Colony collapse disorder

US honey bee hives 1982-2015

The National Agricultural Statistics Service (NASS) reported 2.44 million honey-producing hives were in the United States in February 2008, down from 4.5 million in 1980, and 5.9 million in 1947, though these numbers underestimate the total number of managed hives, as they exclude several thousand hives managed for pollination contracts only, and also do not include hives managed by beekeepers owning fewer than five hives. This under-representation may be offset by the practice of counting some hives more than once; hives that are moved to different states to produce honey are counted in each state's total and summed in total counts.[43]

In 2007 in the US, at least 24 different states[44] had reported at least one case of CCD.[45] In a 2007 survey of 384 responding beekeepers from 13 states, 23.8% met the specified criterion for CCD (that 50% or more of their dead colonies were found without bees and/or with very few dead bees in the hive or apiary).[45] In 2006–2007, CCD-suffering operations had a total loss of 45% compared to the total loss of 25% of all colonies experienced by non-CCD suffering beekeepers.[43][45]

A 2007–2008 survey of over 19% of all US colonies revealed a total loss of 35.8%. Operations that pollinated almonds lost, on average, the same number of colonies as those that did not. The 37.9% of operations that reported having at least some of their colonies die with a complete lack of bees had a total loss of 40.8% of colonies compared to the 17.1% loss reported by beekeepers without this symptom. Large operations were more likely to have this symptom, suggesting a contagious condition may be a causal factor. About 60% of all colonies that were reported dead in this survey died without the presence of dead bees in the hive, thus possibly suffered from CCD.[43]

Between 2007 and 2013 after CCD was described in the US, annual winter colony losses doubled from 15% pre-CCD to 30%. Such loss rates fell to 24% from 2014 to 2017 and CCD symptoms were not as commonly associated with hive losses.[46] While CCD has increased hive losses, honey bee colony numbers in the US have remained stable or grown since the identification of CCD.[47]

Throughout the year in 2017, NASS reported total US hives ranged between 2.63 and 2.99 million throughout the year for operations with more than five colonies, and 35 to 43 thousand hives for those with fewer than five colonies. In the same year, operations with more than 5 colonies lost 77.8 thousand hives (2.6 to 3.0%) with CCD symptoms and those with fewer than 5 colonies lost 6 thousand hives (14 to 17%) with CCD symptoms.[48]

According to the European Food Safety Authority (EFSA), in 2007, the United Kingdom had 274,000 hives, Italy had 1,091,630, and France 1,283,810. In 2008, the British Beekeepers Association reported the bee population in the United Kingdom dropped by around 30% between 2007 and 2008, and an EFSA study revealed that in Italy the mortality rate was 40–50%. However, EFSA officials point out the figures are not very reliable because before the bees started dying, no harmonisation was used in the way different countries collected statistics on their bee populations. At that time (2008), the reports blamed the high death rate on the varroa mite, two seasons of unusually wet European summers, and some pesticides.[49]

In 2009, Tim Lovett, president of the British Beekeepers' Association, said: "Anecdotally, it is hugely variable. There are reports of some beekeepers losing almost a third of their hives and others losing none." John Chapple, chairman of the London Beekeepers' Association, put losses among his 150 members at between a fifth and a quarter. "There are still a lot of mysterious disappearances; we are no nearer to knowing what is causing them." The government's National Bee Unit continued to deny the existence of CCD in Britain; it attributes the heavy losses to the varroa mite and rainy summers that stop bees foraging for food.[50]

In 2010, David Aston of the British Beekeepers' Association stated, "We still do not believe CCD (which is now better defined) is a cause of colony losses in the UK, however we are continuing to experience colony losses, many if not most of which can be explained." He feels recent studies suggest "further evidence to the evolving picture that there are complex interactions taking place between a number of factors, pathogens, environmental, beekeeping practices and other stressors, which are causing honey bee losses described as CCD in the US".[51]

Beekeepers in Scotland also reported losses from 2007 to 2009.[50] Andrew Scarlett, a Perthshire-based bee farmer and honey packer, lost 80% of his 1,200 hives during the 2009/10 winter. He attributed the losses to a virulent bacterial infection that quickly spread because of a lack of bee inspectors, coupled with sustained poor weather that prevented honey bees from building up sufficient pollen and nectar stores.[50]

In Germany, where some of the first reports of CCD in Europe appeared, and where, according to the German national association of beekeepers, 40% of the honey bee colonies died,[52] there was no scientific confirmation; in early May 2007, the German media reported no confirmed CCD cases seemed to have occurred in Germany.[53][50]

In 2012, a report was published stating the first case of CCD, according to more stringent definitions, occurring outside of the US was in Switzerland.[54] At the end of May 2012, the Swiss government reported about half of the bee population had not survived the winter. The main cause of the decline was thought to be the parasite Varroa destructor.[55]

In China, a three-year survey from 2010 to 2013 (using COLOSS questionnaires) showed colony losses of 10.1% on average. Comb renewal and queen problems were identified as significant risk factors.[56]

New Holland TL 90 with a field sprayer on a Narcissus field in Europe.

