Free access
Review
Issue
Apidologie
Volume 41, Number 3, May-June 2010
Honey bee health
Page(s) 312 - 331
DOI http://dx.doi.org/10.1051/apido/2010018
Published online 12 April 2010

© INRA/DIB-AGIB/EDP Sciences, 2010

1. PESTICIDES APPLIED TO CROPS

Despite the dependence on honey bees for the pollination of crops in the USA, colony numbers have declined by 45% over the past 60 years (NAS, 2007). Most honey bee losses from 1966–1979 were attributable to organochlorine, carbamate, organophosphorus, and pyrethroid pesticide exposure (Atkins, 1992). Efforts to restrict pesticide application during bloom provided some relief; however, the residual activity of some pesticides was never effectively addressed (Johansen and Mayer, 1990). Previous reviews and extension publications are available concerning the protection of honey bees from these 4 classes of pesticides (Johansen, 1977; Crane and Walker, 1983; Adey et al., 1986; Johansen and Mayer, 1990; Ellis et al., 1998).

Colony losses were especially severe from 1981 to 2005 with a drop from 4.2 million to 2.4 million (NAS, 2007) although some of the decrease is attributable to changes in how colony numbers were estimated. The introduction of parasitic honey bee mites, Acarapis woodi (1984) and Varroa destructor (1987), contributed to dramatic bee losses. At the same time, the control of crop pests in USA agriculture was rapidly changing. Genetically engineered (GE) crops were developed and extensively deployed, and two new classes of systemic pesticides, neonicotinoids and phenylpyrazoles, replaced many of the older pesticides described above.

The rapid development and deployment of these 2 new insect control techniques distinguish USA agriculture from agriculture in other regions of the world. In Europe a more cautious approach to the adoption of new agricultural practicices has been taken. Since the registration and regulation of GE crops and neonicotinoid and phenylpyrazole pesticides are major shifts in insect control in USA agriculture, they are emphasized in this section of our review.

The recent sequencing of the honey bee genome provides a possible explanation for the sensitivity of bees to pesticides; relative to other insect genomes, the honey bee genome is markedly deficient in the number of genes encoding detoxification enzymes, including cytochrome P450 monooxygenases (P450s), glutathione-S-transferases, and carboxylesterases (Claudianos et al., 2006).

1.1. GE plant varieties

GE (genetically engineered) plant varieties that have herbicide tolerance or insecticidal properties were first introduced into the USA in 1996. Soybeans and cotton are genetically engineered with herbicide-tolerant traits and have been the most widely and rapidly adopted GE crops in the USA, followed by insect-resistant cotton and corn. In 2007 these GE crops were planted on more than 113 million hectares worldwide (USDA-Biotech Crop Data, 2009), and the United States leads the world in acres planted with GE crops with most of the plantings on large farms (Lemaux, 2008). Insect resistance is conferred by incorporating genes coding for insecticidal proteins produced by Bacillus thuringensis (Bt), a widespread soil bacterium (ISB, 2007). While Bt is also delivered through traditional spray application, plants benefit from continuous production of Bt toxins through genetic engineering. Bt δ-endotoxins are activated in the insect gut where they bind to receptor sites on the midgut epithelium to form pores. These pores allow gut contents to leak out of the lumen and cause osmotic stress to midgut cells, leading to the eventual destruction of the midgut and the death of the insect (Soberon et al., 2009). To date, Bt genes have been incorporated into corn (Zea mays), cotton (Gossypium hirsutum), potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), and GE seeds of these crops are available to producers (ISB, 2007). Precommercial field tests of 30 different plant species with Bt genes were conducted in 2008 including apples, cranberries, grapes, peanuts, poplar, rice, soybeans, sunflowers and walnuts (ISB, 2007).

