.

In addition, the public can plant pollinator-friendly plants-plants that are good sources of nectar and pollen such as red clover, foxglove, bee balm, joe-pye weed, and other plants. (For more information, visit www.nappc.org.)
Back to Contents Selected ARS Honey Bee Scientific Publications
Probing the honey bee diet-microbiota-host axis using pollen restriction and organic acid feeding, Insects, May 2020

The miticide thymol in combination with trace levels of the neonicotinoid imidacloprid reduces visual learning performance in European honey bees (Apis mellifera), Apidologie, February 2020

Landscape factors influencing honey bee colony behavior in Southern California commercial apiaries, Nature Scientific Reports, February 2020

Effects of diets containing different concentrations of pollen and pollen substitutes on physiology, Nosema burden, and virus titers in the honey bee (Apis mellifera L.), Apidologie, December 2019

Impact of nutritional stress on honey bee colony health, Nature Scientific Reports, July 2019

Pollen reverses decreased lifespan, altered nutritional metabolism and suppressed immunity in honey bees (Apis mellifera) treated with antibiotics, Journal of Experimental Biology, April 2019

Honey bee colony performance and health are enhanced by apiary proximity to US Conservation Reserve Program (CRP) lands, Nature Scientific Reports, March 2019

Connecting the nutrient composition of seasonal pollens with changing nutritional needs of honey bee (Apis mellifera L.) colonies, Journal of Insect Physiology, July 2018

Honey bees overwintering in a southern climate: longitudinal effects of diet and queen age on colony-level molecular physiology and performance, Nature, July 2018

Routes of Pesticide Exposure in Solitary, Cavity-Nesting Bees, Environmental Entomology, June 2018

Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen, Insect Science, 00:1-12, March 2018

Sensitivity analyses for simulating pesticide impacts on honey bee colonies, Ecological Modelling, May 2018

Gamma irradiation inactivates honey bee fungal, microsporidian, and viral pathogens and parasites, Journal of Invertebrate Pathology, March 2018

Altered serum metabolite profiling and relevant pathways analysis in rats stimulated by honeybee venom: A new insight into allergy to honeybee venom, Journal of Agricultural and Food Chemistry, January 2018

Recent spread of Varroa destructor virus-1, a honey bee pathogen, in the United States, Nature, December 2017

Nectar-inhabiting microorganisms influence nectar volatile composition and attractiveness to a generalist pollinator, New Phytologist, September 2017

Limited impacts of truck-based ultra-low-volume applications of mosquito adulticides on mortality in honey bees (Apis mellifera), Bulletin of Entomological Research, April 2017

Social Immunity and the Superorganism: Behavioral Defenses Protecting Honey Bee Colonies from Pathogens and Parasites, Bee World, April 2017

Sublethal Effects of Imidacloprid on Honey Bee Colony Growth and Activity at Three Sites in the U.S., PLoS One, December 2016

Honey bee gut microbial communities are robust to the fungicide Pristine® consumed in pollen, Apidologie, Nov 2016

Transcriptomic and functional resources for the Small Hive Beetle Aethina tumida, a worldwide parasite of honey bees, Genomics Data, September 2016

Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide Imidacloprid and the organophosphate Acaricide Coumaphos, Journal of Insect Physiology, June 2016

Parasaccharibacter apium, gen. nov., sp. nov., Improves Honey Bee (Hymenoptera: Apidae) Resistance to Nosema, Journal of Economical Entomology, February 2016

Multiyear survey targeting disease incidence in US honey bees, Apidologie, May 2016

The effects of Imidacloprid and Varroa destructor on the survival and health of European honey bees, Apis mellifera, Insect Science, May 2016

Population growth of Varroa destructor (Acari: Varroidae) in honey bee colonies is affected by the number of foragers with mites, Experimental and Applied Acarology, May 2016

The bee microbiome: impact on bee health and model for evolution and ecology of host-microbe interactions, MBio, April 2016

Colony failure linked to low sperm viability in honey bee (Apis mellifera) queens and an exploration of potential causative factors, PLoS One, February 2016

Honey bee colonies provided with natural forage have lower pathogen loads and higher overwinter survival than those fed protein supplements, Apidologie, August 2015

Pesticide exposure in honey bees results in increased levels of the gut pathogen, Naturwissenschaften, February 2012
Back to Contents ARS Bee Research Laboratories

Pollinating Insect-Biology, Management, Systematics Research Unit
Logan, UT

Invasive Species and Pollinator Health Unit
Davis, CA

Southern Horticultural Research Center
Poplarville, MS

Tropical Crop and Commodity Protection Research Unit
Hilo, HI

Agricultural Genetic Resources Preservation Research Unit
Peoria, IL

Crop Bioprotection Research Unit
Peoria, IL

Insect Genetics and Biochemistry Research Unit
Fargo, ND

Soil Management Research Unit
Morris, MN

Vegetable Crops Research Unit
Madison, WI

Integrated Cropping Systems Research Unit
Brookings, SD

U.S. Honey Bee Losses
The total number of managed honey bee colonies has decreased from 5 million in the 1940s to about 2.66 million today, according to a USDA-National Agricultural Statistics Service (NASS) survey. There are many factors that may have contributed to this long-term slide, a number of them economic or cultural through the 1980s. These may have included a drop in the number of farms, especially small farms after World War II, accompanied by increasing opportunities for off-farm jobs for farm wives who often sold honey and honey products. In addition, drops in prices of honey started the downward slide in the number of colonies. In the late 1980s, the onset of Varroa mites and other bee health issues played a role in another drop in numbers of managed colonies. Typical average annual losses jumped to about 15-22 percent of managed colonies.

When Colony Collapse Disorder (CCD) began to be reported in 2006/2007, annual losses of honey bee colonies rose again. CCD has since waned, but high losses have continued, averaging about 30 percent. It is not known if this is because beekeepers are better at ascribing losses to particular causes, if causes have shifted, or if other factors have changed.

There has been a recent increase in the overall total number of managed honey bee colonies. This is being driven mostly by an increasing demand for almond pollination, which is tightly clustered in just a few weeks in late January and early February. California almond acreage grew to 1,020,000 in 2014, up 5 percent from 2013. In 2010, it was 810,000 acres - a 2 percent increase from 2008's 795,000 acres. Almond growers are the largest single users of honey bee pollination and need the colonies all in a short period of time.