According to the USDA, pesticides may be contributing to CCD.[14] A 2013 peer-reviewed literature review concluded neonicotinoids in the amounts typically used harm bees and safer alternatives are urgently needed.[61] At the same time, other sources suggest the evidence is not conclusive,[74] and that clarity regarding the facts is hampered by the role played by various issue advocates and lobby groups.[74]

Scientists have long been concerned that pesticides, including possibly some fungicides, may have sublethal effects on bees, not killing them outright, but instead impairing their development and behavior.[75] Of special interest is the class of insecticides called neonicotinoids, which contain the active ingredient imidacloprid, and other similar chemicals, such as clothianidin and thiamethoxam. Honey bees may be affected by such chemicals when they are used as a seed treatment because they are known to work their way through the plant up into the flowers and leave residues in the nectar. The containment of neonicotinoids to crops and fields designated for treatment is difficult, and many times inefficient. Run-off from treated fields and farm machinery that is not properly cleaned after field treatments can lead to exposure and uptake of pesticides by untreated plants. Therefore, honey bees are not only exposed to neonicotinoids by foraging on treated plants, but also by foraging on plants unintentionally exposed to these chemicals.[76] The doses taken up by bees are not lethal, but possible chronic problems could be caused by long-term exposure.[27] In a laboratory setting, both lethal and sub-lethal effects on foraging behavior, memory, and learning ability have been observed in honey bees exposed to neonicotinoids. However, these effects were not seen in field studies with field-realistic dosages.[77] Most corn grown in the US is treated with neonicotinoids, and a 2012 study found high levels of clothianidin in pneumatic planter exhaust. In the study, the insecticide was present in the soil of unplanted fields near those planted with corn and on dandelions growing near those fields.[78] Another 2012 study also found clothianidin and imidacloprid in the exhaust of pneumatic seeding equipment.

A 2010 survey reported 98 pesticides and metabolites detected in aggregate concentrations up to 214 ppm in bee pollen; this figure represents over half of the individual pesticide incidences ever reported for apiaries. It was suggested that "while exposure to many of these neurotoxicants elicits acute and sublethal reductions in honey bee fitness, the effects of these materials in combinations and their direct association with CCD or declining bee health remains to be determined."[79]

Evaluating pesticide contributions to CCD is particularly difficult for several reasons. First, the variety of pesticides in use in the different areas reporting CCD makes it difficult to test for all possible pesticides simultaneously. Second, many commercial beekeeping operations are mobile, transporting hives over large geographic distances over the course of a season, potentially exposing the colonies to different pesticides at each location. Third, the bees themselves place pollen and honey into long-term storage, effectively meaning a delay may occur from days to months before contaminated provisions are fed to the colony, negating any attempts to associate the appearance of symptoms with the actual time when exposure to pesticides occurred.

Imidacloprid map of use, US, 2012 (estimated)

To date, most of the evaluation of possible roles of pesticides in CCD have relied on the use of surveys submitted by beekeepers, but direct testing of samples from affected colonies seems likely to be needed, especially given the possible role of systemic insecticides such as the neonicotinoid imidacloprid (which are applied to the soil and taken up into the plant's tissues, including pollen and nectar), which may be applied to a crop when the beekeeper is not present. The known effects of imidacloprid on insects, including honey bees, are consistent with the symptoms of CCD;[80] for example, the effects of imidacloprid on termites include apparent failure of the immune system, and disorientation.[81]

In Europe, the interaction of the phenomenon of "dying bees" with imidacloprid has been discussed for quite some time.[82][83][84] A study from the "Comité Scientifique et Technique (CST)" was at the center of discussion, and led to a partial ban of imidacloprid in France. The imidacloprid pesticide Gaucho was banned in 1999 by the French Minister of Agriculture Jean Glavany, primarily due to concern over potential effects on honey bees.[85][86][87] Subsequently, when fipronil, a phenylpyrazole insecticide and in Europe mainly labeled "Regent", was used as a replacement, it was also found to be toxic to bees, and banned partially in France in 2004.[88]