Numerous studies have been conducted to determine the impact of GE crops on honey bees. Canadian scientists found no evidence that Bt sweet corn affected honey bee mortality (Bailey et al., 2005). Studies conducted in France found that feeding Cry1ab protein in syrup did not affect honey bee colonies (Ramirez-Romero et al., 2005). Likewise, exposing honey bee colonies to food containing Cry3b at concentrations 1000 times that found in pollen resulted in no effect on larval or pupal weights (Arpaia, 1996). Feeding honey bees pollen from Cry1ab maize did not affect larval survival, gut flora, or hypopharyngeal gland development (Babendreier et al., 20052007). A 2008 meta-analysis of 25 independent studies concluded that the Bt proteins used in GE crops to control lepidopteran and coleopteran pests do not negatively impact the survival of larval or adult honey bees (Duan et al., 2008).

There is no evidence that the switch to Bt crops has injured honey bee colonies in the USA. To the contrary, it has benefited beekeeping by reducing the frequency of pesticide applications on crops protected by Bt, especially corn and cotton. On the other hand, the switch to GE crops with herbicide resistance has eliminated many blooming plants from field borders and irrigation ditches as well as from the crop fields themselves. The reduction in floral diversity and abundance that has occurred due to the application of Round-UP® Herbicide (glyphosate) to GE crops with herbicide resistance is difficult to quantify. However, there is a growing body of evidence that poor nutrition is a primary factor in honey bee losses. Eischen and Graham (2008) clearly demonstrated that well-nourished honey bees are less susceptible to Nosema ceranae than poorly nourished bees. Because honey bees are polylectic, the adoption of agricultural practices that provide greater pollen diversity has been suggested, including the cultivation of small areas of other crops near monocultures or permitting weedy areas to grow along the edges of fields (Schmidt et al., 1995). A detailed review of management of uncropped farmland to benefit pollinators by Decourtye et al. (2010) is included in this special issue.

1.2. Neonicotinoid and phenylpyrazole pesticides

Another major shift in USA agriculture has been the development and extensive deployment of neonicotinoid and phenylpyrazole pesticides. These pesticides are extensively used in the USA on field, vegetable, turf, and ornamental crops, some of which are commercially pollinated by bees (Quarles, 2008). They can be applied as seed treatments, soil treatments and directly to plant foliage. Neonicotinoids are acetylcholine mimics and act as nicotinic acetychloline receptor agonists. Neonicotinoids cause persistent activation of cholinergic receptors which leads to hyperexcitation and death (Jeschke and Nauen, 2008). One neonicotinoid, imidacloprid, was applied to 788254 acres in California in 2005 (CDPR, 2006), making it the 6th most commonly used insecticide in a state that grows many bee-pollinated crops. The phenylpyrazoles, including fipronil, bind to γ-amino butyric acid (GABA)-gated chloride ion channels and block their activation by endogenous GABA, leading to hyperexcitation and death (Gunasekara et al., 2007).

Neonicotinoid and phenylpyrazole insecticides differ from classic insecticides in that they become systemic (Trapp and Pussemier, 1991) in the plant, and can be detected in pollen and nectar throughout the blooming period (Cutler and Scott-Dupree, 2007). As a consequence, bees can experience chronic exposure to them over long periods of time. While some studies have shown no negative effects from seed-treated crops (Nguyen et al., 2009), acute mortality was the only response measured. Desneux and his colleagues (2007) examined methods that could be used to more accurately assess the risk of neonicotinoid and phenylpyrazole insecticides including a test on honey bee larvae reared in vitro to test for larval effects (Aupinel et al., 2005), a proboscis extension response (PER) assay to access associative learning disruption (Decourtye and Pham-Delegue, 2002), various behavioral effects (Thompson, 2003), and chronic exposure toxicity beyond a single acute dose exposure (Suchail et al., 2001; Decourtye et al., 2005; Ailouane et al., 2009). Pesticide exposure may interact with pathogens to harm honey bee health. Honey bees that were both treated with imidacoprid and fed Nosema spp. spores suffered reduced longevity and reduced glucose oxidase activity (Alaux et al., 2010).

1.3. Registration procedures and risk assessment

In the USA risk assessment related to agrochemical use and registration follow specific guidelines mandated by the Federal Insecticide Fungicide and Rodenticide Act (EPA, 2009a). Despite the importance of honey bees, the effect of pesticide exposure on colony health has not been systematically monitored, and the Environmental Protection Agency (EPA) does not require data on sublethal effects for pesticide registration (NAS, 2007).