To meet the increasing demand, beekeepers are splitting hives and buying more queens to create more colonies, which ends up with greater total numbers of colonies. But they are still losing higher percentages of their colonies now than they were 10 years ago before CCD and all the other bee health problems surfaced. The two numbers are different measures: total colonies vs. percent loss.
Survey Reports Latest Honey Bee Losses
For honey bee colony loss survey results after 2015, please go to USDA/NASS
Back to Contents
Colony Collapse Disorder

One problem plaguing honey bees since 2006 has been Colony Collapse Disorder (CCD), which is a syndrome specifically defined as a dead colony with no adult bees and with no dead bee bodies but with a live queen, and usually honey and immature bees, still present. CCD is not a general term that covers all managed honey bee colonies that are lost due to any reason. No scientific cause for CCD has been proven. Most research has pointed to a complex of factors being involved in the cause of CCD, and possibly not all of the same factors or the same factors in the same order are involved in all CCD incidents.

CCD is far from the only major threat to the health of honey bees and the economic stability of commercial beekeeping in the United States. In fact, the number of managed colonies that beekeepers have reported losing specifically from CCD began to wane in 2010 and has continued to drop. But the beekeeping industry continues to report losing a high percentage of their colonies each year to other causes.
CCD History
In October 2006, some beekeepers began reporting losses of 30-90 percent of their hives. While colony losses are not unexpected, especially over the winter, this magnitude of losses was unusually high.

The main symptom of CCD is very low or no adult honey bees present in the hive but with a live queen and no dead honey bee bodies present. Often there is still honey in the hive, and immature bees (brood) are present. Varroa mites, a virus-transmitting parasite of honey bees, have frequently been found in hives hit by CCD.

This is not the first time that beekeepers are being faced with unexplained losses. The scientific literature has several mentions of honey bee disappearances-in the 1880s, the 1920s, and the 1960s. While the descriptions sound similar to CCD, there is no way to know for sure if those problems were caused by the same agents as CCD.

There have also been unusual colony losses before. In 1903, in the Cache Valley in Utah, 2000 colonies were lost to an unknown "disappearing disease" after a "hard winter and a cold spring." More recently, in 1995-96, Pennsylvania beekeepers lost 53 percent of their colonies without a specific identifiable cause.

In June 2007, ARS and the National Institute of Food and Agriculture (NIFA), USDA's extramural research grants agency, co-chaired a workshop of scientists and stakeholders to develop a Colony Collapse Disorder Action Plan. This plan identified areas where more information was needed and developed a research priority list for additional research projects related to finding the cause/causes of CCD.
Cell Phones and CCD
Despite a great deal of attention having been paid to the idea, neither cell phones nor cell phone towers have been shown to have any connection to CCD or poor honey bee health.

Originally, the idea was provoked by the media making a connection between CCD and a very small study done in Germany. But that study looked at whether a particular type of base station for cordless phones could affect honey bee homing systems. However, despite all the attention that this study has received, the base station has nothing to do with CCD. Stefan Kimmel, the researcher who conducted the study and wrote the paper, e-mailed The Associated Press to say that there is "no link between our tiny little study and the CCD-phenomenon ... Anything else said or written is a lie."

In addition, apiaries are often located in rural areas, where cell phone coverage can be spotty. This makes cell phones or cell towers unlikely culprits.

In addition, apiaries are often located in rural areas, where cell phone coverage can be spotty. This makes cell phones or cell towers unlikely culprits.

Research from ARS and other institutions has provided new management recommendations that beekeepers have begun to adopt. For example, it is now recommended that beekeepers feed honey bees more protein during times of nectar shortage such as during times of drought or in the winter. As part of this, ARS has developed a new bee diet, Megabee, now available to beekeepers. The feeding of supplemental nutrients may help to decrease winter colony losses.
Back to Contents
In 2008, Germany revoked the registration of the neonicotinoid clothianidin for use on seed corn after an incident that resulted in the die-off of hundreds of nearby honey bees colonies. Investigation into the incident revealed that the die-off was caused by a combination of factors, including the failure to use a polymer seed coating known as a "sticker": weather conditions that resulted in late planting of corn while nearby canola crops were in bloom, attracting honey bees; use of a particular type of air-driven equipment used to sow the seeds, which blew clothianidin-laden dust off the seeds and into the air as the seeds were ejected from the machine into the ground; dry and windy conditions at the time of planting, which blew the dust into the nearby canola fields where honey bees were foraging; and a higher application rate than had been authorized was used to treat for a severe root worm infestation.

ARS researchers also have been analyzing samples from healthy and CCD-struck colonies and applying a variety of stressors from the four categories of possible causes to colonies in hopes of provoking a colony response that duplicates CCD.

While a number of potential causes have been championed by a variety of researchers and interest groups, none of them have stood up to detailed scrutiny. Every time a claim is made of finding a "smoking gun," further investigation has not been able to make the leap from a correlation to cause-and-effect. Other times, not even a scientific correlation has been demonstrated in the study claiming to have found "the cause" of CCD.

Researchers have concluded that no one factor is the cause of CCD. Most likely, CCD is caused by multiple factors. It is not possible to know at this time if all CCD incidents are due to the same set of factors or if the factors follow the same sequence in every case.

Studies are being conducted by ARS scientists and collaborators to look at the combined impact of two or more factors on honey bees-most recently the impact of exposure to the neonicotinoid imidacloprid and Nosema. While the dual exposure indicated some sublethal effects on individual honey bees, the overall health of the colony did not show an adverse effect.
Annual Reports of CCD Research Progress 2012 CCD Progress Report 2011 CCD Progress Report 2010 CCD Progress Report 2009 CCD Progress Report 2007-2008 CCD Progress Report
For more information, contact:

Kim Kaplan, Agricultural Research Service
Office of Communications,
(301) 504-1637
Kim.Kaplan@ars.usda.gov.
Back to Contents

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CCD Why Should the Public Care About What Happens to Honey Bees? About one mouthful in three in our diet directly or indirectly benefits from honey bee pollination. Commercial production of many high-value and specialty crops like almonds and other tree nuts, berries, fruits and vegetables depend on pollination by honey bees.