In February 2007, about 40 French deputies, led by Jacques Remiller of the UMP, requested the creation of a parliamentary investigation commission on overmortality of bees, underlining that honey production had decreased by 1,000 tons a year for a decade. By August 2007, no investigation had opened.[89] Five other insecticides based on fipronil were also accused of killing bees. However, the scientific committees of the European Union are still of the opinion "that the available monitoring studies were mainly performed in France and EU-member-states should consider the relevance of these studies for the circumstances in their country."[90]

Around the time when French beekeepers succeeded in banning neonicotinoids, the Clinton administration permitted pesticides that were previously banned,[91] including imidacloprid. In 2004, the Bush administration reduced regulations further and pesticide applications increased.[92][93]

In 2005, a team of scientists led by the National Institute of Beekeeping in Bologna, Italy, found pollen obtained from seeds dressed with imidacloprid contain significant levels of the insecticide, and suggested the polluted pollen might cause honey bee colony death.[94] Analysis of maize and sunflower crops originating from seeds dressed with imidacloprid suggest large amounts of the insecticide will be carried back to honey bee colonies.[95] Sublethal doses of imidacloprid in sucrose solution have also been documented to affect homing and foraging activity of honey bees.[96] Imidacloprid in sucrose solution fed to bees in the laboratory impaired their communication for a few hours.[97] Sublethal doses of imidacloprid in laboratory and field experiment decreased flight activity and olfactory discrimination, and olfactory learning performance was impaired.[98]

Research, in 2008, by scientists from Pennsylvania State University found high levels of the pesticides fluvalinate and coumaphos in samples of wax from hives, as well as lower levels of 70 other pesticides.[69] These chemicals have been used to try to eradicate varroa mites, a bee pest that itself has been thought to be a cause of CCD. Researchers from Washington State University, under entomology professor Steve Sheppard in 2009, confirmed high levels of pesticide residue in hive wax and found an association between it and significantly reduced bee longevity.[99]

The WSU work also focused on the impact of the microsporidian pathogen Nosema ceranae, the build-up of which was high in the majority of the bees tested, even after large doses of the antibiotic fumagillin. Penn State's Dr. Maryann Frazier said, "Pesticides alone have not shown they are the cause of CCD. We believe that it is a combination of a variety of factors, possibly including mites, viruses and pesticides."[99]

In 2010, fipronil was blamed for the spread of CCD among bees, in a study by the Minutes-Association for Technical Coordination Fund in France, which found that even at very low nonlethal doses, this pesticide still impairs the ability to locate the hive, resulting in large numbers of foragers lost with every pollen-finding expedition, though no mention was made regarding any of the other symptoms of CCD;[100] other studies, however, have shown no acute effect of fipronil on honey bees.[101] Fipronil is designed to eliminate insects similar to bees, such as yellowjackets (Vespula germanica) and many other colonial pests by a process of 'toxic baiting', whereby one insect returning to the hive spreads the pesticide among the brood.[102]

Honeycomb of honey bees with eggs and larvae. The walls of the cells have been removed. The larvae (drones) are about 3 or 4 days old.

A large 2010 survey of healthy and CCD-affected colonies also revealed elevated levels of pesticides in wax and pollen, but the amounts of pesticides were similar in both failing and healthy hives. They also confirmed suspected links between CCD and poor colony health, inadequate diet, and long-distance transportation. Studies continue to show very high levels of pathogens in CCD-affected samples and lower pathogen levels in unaffected samples, consistent with the empirical observation that healthy honey bee colonies normally fend off pathogens. These observations have led to the hypothesis that bee declines are resulting from immune suppression.[14]

In 2010, a sequencing of the honey bee genome provided a possible explanation for the sensitivity of bees to pesticides. Its genome is deficient in the number of genes encoding detoxification enzymes, including cytochrome P450 monooxygenases (P450s), glutathione-S-transferases, and carboxylesterases.[103]

In 2012, researchers announced findings that sublethal exposure to imidacloprid rendered honey bees significantly more susceptible to infection by the fungus Nosema, thereby suggesting a potential link to CCD, given that Nosema is increasingly considered to contribute to CCD.[104]

Neonicotinoids may interfere with bees' natural homing abilities, causing them to become disoriented and preventing them from finding their way back to the hive.[105][106][107]