For many years, the classical laboratory method for registering pesticides was to determine the median lethal dose (LD50) of the pest insect. In a second step, the effects of pesticides on beneficial arthropods were examined by running LD50 tests on the beneficial species to identify products with the lowest non target activity (Croft, 1990; Robertson et al., 2007). The honey bee has often served as a representative for all pollinators in the registration process, though the toxicity of pesticides to non-Apis species may be different (Taséi, 2003; Devillers et al., 2003). In the USA this protocol remains the primary basis for risk assessment in pesticide registration. However, this approach to risk assessment only takes into account the survival of adult honey bees exposed to pesticides over a relatively short time frame (OEPP/EPPO, 1992). In Europe, where the standard procedures do not provide clear conclusions on the harmlessness of a pesticide, additional studies are recommended; however, no specific protocols are outlined (OEPP/EPPO, 1992). Acute toxicity tests on adult honey bees may be particularly ill-suited for the testing of systemic pesticides because of the different route of exposure bees are likely to experience in field applications. Chronic feeding tests using whole colonies may provide a better way to quantify the effects of systemics (Colin et al., 2004).

Registration review is replacing the EPA’s pesticide re-registration and tolerance reassessment programs. Unlike earlier review programs, registration review operates continuously, encompassing all registered pesticides. The registration review docket for imidacloprid opened in December 2008. To better ensure a ``level playing field'' for the neonicotinoid class as a whole and to best take advantage of new research as it becomes available, the EPA has moved the docket openings for the remaining neonicotinoids on the registration review schedule (acetamiprid, clothianidin, dinotefuran, thiacloprid, and thiamethoxam) to fiscal year 2012 (EPA, 2009b). The EPA’s registration review document states that ``some uncertainties have been identified since their initial registration regarding the potential environmental fate and effects of neonicotinoid pesticides, particularly as they relate to pollinators (EPA, 2009b)''. Studies conducted in Europe in the late 1990s have suggested that neonicotinoid residues can accumulate in pollen and nectar of treated plants and represent a potential risk to pollinators (Laurent and Rathahao, 2003). Adverse effects on pollinators have also been reported in Europe that have further heightened concerns regarding the potential direct and/or indirect role that neonicotinioid pesticides may have in pollinator declines (Suchail et al., 2000). Recently published data from studies conducted in Europe support concerns regarding the persistence of neonicotinoids. While the translocation of neonicotinoids into pollen and nectar of treated plants has been demonstrated, the potential effect that levels of neonicotinoids found in pollen and nectar can have on bees remains less clear. Girolami et al. (2009) report high levels of neonicotinoids from coated seeds in leaf guttation water and high mortality in bees that consume it. While the frequency of guttation drop collection by bees under field conditions is not documented, the authors describe the prolonged availability of high concentrations of neonicotinoids in guttation water as ``a threatening scenario that does not comply with an ecologically acceptable situation''. The pending EPA review will consider the potential effects of the neonicotinoids on honey bees and other pollinating insects, evaluating acute risk at the time of application and the longer-term exposure to translocated neonicotinoids (EPA, 2009b; Mullin et al., 2010).

2. PESTICIDES USED IN APICULTURE

The Varroa mite, Varroa destructor, is one of the most serious pests of honey bees in the USA and worldwide. It injures both adult bees and brood, and beekeepers are frequently compelled to use varroacides to avoid colony death (Boecking and Genersch, 2008). Varroacides must be minimally harmful to the bees, while maintaining toxicity to mites, which is a challenge given the sensitivity of honey bees to many pesticides (Atkins, 1992). The varroacides used in the USA can be broadly divided into three categories: synthetic organic, natural product and organic acid pesticides.