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Colony Collapse Disorder & Pollinator Health

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Colony Collapse Disorder & Pollinator Health

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New diseases such as gut parasites or Acute Paralysis viruses are becoming an increasing problem that need more time and effort to address. The stress of the colony also plays a massive role in the health of the workers overall which is directly affected by mismanagement and practices that try to provide pollination services. These changes in environment make it much harder for the colony to prosper and oftentimes leads to low nutrition and as a result poorer health.

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Colony Collapse Disorder and Pollinator Health | National Invasive Species Information Center

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Provides selected Colony Collapse Disorder and Pollinator Health resources related to the prevention, control, or eradication of invasive species.

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Economically, bees are one of, if not the most important aspect of modern agriculture with one third of the food that we eat being directly or indirectly pollinated by honey bees and a massive portion of our almond trees alone are supported by honey bee pollination. A collapsed colony will most often show itself as a nearly empty hive devoid of even worker corpses with just a queen, her brood, and maybe some leftover honey. While varroa mites that are found inside the hive are thought to be the main suspect, a wide variety of problems working together is more likely as many pesticides that include neonics made a bee 2-3 times more likely to die messing with their homing capabilities.

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While Colony Collapse Disorder has been researched for years, the common understanding that Varroa mites and their increasing resistance to chemical treatment was the main culprit, however bloodborne diseases spread by the mites seem to be playing a much bigger role in more recent studies. These diseases and worsening problems caused by climate change such as hurricanes, droughts, and floods increasingly seen all over the world is not helping bees cope. Losses of hives all around the country during the annual almond pollination in California seemed to have doubled, and Hurricane Ian in Florida gave the state it's own array of problems. Yet, he recent cause for the Winter damage of early 2023 is not entirely known despite the growing understanding of CCD.

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2023 is looking like one of the worst years for honey bees in nearly two decades | WSU Tree Fruit | Washington State University

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Written by Tim Lawrence, Washington State University. February 21, 2023

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A study centered around the differences of colonies affected by CCD and healthy colonies with intense focus on the symptoms of ailing bees in contrast to normal aging. Water regulation was shown to be greatly impacted by CCD affected colonies with a variety of other symptoms that are unique compared to aging such as the fecal quality, venom sac color, and other various anatomical differences. These unique biomarkers can help identify which colonies are in the early stages of CCD to make management of the problem easier and more effective.

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PLoS One. 2017; 12(7): e0179535.

Abstract

Honey bee ( Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD) and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions), and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees), we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence) and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and factors impacting bee health.

Introduction

Honey bee ( Apis mellifera) health has been in decline, with winter losses in the United States averaging 30% since Colony Collapse Disorder (CCD) was first identified in 2006 and annual losses approaching 50% [ 1- 8]. Numerous interacting factors have been implicated in colony declines including poor nutrition, increased pressure from ecto- and endoparasites, increased bacterial and/or viral loads, and synergistic pesticide interactions [ 9- 13]. Failure to recognize the early symptoms of disease in honey bee colonies could hamper the treatment and containment. The first stages of honey bee colony decline often go unrecognized, as healthy colonies have excess populations, which act as buffers against sudden losses in the work force [ 14, 15]. When large numbers of the older cohort of forager bees die, younger in-hive bees accelerate maturation and become precocious foragers to replace foraging work force losses[ 16]. The subtle changes in a colony's population, as a result, are hard to detect, meaning that beekeeper intervention may not occur until the colony is already weak or has died. An ability to look at adult honey bee anatomical and physiological characteristics to predict poor honey bee health could allow for early disease detection, enabling timely intervention.

Pathophysiology, an approach that studies the structural and functional changes in tissues that accompany disease, is a valuable tool for early detection of disease in human subjects [ 17, 18]and may also have potential application when monitoring honey bee health. Linking physiological symptoms with a disease can elucidate the pathways in dysfunction that culminate in disease outbreak and/or symptomology[ 19]. Because managed pollinators frequently don't show external indications of ill health until they are near death, the use of pathophysiology has particular promise towards predicting and understanding bee disease in general and CCD specifically. The U.S. experienced elevated colony losses in 2006-2008, many of which were characterized by a common set of specific symptoms: (1) the rapid loss of adult worker bees from affected beehives, resulting in weak or dead colonies with excess brood populations relative to adult bee populations; (2) a noticeable lack of dead worker bees both within and surrounding the affected hives; and (3) the delayed invasion of hive pests (e.g., small hive beetles and wax moths) and cleptoparasitism from neighboring honey bee colonies. Subsequently, this syndrome was termed CCD, and its case definition was revised to include (4) the absence of Varroa destructor and Nosema spp. loads at levels that cause economic damage [ 20]. Though affected colonies shared this specific set of symptoms, no single variable has emerged as the most likely cause of CCD. The bees do often have relatively high pathogen loads, suggesting an increased exposure or a weakened immune response [ 8, 21- 24].

For insects, the diagnosis of pathogens and a disease state begins with dissection to examine the internal tissues and structures for abnormal pathologies [ 25]. For all insects, preserving as much of the tissue without degradation is important, thus freezing and transferring the cadavers to 70% ethanol permits short term storage and dissection at a later date. For honey bees, many references and manuals have been created on how to diagnose pathogens and pests that might be found in the colony [ 26- 30]. Most of these manuals discuss commonly found pests and pathogens that are diagnosed easily by external examination of the brood and adult bees. For tracheal mites and Nosema, methods for diagnosis require the dissection of adult bees [ 31- 33]\. To perform these dissections, a good understanding of bee internal anatomy is required; for Apis mellifera, the anatomy is well described [ 34, 35]and methods on how to perform dissections outlined [ 36]. To date, these methods have not allowed for the diagnosis of complex pathologies or disease states such as CCD. This manuscript puts forward methods using dissections with dissecting microscopes to find pathologies and their descriptions. These pathologies were described both for adult bees of different ages in healthy colonies and also for adult bees from collapsing colonies and bees in apiaries with CCD. Previous research has found that colonies located near collapsing colonies are at high risk of dying [ 20].