Also, in 2012, researchers in Italy published findings that the pneumatic drilling machines that plant corn seeds coated with clothianidin and imidacloprid release large amounts of the pesticide into the air, causing significant mortality in foraging honey bees. According to the study, "Experimental results show that the environmental release of particles containing neonicotinoids can produce high exposure levels for bees, with lethal effects compatible with colony losses phenomena observed by beekeepers."[108] Commonly used pesticides, such as the imidacloprid, reduce colony growth and new queen production in experimental exposure matched to field levels.[105] Lu et al (2012) reported they were able to replicate CCD with imidacloprid.[109] Another neonicotinoid, thiamethoxam, causes navigational homing failure of foraging bees, with high mortality.[107]

A 2012 in situ study provided strong evidence that exposure to sublethal levels of imidacloprid in high fructose corn syrup (HFCS) used to feed honey bees when forage is not available causes bees to exhibit symptoms consistent to CCD 23 weeks after imidacloprid dosing. The researchers suggested, "the observed delayed mortality in honey bees caused by imidacloprid in HFCS is a novel and plausible mechanism for CCD, and should be validated in future studies."[109][110]

In March 2013, two studies were published showing that neonicotinoids affect bee long-term and short-term memory, suggesting a cause of action resulting in failure to return to the hive.[111][112] In another study done in 2013, scientists reported that experiments suggested that exposure to the neonicotinoid pesticides clothianidin and imidicloprid results in increased levels of a particular protein in bees that inhibits a key molecule involved in the immune response, making the insects more susceptible to attack by harmful viruses.[113] Growth in the use of neonicotinoid pesticides has roughly tracked rising bee deaths.[11][114] In 2015, an 11-year British study showed a definitive relationship between increasing agricultural use of neonicotinoid and escalating honey bee colony losses at a landscape level.[115] This is the first field study to establish a link between neonicotinoids and CCD.[116]

Bee collecting pollen

In July 2013, scientists from the University of Maryland and the US Department of Agriculture found that a combination of pesticides has been contaminating the pollen bees use to feed their hives. When researchers collected pollen from hives on the east coast, they discovered that it was contaminated (on average) with 9 different fungicides and pesticides, although scientists found a blend of 21 different agricultural chemicals in one sample of pollen. Eight ag chemicals were identified to be associated with increased risk of infection by Nosema ceranae.[65]

A meta-analysis study published in February 2016 strongly suggests a pattern linking imidacloprid to sublethal effects on honey bees, stating: "trace dietary imidacloprid at field-realistic levels in nectar will have no lethal effects, but will reduce expected performance in honey bees by between 6 and 20%. Statistical power analysis showed that published field trials that have reported no effects on honey bees from neonicotinoids were incapable of detecting these predicted sublethal effects with conventionally accepted levels of certainty."[117]

In 2012, several peer-reviewed independent studies were published showing that neonicotinoids had previously undetected routes of exposure affecting bees including through dust, pollen, and nectar[108] and that subnanogram toxicity resulted in failure to return to the hive without immediate lethality,[118] one primary symptom of CCD.[104] Research also showed environmental persistence in agricultural irrigation channels and soil.[119] These reports prompted a formal peer review by the European Food Safety Authority, which stated in January 2013 that some neonicotinoids pose an unacceptably high risk to bees, and identified several data gaps not previously considered. Their review concluded, "A high acute risk to honey bees was identified from exposure via dust drift for the seed treatment uses in maize, oilseed rape and cereals. A high acute risk was also identified from exposure via residues in nectar and/or pollen."[120][121] Dave Goulson, an author of one of the studies that prompted the EFSA review, has suggested that industry science pertaining to neonicotinoids may have been deliberately deceptive, and the UK Parliament has asked manufacturer Bayer Cropscience to explain discrepancies in evidence they have submitted to an investigation.[122]

Early in 2013, the European Food Safety Authority issued a declaration that three specific neonicotinoid pesticides pose an acute risk to honey bees, and the European Commission (EC) proposed a two-year ban on them.[123] David Goulson, who led one of the key 2012 studies at the University of Stirling, said the decision "begs the question of what was going on when these chemicals were first approved." The chemical manufacturer Bayer said it was "ready to work with" the EC and member states.[124] In April 2013, the European Union voted for a two-year restriction on neonicotinoid insecticides. The ban will restrict the use of imidacloprid, clothianidin, and thiamethoxam for use on crops that are attractive to bees. Eight nations voted against the motion, including the British government, which argued that the science was incomplete.[125] The ban can be seen as an application of the "precautionary principle", established at the 1992 Rio Conference on the Environment and Development, which advocates that "lack of full scientific certainty shall not be used as a reason for postponing cost-effective measures to prevent environmental degradation."[74][126]