2.1. Synthetic organic pesticides

The pyrethroid tau-fluvalinate, a subset of isomers of fluvalinate, was the first synthetic varroacide registered for use in honey bee colonies in the USA. It was first registered as a Section 18 (emergency use label, state by state approval) in 1987 (Ellis et al., 1988). The Section 18 label allowed plywood strips to be soaked in an agricultural spray formulation of tau-fluvalinate, (Mavrik®), and treatment was made by suspending the strips between brood frames. In 1990 plastic strips impregnated with tau-fluvalinate (Apistan®) replaced homemade plywood strips (PAN, 2009) with a Section 3 label (full registration for use in all states). According to the label, a single strip contains 0.7 g tau-fluvalinate, as much as 10% of which may diffuse from the plastic strip formulation into hive matrices over the course of an 8 week treatment (Bogdanov et al., 1998; Vita Europe Ltd., 2009). While the agricultural spray formulation of tau-fluvalinate (Mavrik®) is no longer legal to use in the USA, its low cost and history of legal use in beehives make it vulnerable to misuse and may contribute to tau-fluvalinate residues detected in beeswax (Bogdanov, 2006; Wallner,1999; Berry, 2009; Mullin et al., 2010).

As a pyrethroid, tau-fluvalinate kills mites by blocking the voltage-gated sodium and calcium channels (Davies et al., 2007). While most pyrethroids are highly toxic to honey bees, tau-fluvalinate is tolerated in high concentrations due in large part to rapid detoxification by cytochrome P450 monooxygenases (P450s) (Johnson et al., 2006). However, tau-fluvalinate is not harmless to bees and does affect the health of reproductive castes. Queens exposed to high doses of tau-fluvalinate were smaller than untreated queens (Haarmann et al., 2002). Drones exposed to tau-fluvalinate during development were less likely to survive to sexual maturity relative to unexposed drones, and they also had reduced weight and produced fewer sperm (Rinderer et al., 1999). However, the practical consequence of tau-fluvalinate exposure on drones may be limited, as drones exposed to tau-fluvalinate produced as many offspring as unexposed drones (Sylvester et al., 1999).

Tau-fluvalinate was initially very effective at controlling Varroa mites, but many Varroa populations now exhibit resistance (Lodesani et al., 1995). Resistance to tau-fluvalinate in Varroa is due, at least in part, to a mutation in the voltage-gated sodium channel which confers reduced binding affinity for tau-fluvalinate (Wang et al., 2002). Despite diminished effectiveness, tau-fluvalinate continues to be used for Varroa control in the USA (Elzen et al., 1999; Macedo et al., 2002).

As the efficacy of tau-fluvalinate against Varroa was beginning to wane, coumaphos, an organophosphate pesticide, was granted Section 18 approval in the USA in 1999 as varroacide (Federal Register, 2000), and as a treatment for the small hive beetle, Aethina tumida Murray. Coumaphos is administered as Checkmite+® strips, each containing approximately 1.4 g coumaphos, which are hung between brood frames for 6 weeks. Coumaphos, or its bioactivated oxon metabolite, kills through the inactivation of acetylcholinesterase, thereby interfering with nerve signaling and function. While coumaphos initially proved effective at killing tau-fluvalinate resistant Varroa populations (Elzen et al., 2000), coumaphos resistant mite populations were found as early as 2001 (Elzen and Westervelt, 2002). The mechanism of resistance to coumaphos in Varroa is unknown, though esterase-mediated detoxification may be involved (Sammataro et al., 2005). Resistance likely follows the mechanisms of coumaphos resistance observed in the southern cattle tick, Boophilus microplus, which include acetylcholinsesterase insensitivity and enhanced metabolic detoxification (Li et al., 2005). Honey bees tolerate therapeutic doses of coumaphos, at least in part, as a consequence of detoxicative P450 activity (Johnson et al., 2009). However, honey bees can suffer negative effects from coumaphos exposure; queens exposed to coumaphos were smaller, suffered higher mortality and were more likely to be rejected when introduced to a colony (Haarmann et al., 2002; Collins et al., 2004; Pettis et al., 2004). Drone sperm viability was lower in stored sperm collected from drones treated with coumaphos (Burley et al., 2008).