Here we apply pathophysiology to honey bees to identify gross abnormalities that may be useful in diagnosing colonies before they develop the late-stage disease symptoms typical of CCD. We first developed a standard ranking system to score the gross lesions and other symptoms seen in individual honey bees. We then statistically analyzed these symptoms and their distributions using a CART analysis and found differences in symptom frequencies between CCD and non-CCD bees. We remained concerned that these differences were due to bee age, as typically only young bees remain behind in colonies collapsing from CCD [ 20, 37]. To determine which differences could be explained solely by age, we compared gross symptoms in bees performing different age-related tasks. In the end, we developed a list of physio-pathological traits that serve as biomarkers to predict colony health.

Results

Development of standard scoring system for lesions

Following Snodgrass [ 35]as a guide for normal anatomy in the honey bee abdomen, we carefully examined each bee's abdominal cavity, gastro-intestinal tract, and sting region for gross lesions and other symptoms, and scored 17 different visible conditions as listed in Table 1 and shown in Figs ​Figs1 1 - 8 . Dissected bees were individually scored for these 17 conditions. Due to limited funds and personnel, we confined our examinations to the aforementioned regions. However, we recognize that additional examination of important honey bee glands and fat body may add further insight which was beyond the scope of this current work.

Table 1

Variable/ Characteristic

Categorization (score)

Description

Present (1)

Spots of discolored muscle or connective tissue present in the abdominal cavity

Present (1)

Opaque white nodules can be found in or on abdominal tissues

Medium (1)

1-1.5mm wide by 4.5-5.5 mm long

Large (2)

1.5-2.5mm wide by 5.5-6.5mm long

Medium (1)

Sheath mostly tan to brown

Dark (2)

Sheath almost entirely brown or black

Present (1)

Dark band (scar) running partially or entirely around the perimeter of the pylorus region

Slight discoloration (1)

≥ 2/3 of the tubules tan to brown

Severe discoloration (2)

≥ 2/3 of the tubules brown to black

Reduced (1)

< ~50 tubules present

Iridescent spots present (1)

Small iridescent spots seen along the Malpighian tubules

Medium (1)

Orange, deep yellow, red, or light brown

Dark (2)

Dark brown

½ full (1)

Fills 33-66% of the abdominal cavity

full (2)

Fills 66-100% of the abdominal cavity

Semi-hard (1)

Breaks apart in clumps when probed

Hard (2)

Remains completely solid when probed

Present (1)

Hard concretions that look like small grains of rice present

Discolored (1)

Any amount of discoloration visible

Present (1)

Any sort of solid debris is present in the venom sac

Intermediate (1)

Sting gland is slightly larger than normal, and appears to have a second layer

Very swollen (2)

Sting gland is extremely oversized

Present (1)

Sting gland contains areas of melanin

Medium (1)

Tan to brown

Dark (2)

Brown to dark brown

We first examined all exposed abdominal tissue to detect black or melanized tissue and white nodules ( Fig 1 ). These pathologies are not tissue specific, as melanization of tissues is the result of phenol oxidase activation in tissue and indicative of activation of the humoral immune responses [ 38]. We found white nodules present inside the abdominal cavity ( Fig 1 , panel 1.2.1), attached ( Fig 1 , panel 1.2.2) or embedded ( Fig 1 , panel 1.2.3) in tissue in the bees kept in 70% ethanol; these nodules are not apparent in freshly killed bees, indicating that tissue dehydration is needed for visualization[ 39]. These nodules are reported to consist primarily of nodules of hemocytes rich in the amino acid tyrosine [ 39]and can form in response to bacterial infection[ 40]. Similar nodules were previously found in pupae and adult bees parasitized by Varroa, implying that they are an immunological response to parasitic feeding[ 39].

We next examined the ventriculus (midgut) by measuring ventriculus color and size ( Fig 2 ). The ventriculus is located between the posterior end of the proventriculus (a valve at the posterior portion of the foregut) and the anterior end of the proctodeum (hindgut), where the Malpighian tubules connect. The ventriculus is the principal site of digestion and absorption and is where the microsporidian parasite Nosema germinates[ 34]. Ventriculus color is dictated by the color of the surrounding muscle fibers and the outer connective tissue sheath[ 41, 42]. In healthy bees, the ventriculus, when removed in saline, is translucent with reddish-brown appearance that may turn milky white or chalky upon Nosema infection[ 43]; although, careful research has demonstrated that this is not always the case in worker bees infected with either Nosema apis or Nosema ceranae [ 44- 46]. When examining bees stored in alcohol, Snodgrass describes the healthy ventriculus as having an opaque white appearance[ 35]. We observed that after extended storage in alcohol or under poor storage conditions with high temperature, many of the bee's tissues darken; to ensure that we accounted for natural darkening over time in storage, we scored each bee's ventriculus color as light, medium, or dark relative to the color of the connective tissue and muscle (sheath) that is light or white in color in healthy newly autopsied bee. We also measured ventriculus size, which is dependent, in part, on how much the bee recently ate and/or the degree of Nosema infection [ 47].

Examination of the proctodeum included the pylorus region of the anterior intestine and the posterior intestine (rectum). The pyloric valve regulates passage from the ventriculus to the anterior intestine [ 34]. The anterior intestine is involved in ion regulation [ 48]and the rectum stores waste matter, while rectal pads in the rectum can absorb water from the feces[ 49]. We examined the pyloric valve for the presence or absence of a dark banded "scar" ( Fig 3 , red arrows). This symptom was first described in 1946 [ 50]and is a melanization response, which can be triggered by colonization by the gammaproteobacterium Frischella perrara [ 51]. The scarring is apparent even when looking through the outer layer of circular muscle fibers that surround this valve and is thus not due to tissue manipulation during dissection. The scar is typically 100-400 microns wide and partially or completely encircles the pyloric valve.

The Malpighian tubules (located at the anterior end of the intestine) open into the pyloric lumen immediately behind the pyloric valve region[ 35]. These are the principal organs of excretion and osmoregulation in insects, analogous to vertebrate kidneys [ 34, 52]. In healthy honey bee adults, Malpighian tubules number 100 or more and are long, slender, translucent tubes [ 34]. Diseases such as Malpighamoeba mellificae [ 53]can cause tubule damage. We scored the Malpighian tubules for variations in overall color ( Fig 3 ), number ( Fig 4 ), and the presence or absence of iridescence ( Fig 4 , black arrows). Iridescent spots, also called "concretions" and "mineralized granules," are dense bodies found within the Malpighian tubule epithelium or lumen[ 54]. These concretions often appear iridescent and result from mineral storage, xenobiotic detoxification, or an encapsulating bacterial immune response [ 52, 55, 56].