Pollination

In March 2013, professional beekeepers and environmentalists jointly filed a lawsuit against the United States Environmental Protection Agency (EPA) for continuing to allow the use of neonicotinoids in the United States. The suit specifically asks for suspension of clothianidin and thiamethoxam. The lawsuit follows a dramatic die off of bees in the United States, with some beekeepers losing 50% of their hives.[127] The EPA responded to the suit by issuing a report blaming the Varroa mite for the decline in bees and claiming the role of neonicotinoids in bee extinction has been overstated.[128]

Also in 2013, the Save America's Pollinators Act of 2013 (H.R. 2692) was introduced in Congress.[129][130] The proposed act asks that neonicotinoids be suspended until a full review of their impacts has occurred. The bill was reintroduced on 4 March 2015 as the Saving America's Pollinators Act (H.R. 1284), where it is currently being debated by the House Subcommittee on Biotechnology, Horticulture, and Research.

Early researchers commented that the pathway of propagation functions in the manner of a contagious disease; however, some sentiment existed that the disorder may involve an immunosuppressive mechanism,[131] potentially linked to "stress" leading to a weakened immune system. Specifically, according to research done in 2007 at the Pennsylvania State University: "The magnitude of detected infectious agents in the adult bees suggests some type of immunosuppression". These researchers initially suggested a connection between Varroa destructor mite infestation and CCD, suggesting that a combination of these bee mites, deformed wing virus (which the mites transmit) and bacteria work together to suppress immunity and may be one cause of CCD.[3][132] Parasites, such as varroa mites (Varroa destructor), honey bee tracheal mites (Acarapis woodi), fungal, bacterial and viral diseases, and kleptoparasites such as small hive beetles (Aethina tumida), are all problems that have been introduced within the last 20 years in the continental U.S., and are faced by beekeepers.[43]

When a colony is dying, for whatever cause, and other healthy colonies are nearby (as is typical in a bee yard), those healthy colonies often enter the dying colony and rob its provisions for their own use. If the dying colony's provisions were contaminated (by natural or man-made toxins), the resulting pattern (of healthy colonies becoming sick when in proximity to a dying colony) might suggest to an observer that a contagious disease is involved. However, in typical CCD cases, provisions of dying colonies are not robbed, suggesting that toxins do not spread via robbing, thereby mimicking a disease.

Additional evidence that CCD is an infectious disease came from the following observations: the hives of colonies that had died from CCD could be reused with a healthy colony only if they were first treated with DNA-destroying radiation,[133] and the CCD Working Group report in 2010 indicated that CCD-exhibiting hives tended to occur in proximity to one another within apiaries.[69]

Coumaphos, an organophosphate, is lipophilic, and so accumulates in wax. Increased levels of compound in wax have been shown to decrease survivorship of developing queens.[134]

Varroa destructor on a honey bee host

According to a 2007 article, the mite Varroa destructor remains the world's most destructive honey bee killer, due in part to the viruses it carries, including deformed wing virus and acute bee paralysis virus, which have both been implicated in CCD.[132][135] Affliction with Varroa mites also tends to weaken the immune system of the bees. Dr. Enesto Guzman, an entomological researcher at the University of Guelph in Canada, studied 413 Ontario bee colonies in 2007–08. The presence of Varroa mites within colonies before winter was observed to weaken the immune systems of bees and introduce viruses that led to colony death during the winter. About 27% of hives did not survive the winter, and the Varroa mite was identified as the cause in 85% of the cases.[136] Varroa mites also affect the queen's ability to reproduce, which is detrimental to the survival of the hive.[137] As such, Varroa mites have been considered as a possible cause of CCD, though not all dying colonies contain these mites.[138]

Varroa destructor is a parasitic mite that colonizes beehives and preys on honey bees by consuming their hemolymph. Varroa mites parasitize all types of honey bees (workers, nurse bees, larvae) depending on their life cycle stage. During the phoretic stage, Varroa prefer to attach to nurse bees as this results in higher fitness leading into the reproductive stage. The mites then feed on larvae during their reproductive stage and increased fitness leads to an increase in mite fecundity (number of female offspring).[139] Due to Varroa's ability to feed on all types of honey bees, they are one of the biggest threats to colonies, especially over winter.