Amitraz, a formamidine pesticide, was once registered (1992–Section 18 label) in the USA under the trade name Miticur® with the active ingredient incorporated in a plastic strip that was suspended between brood frames (PAN, 2009). However, the product was withdrawn from the market in 1994 when some beekeepers reported colony losses following treatment (PAN, 2009). While no conclusive evidence was presented that the product had harmed bees, the registrant decided to withdraw the product from the market (PAN, 2009). Amitraz is available in the USA as a veterinary miticide (Taktic®), but the label does not allow for use in honey bee colonies; however, the frequency with which amitraz metabolites are found in beeswax suggests that it continues to be used (Mullin et al., 2010; Berry, 2009). Amitraz strips (Apivar®) were granted an emergency registration for Varroa control by the Canadian PMRA for 2009 (PMRA, 2009), but they are not available to beekeepers in the USA.

Amitraz is an octopaminergic agonist in arthropods (Evans and Gee, 1980) and as such has the potential to influence honey bee behavior. High levels of octopamine in the honey bee brain are associated with increased foraging behavior and young bees which are fed octopamine are more likely to begin foraging than untreated bees (Schulz and Robinson, 2001). Forager honey bees treated with octopamine increased the reported resource value when communicating via the dance language (Barron et al., 2007). Amitraz has also shown acute toxicity, with larvae showing increased apoptotic cell death in the midgut when exposed to an amitraz solution (Gregorc and Bowen, 2000).

Despite the status of amitraz as an unregistered varroacide, Varroa mite populations in the USA exhibit resistance to amitraz, possibly through elevated esterase-mediated detoxification (Sammataro et al., 2005). The mechanism of Varroa resistance may be similar to the detoxicative resistance to amitraz that has been observed in some populations of Southern cattle ticks (Li et al., 2004).

Fenpyroximate is a pyrazole acaricide that was introduced for use in the USA in 2007 as Hivastan® under Section 18 registration (Wellmark, 2009). Hivistan® is formulated as a patty containing 675 mg of fenproximate. Fenpyroximate presumably kills mites through the inhibition of electron transport in the mitochondria at complex I, thereby interfering with energy metabolism (Motoba et al., 1992). While resistance to fenpyroximate in Varroa has not yet been observed, the eventual emergence of resistance is likely as it has been observed in other mites including the 2-spotted spider mite (Tetranychus urticae) which achieved resistance through elevated detoxicative P450 and esterase activity (Kim et al., 2004). The mechanism of tolerance to fenpyroximate in honey bees is unknown, but it is likely through the same detoxicative mechanisms, P450-mediated hydroxylation followed by transesterification, that occurs in vertebrates and other insects (Motoba et al., 2000). One potential consequence of chronic exposure to fenpyroximate, as an inhibitor of complex I mitochondrial activity, is the increased generation of reactive oxygen species (Sherer et al., 2007). Increased adult mortality has been observed with fenpyroximate use during the first days after application (CDPR, 2008).

2.2. Natural product pesticides

Natural product based varroacides have come into widespread use as synthetic pesticides have dwindled in effectiveness. Thymol and menthol, monoterpenoid constituents of plant-derived essential oils, are used for control of Varroa and tracheal mites, respectively. Thymol is the chief constituents in the fumigants Apilife Var® (tablets) and Apiguard® (gel), both of which are registered under Section 3. Essential oil-based varroacides were exempted from extensive testing for EPA registration because they are common food additives and ``generally recognized as safe'' for human consumption (Quarles, 1996). However, monoterpenoids such as thymol and menthol may not necessarily be safe for honey bees, since these compounds play a role in plants as broad spectrum pesticides (Isman, 2006). Indeed, thymol and menthol were found to be among the most toxic of all terpenoids tested when applied to honey bees as a fumigant (Ellis and Baxendale, 1997). These monoterpenoids likely kill Varroa by binding to octopamine (Enan, 2001) or GABA receptors (Priestley et al., 2003). Despite being naturally derived, these compounds may harm honey bees: thymol treatment can induce brood removal (Marchetti and Barbattini, 1984; Floris et al., 2004) and may result in increased queen mortality (Whittington et al., 2000).