The remainder of the anterior intestine plays a crucial role in the passage of waste into the rectum[ 34]; however, we did not score this area as preliminary work revealed no obvious pathologies in this region.

Rectal fecal matter includes pollen exines, pollen lipid globules, and the sloughed epithelial cells from the ventriculus[ 34]. The rectum also contains a large community of presumably symbiotic microorganisms that may help decompose and detoxify undigested food [ 57, 58]. These organisms may also provide benefit to their host by conferring immunity to some common bee diseases. Pesticides and veterinary honey bee medicines can influence the composition of honey bee microflora[ 59]. For instance, Lactobacillus resident in the hindgut may protect their host against Paenibacillus larvae, the causal agent of American foulbrood[ 60][ 61]. The rectum reabsorbs water from waste [ 62]and stores the waste until honey bees can take a cleansing flight[ 63]. If bees are unable to empty their rectums for extended periods of time, indigestible food components such as starch may ferment, enabling a proliferation of bacteria, yeasts, and microfungi, which can result in obvious signs of dysentery on frames and at the hive entrance [ 34]. The rectum's ability to contract and distend allows for extensive fecal accumulation during winter and inclement weather[ 34].

For the purposes of this study, several components of the bees' rectum and fecal matter were examined and scored: color, rectum size (which depends on fecal amount accumulated), content consistency, and the presence or absence of stone-like "enteroliths" (Figs ​(Figs5 5 and ​and6). 6 ). We use the term enteroliths, since these are hard, rounded and similar to the structures (enteroliths) described in the rectums of honey bee queens [ 64]or the mineral concretions called enteroliths that are sometimes found in the intestines of humans and horses[ 65]. In our studies, the contents of the rectum were visible through its outer layers of muscles. We quantified fecal matter accumulation by estimating the percentage of the abdomen a given rectum filled the abdomen; large 75-100% full, medium 30-75% full, and small 0-30% full ( Fig 5 , panel 1.10). To measure fecal matter consistency, we used forceps to break through the rectum and probed the fecal matter held inside. When probing, we noted if the contents were hard, semi-soft or very soft ( Fig 6 , panel 1.11). We also noted the presence/absence of enteroliths that looked like small grains of rice contained in the rectum ( Fig 6 , panel 1.12).

Next, we examined the sting apparatus for abnormal pathologies. The sting of Apis mellifera is contained within the abdomen in segment VII, and consists of the venom gland (also called poison or acid gland) and a sting gland. The venom gland consists of a sac that opens by a thin neck into the base of a bulbous cavity near the stinger[ 35]. The sting gland is long and strand-like. Investigation began with the venom sac, noting variations in color ( Fig 7 , panel 1.13). We assessed the sting gland for discoloration, swelling, and the presence of the black-pigment melanin (melanosis) ( Fig 8 , panels 1.14-1.16). The color of sting glands ranged from translucent to tan to dark brown, with either solid or patchy melanization.

Comparison of bees from CCD vs. control apiaries

Colonies in an apiary with CCD-symptomatic hives may not have started to collapse but may already have been suffering from ill health without yet expressing symptoms. For this reason, we compared all bees collected from colonies in apiaries suffering from CCD with bees collected from colonies in apiaries with no sign of the disorder ( Table 2 ). Seven pathologies showed different disease manifestation patterns in apiaries with and without CCD: Malpighian tubule iridescence, rectum distension, fecal matter consistency, rectal enteroliths, venom sac color, sting gland swelling, and sting-gland melanosis. CCD apiaries were more likely to have bees with rectums that were less than half-full as compared to bees that came from non-CCD apiaries. The content of the rectums of bees from CCD apiaries also was softer. Bees collected from CCD apiaries were 7.7 times more likely to have rectal enteroliths when compared to bees collected from control apiaries. Bees from CCD apiaries were more likely to have sting gland tissue melanosis. In contrast, bees from a CCD apiary were less likely to have discolored venom sacs and swollen sting glands than bees from control apiaries.

Table 2

Bees from CCD apiary

Bees from non-CCD apiary

Likelihood ratio test (CCD vs. non-CCD)

Variable

N

Score 0 (%)

Score 1 (%)

Score 2 (%)

N

Score 0(%)

Score 1(%)

Score 2(%)

χ²

P

CART analysis highlighted four of the physio-pathological traits described above as having high predictive power (i.e. relative importance) for the presence of CCD in an apiary ( Fig 9 ). The most informative with a power = 100 was rectum distension (size), followed by Malpighian tubule iridescence (power = 83.65), fecal matter consistency (power = 74.49), and venom sac color (power = 53.02).

Comparison of bees from CCD symptomatic colonies vs. non-symptomatic colonies

We compared bees collected from colonies showing CCD symptoms with bees taken from non-symptomatic colonies. A subset of the physio-pathological traits (Malpighian tubule iridescence, rectum distension and the presence of rectal enteroliths) was differentially expressed in CCD and non-CCD colonies ( Table 3 ). Iridescent spots in the Malpighian tubules were more prevalent in bees from CCD symptomatic colonies than in bees from non-symptomatic colonies. Compared to bees from colonies without CCD, bees from CCD colonies were likely to have rectums that were less than ½ full. Rectal enteroliths occurred more than 3 times as frequently in CCD suffering bees compared to non-CCD suffering bees. This subset of physio-pathological traits (rectal size and presence of enteroliths) was similarly differentially expressed in CCD positive apiaries and colonies.

Table 3

Bees from CCD Symptomatic colony

Bees from non-symptomatic colony

CCD vs. non-CCD

Variable

N

Score 0 (%)

Score 1 (%)

Score 2 (%)

N

Score 0 (%)

Score 1 (%)

Score 2 (%)

χ²

P

CART analysis revealed that these four characteristics continued to strongly influence the decision tree when discriminating between symptomatic and non-symptomatic colonies ( S1 Fig), but the order of importance shifted when compared to the CART analysis that distinguished between CCD apiaries and non-CCD apiaries (see Fig 9 ). Rectal fecal-matter consistency had the greatest relative importance (power = 100), followed closely by rectum size (98.84). An additional 8 factors contributed to the most parsimonious decision tree, including sting gland color (66.96), venom sac color (58.72), sting gland melanosis (54.95) Malpighian tubule iridescence (54.51), rectal enteroliths (51.00), rectum color (50.74) and Malpighian tubule color (50.65). The resulting decision tree had a specificity of 59.3% (95% CI: 54.0-64.4) and sensitivity of 60.3% (95% CI: 54.9-65.4). The lower overall tree specificity and sensitivity indicated that one can correctly distinguish between CCD and control apiaries with greater certainty than one can distinguish between CCD and non-CCD colonies that may or may not share the same apiary.