In 2004, Israeli acute paralysis virus (IAPV), was discovered in Israel and at one time it was considered the cause of CCD. It was named after the place it was first identified; its place of origin is unknown. In September 2007, results of a large-scale statistical RNA sequencing study of afflicted and unafflicted colonies were reported. RNA from all organisms in a colony was sequenced and compared with sequence databases to detect the presence of pathogens. All colonies were found to be infected with numerous pathogens, but only the IAPV virus showed a significant association with CCD: the virus was found in 25 of the 30 tested CCD colonies, and only in one of the 21 tested non-CCD colonies.[133][140]

Research in 2009 has found that an indicator for an impaired protein production is common among all bees affected by CCD, a pattern consistent with IAPV infection. It is conjectured that Dicistroviridae, like the IAPV, cause degradation of the ribosomes, which are responsible for protein production of cells, and that this reduced ribosomal function weakens the bees, making them more vulnerable to factors that might not otherwise be lethal.[41][141]

Some have suggested the syndrome may be an inability by beekeepers to correctly identify known diseases such as European foulbrood or the microsporidian fungus Nosema apis. The testing and diagnosis of samples from affected colonies (already performed) makes this highly unlikely, as the symptoms are fairly well known and differ from what is classified as CCD. A high rate of Nosema infection was reported in samples of bees from Pennsylvania, but this pattern was not reported from samples elsewhere.[3]

When healthy bees are fed pollen filled with fungicides, insecticides and other agriculture chemicals, they are more likely to be infected by Nosema ceranae, a parasitic microsporidian fungus associated with widespread death of honey bees.[12] Hives of western honey bees infected with Nosema ceranae are wiped out within eight days[142] indicating that CCD may be caused by N. ceranae. A research team claim to have ruled out many other potential causes,[143][89] however, a 2009 survey of US CCD-affected bee populations found only about half of the colonies sampled, both in CCD and control populations, were infected with N. ceranae.[69]

A hyperparasitic microsporidian, Nosema podocotyloidis, a parasite of a digenean, Podocotyloides magnatestis, which is itself a parasite of the fish Parapristipoma octolineatum (Teleostei)

The primary antifungal agent used against Nosema is fumagillin, which has been used in a German research project to reduce the microsporidian's impact, and is mentioned as a possible remedy by the CCDWG.[144] Higes also claims to have successfully cured colonies with fumagillin.[145][146] A review of these results described these results as promising, but cautioned "N. ceranae may not be to blame for all cases of colony collapse".[147] Various areas in Europe have reported this fungus, but no direct link to CCD has yet been established.[148][149]

In 2007, N. ceranae was reported in a few hives in California.[150] The researcher did not, however, believe this was conclusive evidence of a link to CCD; "We don't want to give anybody the impression that this thing has been solved".[151] A USDA bee scientist has similarly stated, "while the parasite Nosema ceranae may be a factor, it cannot be the sole cause. The fungus has been seen before, sometimes in colonies that were healthy".[152]

N. ceranae has been detected in honey bees from several states using PCR of the 16S gene.[153][154] In New York, N. ceranae was detected in 49 counties, and of the 1,200 honey bee samples collected, 528 (44%) were positive for Nosema, from which, PCR analysis of 371 spore positive samples revealed 96% were N. ceranae, 3% had both N. ceranae and N. apis, and 1% had N. apis only.[155]

A University of Montana and Montana State University team of scientists headed by Jerry Bromenshenk and working with the US Army's Edgewood Chemical Biological Center published a paper in October 2010 saying that a new DNA virus, invertebrate iridescent virus type 6 (IIV-6), and the fungus Nosema ceranae were found in every killed colony the group studied. In their study, they found neither agent alone seemed deadly, but a combination of the virus and N. ceranae was always 100% fatal.[156][157][158] Information about the study was released to the public in a front-page article in The New York Times.[159] A few days later, an article was published in Fortune Magazine with the title, "What a scientist didn't tell the New York Times about his study on bee deaths". Professor of entomology at Penn State University James Frazier, who was researching the sublethal impact of pesticides on bees, said that while Bromenshenk's study generated some useful data, Bromenshenk has a conflict of interest as CEO of a company developing scanners to diagnose bee diseases.[160] A few months later, the methods used to interpret the mass spectrometry data in the Bromenshenk study were called into question, raising doubts as to whether IIV-6 was ever correctly identified in any of the samples examined.[161][162]