2.3. Organic acid pesticides

Two organic acids, formic acid and oxalic acid, are attractive options as varroacides because both are naturally present in honey (Bogdanov, 2006; Rademacher and Harz, 2006). Formic acid is registered with Section 3 approval in the USA under the trade name MiteAway II® (NOD, 2009). MiteAway II® is a fumigant varroacide that is packaged in a slow release pad. Formic acid likely kills Varroa by inhibiting electron transport in the mitochondria through binding of cytochrome c oxidase, thereby inhibiting energy metabolism (Keyhani and Keyhani, 1980) and may produce a neuroexcitatory effect on arthropod neurons (Song and Scharf, 2008). Formic acid can harm honey bees by reducing worker longevity (Underwood and Currie, 2003) and harming brood survival (Fries, 1991).

Oxalic acid is registered for use as a varroacide in Canada and Europe, but not in the USA. In Canada it is trickled over honey bees in a sugar syrup solution (Canadian Honey Council, 2005) or sublimated using a vaporizer (Varrox, 2007). Research has shown it to be highly effective against Varroa in cool climates when brood is not present (Aliano and Ellis, 2008). The mode of action of oxalic acid against Varroa is unknown, but direct contact between Varroa and oxalic acid is required (Aliano and Ellis, 2008). Oxalic acid treatments administered in water are ineffective (Charrière and Imdorf, 2002), but administration in sugar water improves efficacy by adhering the active ingredient to the bees (Aliano and Ellis, 2008). In mammals, oxalic acid interferes with mitochondrial electron transport, probably through interaction with complex II or IV, leading to increased production of reactive oxygen species and to kidney toxicity (Cao et al., 2004; Meimaridou et al., 2005). Repeated treatment of colonies with oxalic acid can result in higher queen mortality and a reduction in the amount of sealed brood (Higes et al., 1999). The midguts of honey bees fed oxalic acid in sugar water exhibited an elevated level of cell death (Gregorc and Smodisskerl, 2007), though in field conditions bees will generally avoid consuming syrup with oxalic acid (Aliano and Ellis, 2008).

Oxalic acid is readily available and inexpensive in the USA for use as a wood bleach, but it is not labeled for use in controlling Varroa. Its easy availability from many sources has limited the willingness of suppliers to undergo the expensive and time-consuming registration process.

2.4. Interactions

With the large number of varroacides available to beekeepers in the USA, there is potential for interactions between compounds, a problem compounded by the fact that many synthetic varroacides are lipophilic and may remain in the wax component of hives for years following treatment (Bogdanov et al., 1997; Wallner, 1999; Mullin et al., 2010). The overlapping modes of action and mechanisms of tolerance in honey bees are also cause for concern. Interactions have been observed between tau-fluvalinate and coumaphos at the level of P450 detoxification (Johnson et al., 2009), and it seems likely that all varroacides depending on P450-mediated detoxification will display similar interactions. Fenpyroximate and the organic acids all interact with components of the mitochondrial electron transport chain (Keyhani and Keyhani, 1980; Motoba et al., 1992), where interactions could also be possible. Synergistic interactions between formamadines and pyethroids occur in other insects (Plapp, 1979) and may occur in honey bees between amitraz and tau-fluvalinate.

Interactions between in-hive varroacides and out-of-hive insecticides and fungicides are also of concern, particularly interactions between the P450-detoxified varroacides and the P450-inhibiting ergosterol biosynthesis inhibiting fungicides (Pilling et al., 1995).

3. PESTICIDE RESIDUES IN HIVE PRODUCTS

3.1. Need for sensitive pesticide analysis

Pesticide contamination of hive products is expected when honey bee colonies perish due to pesticide exposure. Colony mortality is often accompanied by part-per-million (ppm) residues in wax, beebread, honey and dead bee samples. However, part-per-billion (ppb) and occasionally ppm residues levels can be detected in hive matrices when honey bees forage in any conventional agricultural or urban setting. Since honey or pollen contaminated at ppb levels with newer classes of insecticides such as neonicotinoids (e.g. imidacloprid) or phenylpyrazoles (e.g. fipronil) are known to impair honey bee health (Decourtye et al., 2004; Halm et al., 2006; Desneux et al., 2007), it is important to use sensitive analytical technologies. Many pesticide contaminants, such as lipophilic pyrethroids and organophosphates, can be monitored in the hive using gas chromatography-mass spectrometry (GC-MS). The more recently developed liquid chromatography-tandem mass spectrometry (LC/MS-MS) analytical capability is essential for newer systemic pesticides, particularly the neonicotinoids (Bonmatin et al., 2005; Chauzat et al., 2006). Older systemic residues, like the toxic sulfur-oxidation metabolites of aldicarb, many modern fungicides and herbicides, and the polar degradates of newer fungicides and herbicides cannot be analyzed at ppb limits of detection without LC/MS-MS (Alder et al., 2006).