Effects of age on physio-pathological traits

Adult bee task is closely associated with age, with newly-emerged bees maturing into nurses and then transitioning to foraging. We compared pathologies across four age-related tasks (newly emerged, nurse, non-pollen forager, and pollen forager) and found significant differences among six of the seven physio-pathological traits we investigated (Figs ​(Figs10 10 and ​and11): 11 ): white nodules (χ ² = 552.9, df = 3, p < 0.0001), pyloric scarring (χ ² = 213.6, df = 3, p < 0.0001), Malpighian tubule quantity (χ ² = 70.1, df = 3, p < 0.0001), sting gland swelling (χ ² = 63.3, df = 3, p < 0.0001), sting gland tissue melanosis (χ ² = 193.2, df = 3, p < 0.0001), and sting gland color (χ ² = 56.4, df = 3, p < 0.0001). Malpighian tubule color did not differ between bees of different ages (χ ² = 0.8, df = 3, p = 0.84). The prevalence of white nodules and sting-gland swelling declined with age. White nodules were found more often in newly-emerged workers than in any other age group. Sting gland discoloration also declined with age but was equally prevalent in newly-emerged workers and nurse bees. Pyloric scarring and melanized sting glands had the opposite pattern, increasing in prevalence with age. Malpighian tubule quantity was significantly reduced in newly-emerged workers as compared to all other worker age classes. Pathology frequencies were always equally distributed between pollen and non-pollen foragers.

We further classified the cohorts of healthy bees into in-hive bees (young) and foragers (old), to determine if physio-pathological traits observed in CCD colonies were a factor of the bees being young instead of ill. Bees left in CCD colonies are assumed to be young, given the presence of developing brood and newly-emerged bees, but the exact age or age-related task is undeterminable given the highly-reduced worker-bee populations. The transition in healthy bees between in-hive nursing and outside foraging is accompanied by a suite of physiological changes [ 16, 66, 67]and these may be reflected in changes in internal tissues. Five of the above physio-pathological traits differed significantly between in-hive bees and foragers. Younger bees were more associated with these: white nodules (χ ² = 376.3, df = 1, p < 0.0001), pyloric scarring (χ ² = 125.2, df = 1, p < 0.0001), sting gland swelling (χ ² = 48.7, df = 1, p < 0.0001), sting gland tissue melanosis (χ ² = 175.8, df = 1, p < 0.0001), and sting gland color (χ ² = 50.2, df = 1, p < 0.0001). Malpighian tubule quantity, which was significantly reduced in newly-emerged bees, did not differ when we grouped bees by in-hive bees vs. forager bees (χ ² = 2.12, df = 1, p = 0.15). Malpighian tubule color was also independent of this transition between in-hive tasks and foraging (χ ² = 0.4, df = 1, p = 0.53).

CART analysis revealed that white nodules had the greatest predictive power to distinguish between in-hive bees and foragers (power = 100), while sting gland melanosis also contributed (power = 28.58). Consistent with regressions, our classification tree found that in-hive bees were more likely to have white nodules and less likely to show sting gland melanosis ( Fig 12 ). Overall the tree had 72.2% (95% CI: 68.4-75.7) specificity and 82.0% (95% CI: 78.8-84.9) sensitivity. CART analysis could not distinguish between different age-associated tasks (i.e., distinguish between newly-emerged workers and in-hive nurse bees or between non-pollen foragers pollen foragers).

Discussion

Our results demonstrate that physio-pathological traits associated with collapsing colonies are present even before all colonies in an apiary become symptomatic, suggesting that studying bees from apparently healthy colonies can be used to detect diseased colonies prior to collapse. A set of physical characteristics (rectum distension, Malpighian tubule iridescence, fecal matter consistency, and venom sac color (Figs ​(Figs5 5 - 7 )) can identify CCD prior to colony demise. Comparing symptoms between bees from CCD apiaries and non-CCD apiaries revealed that CCD apiaries tended to have partially filled rectums, soft fecal matter, rectal enteroliths and sting gland melanosis. Sting gland melanosis was associated more often with older foragers and not young bees, so it was surprising to see it more frequently in bees from CCD apiaries than non-CCD apiaries, given that it is assumed that bees in CCD apiaries are recently emerged bees.

Using these physio-pathological traits to distinguish between bees from CCD-symptomatic versus non-symptomatic colonies was more difficult, since the decision trees resulting from CART analysis had significantly lower sensitivity and specificity. The rectum's characteristics played an important role in this decision tree, with fecal matter consistency and rectal distension (size) contributing the greatest power. Regressions revealed that Malpighian tubule iridescence was significantly more common in symptomatic bees, as were rectal enteroliths.

Physio-pathological traits associated with CCD were not the result of the collapsed colonies containing only young bees. The majority of physio-pathological traits that differed among bee castes (white nodules, pyloric scarring, Malpighian tubule quantity, and sting gland color) were not the same as those associated with CCD colonies. In fact, younger bees consistently had a very different appearance of their internal tissues as compared to those of bees from CCD-symptomatic apiaries and colonies. For example, the sting glands in bees in CCD-symptomatic colonies and apiaries were more likely to be melanized, while younger in-hive bees were less likely to show sting gland melanization, suggesting an active immune response was occurring in this tissue in the CCD bees. Not all characteristics that were examined in the CCD analysis were investigated in the age study, since preliminary dissections across age groups revealed no changes in the rectal characteristics and rectal enteroliths were never detected in the bees from healthy colonies. Now that pathological symptoms tied to CCD are identified, the mechanisms that underlie these physio-pathological traits beg investigation.