In 2013, researchers collected pollen from hives and fed it to healthy bees. The pollen had an average of nine different pesticides and fungicides. Further, the researchers discovered that bees that ate pollen with fungicides were three times more likely to be infected by parasites. Their study shows that fungicides, thought harmless to bees, may actually play a significant role in CCD. Their research also showed that spraying practices may need to be reviewed because the bees sampled by the authors foraged not from crops, but almost exclusively from weeds and wildflowers, suggesting that bees are more widely exposed to pesticides than thought.[163]

Dennis vanEngelsdorp, an entomologist at the University of Maryland, has been quoted as saying "Fungicides, which we didn't expect to harm insects, seem to have a sub-lethal effect on bee health". He went on further to state this is important because fungicides are not heavily regulated.[12]

Most beekeepers affected by CCD report that they use antibiotics and miticides in their colonies, though the lack of uniformity as to which particular chemicals they use[3] makes it unlikely that any single such chemical is involved. However, it is possible that not all such chemicals in use have been tested for possible effects on honey bees, and could therefore potentially be contributing to the CCD phenomenon.[2][164] Beekeepers use miticides to rid colonies of Varroa infestations; however, treatment can lead to higher levels of viral infections in colonies. High doses of treatment or the use of miticides for an extended period of time can lead to immune-suppression in honey bees, making them more susceptible to viruses. A study at the University of Texas in Austin found that commonly used antibiotics found in beekeeping to prevent disease lower the gut microbial levels in honeybees, making them more susceptible to disease. A widespread occurrence of viral infections within a beehive can lead to colony collapse. Researchers state that although the antibiotics are necessary, beekeepers should exercise caution when determining amount and length of use.[165] A widespread occurrence of viral infections within a beehive can lead to colony collapse. Although miticides are not a direct cause of CCD they do play a role in it.[166]

In 2008 high levels of the pesticides fluvalinate and coumaphos were found in samples of wax from hives, as well as lower levels of 70 other pesticides.[69] These chemicals have been used to try to eradicate varroa mites, a bee pest that itself has been thought to be a cause of CCD. A 2009 study confirmed high levels of pesticide residue in hive wax and found an association between the pesticide and reduced bee longevity.[99] Nosema ceranae, was found in high concentrations in the majority of the bees tested, even after administering large doses of the antibiotic fumagillin. Maryann Frazier commented, "Pesticides alone have not shown they are the cause of CCD. We believe that it is a combination of a variety of factors, possibly including mites, viruses and pesticides."[99]

Environmental changes may have an effect on honey bee development, but the precise impact of potential environmental changes on honey bees as a result of climate change is unknown.[167]

Moving spring bees from South Carolina to Maine for blueberry pollination

Since U.S. beekeeper Nephi Miller first began moving his hives to different areas of the country for the winter of 1908, migratory beekeeping has become widespread in America. Bee rental for pollination is a crucial element of U.S. agriculture, which could not produce anywhere near its current levels with native pollinators alone.[168] U.S. beekeepers collectively earn much more from renting their bees out for pollination than they do from honey production.

Researchers are concerned that trucking colonies around the country to pollinate crops, where they intermingle with other bees from all over, helps spread viruses and mites among colonies. Additionally, such continuous movement and re-settlement is considered by some a strain and disruption for the entire hive, possibly rendering it less resistant to all sorts of systemic disorder.[169]

Most of the focus on CCD has been toward environmental factors. CCD is a condition recognised for greatest impact in regions of 'industrial' or agricultural use of commercially bred bee colonies. Natural breeding and colony reproduction of wild bees is a complex and highly selective process, leading to a diverse genetic makeup in large within-colony populations of bees,[170] which might not be reproduced in commercially bred colonies.

In 2007, one of the patterns reported by the CCD Study Group at Pennsylvania State was that all producers in a preliminary survey noted a period of "extraordinary stress" affecting the colonies in question prior to their die-off, most commonly involving poor nutrition and/or drought.[3] This was the only factor that all of the cases of CCD had in common in the report; accordingly, there appeared to be at least some significant possibility that the phenomenon was correlated to nutritional stress that may not manifest in healthy, well-nourished colonies. This was similar to the findings of another independent survey done in 2007 in which small-scale beekeeping operations (up to 500 colonies) in several states reported their belief that malnutrition and/or weak colonies was the factor responsible for their bees dying in over 50% of the cases, whether the losses were believed to be due to CCD or not.[45]