The importance of the size and contents of the rectum when distinguishing between both CCD and control apiaries and between symptomatic and non-symptomatic colonies suggests possible nutritional or water-balance disruptions, as the rectum serves as a nutritional waste storage vessel during confinement. Our results suggest CCD bees may be unable to either produce or retain fecal waste in the rectum during extended periods of confinement, such as over winter or during rainy weather. Bees in both apiaries and colonies having CCD symptoms had significantly less fecal matter in the rectum. There was no reason to expect that these bees would retain fecal waste since bees had been in located in Southern states for several weeks prior to collapse and should have been taking cleansing flights in warm weather. It is more likely that bees from CCD colonies may have been underfed, especially with pollen, resulting in less fecal matter than was found in healthy bees (less food = less feces). Rectal enteroliths, which were more common in bees from CCD-positive apiaries and almost never detected in healthy bees, may indicate a disrupted excretory physiology. Rectal enteroliths have previously been reported in queens, where they were associated with egg-laying cessation, abnormal eggs, and swollen rectums. In the 1960's, the researcher Fyg performed chemical analyses and found uric acid and minerals in the enterolith concretions in queens; he hypothesized these were indicative of constipation and poor osmoregulation [ 64, 68].

Malpighian tubule iridescence was more common in bees from CCD apiaries and symptomatic colonies. This observed pathophysiology may have several causes, including exposure to pathogens and/or different environmental toxins[ 52, 69, 70]. In wild-type Drosophila, exposure to environmental toxins such as ethylene glycol or melamine can result in concretions that are iridescent[ 70]. These concretions indicate a disruption in osmoregulation via the ion and organic solute transporters[ 52, 70]. Besides involvement in osmoregulation, Malpighian tubules also have important roles in detoxification with an expression of P450 genes [ 52]used for effective xenobiotic metabolism[ 71]. The Malpighian tubules can also mount an immune response independent of the fat body[ 72]. No pathogen has a known association with iridescent Malpighian tubules; however, this needs further exploration. The Malpighian tubules play a variety of protective roles [ 73]in addition to excretion, and their impairment could help contribute to increased colony losses in the face of environmental stressors.

White nodules observed in the hemocoel were common to newly-emerged and nurse bees. CART analysis demonstrated that these white nodules had the greatest predictive power when distinguishing between in-hive bees and foragers. Nodulation/encapsulation is a highly conserved physiological response that entraps micro-organisms through the aggregation of hemocytes, a key component of the insect cellular immune system [ 39, 74]. The presence of the nodules suggests a highly active immune system. Given that the nodules were common to newly-emerged bees, they may be a remnant from tissue remodeling that occurs during metamorphosis and pupation [ 75]. In Drosophila and stingless bees, the larval fat body disassociates into single cells; some of the cells persist into the newly-emerged adult and then undergo cell death [ 75, 76]. It is possible that this process is slower in honey bees, with the majority of dying cells being recognized and engulfed in hemocyte aggregations in newly-emerged bees. Some white nodules were still present in nurse bees. Alternatively, the nodulation may result from hemocyte aggregation in response to parasitization by varroa mites, which feed predominantly on developing pupae and damage may be apparent in newly-emerged bees.

The pathology of the rectum and Malpighian tubules of CCD bees suggests that osmoregulation and/or ion balance are key processes worthy of further investigation. These disorders may indicate that stresses are affecting osmoregulation and ion balance, both at an individual and colony level [ 77]. As brood rearing and colony homeostasis require constant temperatures regulated via water evaporation [ 78], individual workers suffering from poor osmoregulation could leave the colony susceptible to temperature fluctuations and desiccation with detrimental effects on the quality of brood rearing, food storage, and colony survival. Malpighian tubule disturbances have been linked to environmental toxins in Drosophila [ 70], and the role of xenobiotics in the development of Malpighian tubule pathologies in honey bees needs further investigation.

Our data clearly demonstrate that pathophysiology is a useful tool in evaluating colony health prior to demise; and that particular pathologies will likely serve as biomarkers for diagnosis or prediction of colony health. Additional research is needed to determine if these physio-pathological traits are seen in other colony losses beyond those with CCD symptoms. The addition of our necropsy methods to the toolbox used by researchers and beekeepers will be valuable in evaluating colony health and in predicting the future of their bees.

Methods

Selecting CCD and "healthy" colonies

Colonies used were sampled in 2007 as part of an epidemiological study that sought to determine potential causes of CCD[ 20]; we collected bees from 13 apiaries located in either Florida or California during January and February 2007. Colonies were classified in the field as coming from apiaries lacking CCD (18 control colonies from "non-CCD apiaries") or apiaries having colonies with CCD (55 colonies from "CCD apiaries"). Out of the 55 colonies in CCD apiaries, 36 were in apparent decline ("symptomatic CCD"), while 19 appeared healthy ("non-symptomatic CCD") at the time of collection. For CCD colonies, we examined approximately 10 bees from each colony, with samples taken randomly from bees collected from the brood nest (when brood was present) [ 20].

For healthy colonies, these were from one of the beekeeping operations originally sampled; the colonies were transferred to Pennsylvania and kept in one of the author's backyards in 2008. The colonies were observed for several months to verify health. From these colonies, bees from various age groups were sampled, with approximately 50 bees of each group taken from each of six healthy colonies. All of the bee samples were initially frozen at -80°C and then stored in 70% ethanol at room temperature.

Bee dissections and gross lesion standardized scoring

Bees necropsied (usually 10 at a time) were removed from their storage bottles and allowed to dry slightly on a paper towel. We made two shallow incisions along the lateral sides of a bee's abdomen, in the pleural region, using forceps and micro-scissors (EMS high-precision tweezers, style 5B with extra fine and bent tips; straight Dumont positive-action tweezers, Style SS; Vannas Capsulotomy scissors with 5 mm straight blade). Incisions started at the posterior end of the abdomen and ended near the petiole. Unless otherwise noted, once the incisions were made, we detached the abdomen from the thorax and placed it in a Petri-dish containing 70% ethanol to facilitate tissue separation. Under a stereo microscope (Leica MZ16 A), we removed abdominal sclerites, clearing away the sternites and tergites from around the gastro-intestinal tract until the internal tissues were completely visible. Following Snodgrass[ 35], we carefully examined each bee's abdominal cavity, gastrointestinal tract, and sting region for gross lesions and other symptoms, and scored 17 different visible conditions as listed in Table 1 .

Effects of bee age and task on gross lesions and symptoms

To determine whether pathologies seen at greater frequency in bees from CCD colonies (assumed to contain primarily young bees performing in-nest tasks) are due to CCD or are the result of skewed cohort composition of the colony, we performed autopsies on healthy worker bees taken from healthy colonies that were performing distinctive tasks that link to age. We chose six CCD-negative Apis mellifera colonies for cohort-specific sampling. From each of these colonies we sampled and dissected 50 bees from each of the following cohorts: newly-emerged workers (N.E.W.), nurses, pollen foragers, and non-pollen foragers (carrying nectar, water, or returning empty). Bees transition among these cohorts following an age-dependent polyethism, with bees from healthy colonies transitioning from newly-emerged adult bees to nurses, then to foraging. Newly-emerged workers are distinguished by their fluffy thorax hairs, which remain distinctive for a short time after emergence. These bees were removed from frames with visible emerging bees. Nurse bees feed larvae and the queen and are found in the interior portions of the nest actively engaged in attending the brood. We collected foragers by placing a screen over the hive entrance and sampled bees attempting to re-enter the nest. Bees carrying pollen on their corbiculae were deemed pollen foragers, while those without pollen loads were labeled as non-pollen foragers. Necropsy protocols followed those described above. We did not color mark bees and allow them to age into their respective tasks, because we wanted minimal disturbance to the colony. We sought to capture the normal age distribution for each of these worker task roles as the comparative bees sampled from CCD colonies were of unknown, but presumably mixed ages. These bees were frozen and immersed in 70% ethanol in the same manner as the bees taken from CCD colonies. We limited our investigations to seven physio-pathological traits: white nodules, pyloric scarring, Malpighian tubule color, Malpighian tubule quantity, sting gland swelling, sting gland tissue melanosis, and sting gland color. Based upon preliminary studies with more than 50 healthy bees, we did not examine the rectum, given that all bees had full rectums containing soft contents and no enteroliths.

Data analysis

Statistical analyses were carried out in R[ 79]. We first assessed whether each individual gross lesion or pathology predicted CCD status. We compared the pathologies diagnosed in bees as either coming or not coming from CCD-effected colonies or apiaries using generalized linear mixed models (GLMMs) with pathology scores as the response variable, the apiary's or colony's CCD status as the fixed effect and the bee's colony as a random effect. The apiary-level analyses permit detection (if present) of CCD-related traits when colonies are suffering from CCD without yet expressing symptoms of collapse. Colonies categorized as non-symptomatic included those from both CCD and control apiaries. Binary physio-pathological traits were analyzed via logistic regressions with the lme4 R package [ 80], and ordinal pathologies via cumulative logistic regressions with the ordinal R package[ 81].

When CCD status significantly affected pathology scores, we calculated odds ratios comparing pathology scores in colonies or apiaries with and without CCD. For two pathology types, Malpighian tubule iridescence and sting gland swelling, the pathology or its absence was sufficiently rare that GLMM results were too imprecise. For these, we employed chi-squared tests to assess whether pathology prevalence differed between bees from colonies or apiaries with and without CCD.

We analyzed the effects of bee age on gross lesions and pathologies using GLMMs similar to those described above. Here, the fixed effect was bee age rather than CCD status. We followed the common practice of analyzing the ordinal response variable (Malpighian tubule color) via Poisson regression to facilitate post-hoc testing within the same framework that we used for logistic regression. To determine significant differences between age class pairs, we employed Tukey HSD tests using the Multcomp R package [ 82].

To better understand how combinations of pathologies or gross lesions are related to CCD, we conducted classification and regression tree (CART) analyses. A CART analysis is a non-linear and non-parametric model that is fitted by binary recursive partitioning of multidimensional covariate space[ 83]. Using Salford Predictive Modeler software (Salford Systems, San Diego, CA, USA), the analysis successively splits the dataset into increasingly homogeneous subsets until it is stratified to meet specified criteria. The Gini index was used as the splitting method, and 10-fold cross-validation was used to test the predictive capacity of the obtained trees. CART performs cross validation by growing maximal trees on subsets of data then calculating error rates based on unused portions of the data set. To accomplish this, CART divides the data set into 10 randomly selected and roughly equal parts, with each "part" containing a similar distribution of data from the populations of interest (e.g., CCD apiaries and non-CCD apiaries). CART then uses the first 9 parts of the data, constructs the largest possible tree, and uses the remaining 1/10 of the data to obtain initial estimates of the error rate of the selected sub-tree. The process is repeated using different combinations of the remaining 9 subsets of data and a different 1/10 data sub-set to test the resulting tree. This process is repeated until each 1/10 sub-set of the data has been used to test a tree that was grown using a 9/10 data sub set. The results of the 10 mini-tests are then combined to calculate error rates for trees of each possible size; these error rates are applied to prune the tree grown using the entire data set. The consequence of this process is a set of fairly reliable estimates of the independent predictive accuracy of the tree, even when some of the data for independent variables are incomplete and/or comparatively small. Further details about CART are presented in previously published articles (e.g.,[ 84- 87][ 88].

We constructed three classification trees using CART analysis. The first tree differentiated between CCD apiaries and non-CCD apiaries. The second tree distinguished CCD symptomatic bees from non-symptomatic bees. The final tree analyzed characteristics due to honey bee task, thus allowing determination of factors that change with bee age.

Supporting information

S1 Fig
CART analysis of bees from CCD symptomatic and non-symptomatic colonies from all investigated apiaries.
(PDF)

S1 Data Set
Autopsy data from individual bees from CCD and non-CCD colonies in CCD and non-CCD apiaries.
(CSV)

S2 Data Set
Autopsy data from cohorts of bees of different metamorphic stages, sexes, and age-related cohorts.
(CSV)

Funding Statement

The following provided funding for this study: Pennsylvania Department of Agriculture (DVE, DCF), US National Honey Board (DVE, DCF), and the Pennsylvania State University, Department of Entomology (DCF, DVE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

All relevant data are within the paper and its Supporting Information files.

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# data[].attributes.attachment_link.title
Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology

# data[].attributes.attachment_link.description
Honey bee (Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important ...