Some researchers have attributed the syndrome to the practice of feeding high-fructose corn syrup (HFCS) to supplement winter stores. The variability of HFCS may be relevant to the apparent inconsistencies of results. One European writer has suggested a possible connection with HFCS produced from genetically modified corn.[171] However, at least one researcher states that if this were the sole factor involved, this should also lead to the exclusive appearance of CCD in wintering colonies being fed HFCS, but many reports of CCD occur in other contexts with beekeepers who do not use HFCS.[172]

Other researchers state that colony collapse disorder is mainly a problem of feeding the bees a monoculture diet when they should receive food from a variety of sources/plants. In winter, these bees are given a single food source such as corn syrup (high-fructose or other), sugar and pollen substitute. In summer, they may only pollinate a single crop (e.g., almonds, cherries, or apples).[173] The monoculture diet is attributed to bee rentals and migratory bee keeping. Honey bees are only being introduced to select commercial crops such as corn. These single pollen diets are greatly inferior to mixed pollen diets. However, there are a few pollens that are acceptable for honey bees to be introduced to exclusively, including sweet clover and mustard.[174]

Pupae of honeybee drones in opened cells at both sides of a honeycomb. The drones at the right side are some days older and more developed.

A study published in 2010 found that bees that were fed pollen from a variety of different plant species showed signs of having a healthier immune system than those eating pollen from a single species. Bees fed pollen from five species had higher levels of glucose oxidase than bees fed pollen from one species, even if the pollen had a higher protein content. The authors hypothesised that CCD may be linked to a loss of plant diversity.[175] Researches found a proper diet that does lead to a healthy honey bee population. "The authors recommended a diet containing 1000 ppm potassium, 500 ppm calcium,300 ppm magnesium and 50 ppm each of sodium, zinc, manganese, iron and copper."[174] A 2014 studies found that bees fed high-fructose corn syrup or sugar shows downregulation in several genes related to protein metabolism and oxidation reduction as compared to those fed the similarly low-protein honey.[176]

A 2013 study found that p-Coumaric acid, which is normally present in honey, assists bees in detoxifying certain pesticides. Its absence in artificial nutrients fed to bees may therefore contribute to CCD.[177]

Despite considerable discussion on the Internet and in the lay media, there have been almost no careful studies, published in peer reviewed scientific literature, on effects of electromagnetic field exposure on honeybees.[178][179] One of the few peer-reviewed studies was published in 1981 and found that even at microwave radiation powers far higher than used in communication, that bees were not significantly affected.[180]

A study on the non-thermal effects of radio frequency (RF) on honey bees (Apis mellifera carnica) reported there were no changes in behavior due to RF exposure from DECT cordless phone base stations operating at 1,880–1,900 MHz.[181] A later study established that close-range electromagnetic field (EMF) may reduce the ability of bees to return to their hive.[182] In the course of their study, one half of their colonies broke down, including some control hives that did not have embedded DECT base stations. In April 2007, news of this study appeared in various media outlets, beginning with an article in The Independent, which stated that the subject of the study included mobile phones and had related them to CCD.[183] Although cellular phones were implicated at the time by other media reports, they were not covered in the quoted study. The researchers involved have since stated that their research did not include findings on cell phones, nor their relationship to CCD, and indicated that the Independent article had misinterpreted their results and created "a horror story".[178][179]

A review of 919 peer-reviewed scientific studies investigating the effects of EMF on wildlife, humans, and plants included 7 studies involving honey bees; 6 of which reported negative effects from exposure to EMF radiation, but none demonstrated any specific link to CCD.[184] A 2004 exploratory study was conducted on the non-thermal effects of electromagnetic exposure and learning. The investigators did not find any change in behavior due to RF exposure from the DECT base station operating at 1880–1900 MHz.[181]

Honeybees can detect weak static, or low-frequency magnetic fields, which they use as one of several cues in navigation. However, no mechanism has been established by which weak radio frequency energy can affect the behavior of insects, apart from minor heating effects.[185]

GM crops are not considered to be a cause. In 2008 a meta-analysis[186] of 25 independent studies assessing effects of Bt Cry proteins on honeybee survival (mortality) showed that Bt proteins used in commercialized GE crops to control lepidopteran and coleopteran pests do not negatively impact the survival of honeybee larvae or adults. Additionally, larvae consume only a small percent of their protein from pollen, and there is also a lack of geographic correlation between GM crop locations and regions where CCD occurs.[187]