During the last decade, some European beekeepers have reported heavy losses of honey bee colonies located near crops treated with the neonicotinoid imidacloprid (Rortais et al., 2005). Although Bonmatin et al. (2005) and Chauzat et al. (2006) found low ppb levels of the imidacloprid in a high percentage of pollen samples collected from maize, sunflower and canola, when pesticide residues from all matrices were pooled together, analysis did not show a significant relationship between the presence of pesticide residues and the abundance of brood and adults, and no statistical relationship was found between colony mortality and pesticide residues (Chauzat et al., 2009). Another way to associate pesticides and honey bee mortality is to examine dead bees, but obtaining samples can be difficult if bees die away from the hive as it is necessary to use recently dead or dying bees (Johansen and Mayer, 1990).

3.2. Major incidences of pesticide residues in the beehive

This review will focus on pesticide residues studies done during the past 20 years. Smith and Wilcox (1990) report residues found in beehives in the USA prior to the period covered by this review. Over 150 different pesticides have been found in colony samples (Mullin et al., 2010). In recent years, the highest residues of pesticides in colonies are from varroacides that accumulate in the wax (Mullin et al., 2010). Varroacides found in beeswax, pollen and bee bread include amitraz, bromopropylate, coumaphos, flumethrin and tau-fluvalinate. Residue levels of these varroacides generally increase from honey to pollen to beeswax (Lodesani et al., 1992; Wallner, 1995; Bogdanov et al., 1998; Bogdanov, 2004; Tremolada et al., 2004; Martel et al., 2007; Frazier et al., 2008).

Varroacide residues in honey have been found to reach as high as 2.4 ppm acrinathrin, based on its 3-phenoxybenzaldehyde degradate (Bernal et al., 2000), 0.6 ppm of amitraz (Mullin et al., 2010), 2 ppm of coumaphos (Martel et al., 2007), and 0.75 ppm fluvalinate (Fernandez et al., 2002). Bee bread was also found to be contaminated with up to 1.1, 0.01, 5.8 and 2.7 ppm, respectively of amitraz, bromopropylate, coumaphos and fluvalinate (vanEngelsdorp et al., 2009b; Mullin et al., 2010). Levels in brood and adult bees can be higher than in the food, with 14 ppm amitraz, 5.9 ppm fluvalinate (vanEngelsdorp et al., 2009b; Mullin et al., 2010), 2.8 ppm of coumaphos (Ghini et al., 2004), and 2.2 ppm bromopropylate (Lodesani et al., 1992) being reported. Nevertheless, wax remains the ultimate sink for these varroacides reaching 46, 94 and 204 ppm, respectively, of amitraz, coumaphos and fluvalinate (vanEngelsdorp et al., 2009b; Mullin et al., 2010), 135 ppm of bromopropylate (Bogdanov et al., 1998), and 7.6 and 0.6 ppm, respectively, of the miticides chlorfenvinophos and acrinathrin (Jimenez et al., 2005).

Pesticide residues of agrochemicals acquired by foragers are equivalent or higher in pollen (stored and trapped at the hive entrance), adult bees and occasionally honey, than in wax. Major pollen detections include the insecticides aldicarb (1.3 ppm), azinphos methyl (0.6 ppm), chlorpyrifos (0.8 ppm), and imidacloprid (0.9 ppm); fungicides boscalid (1 ppm), captan (10 ppm), and myclobutanil (1 ppm), and herbicide pendimethalin (1.7 ppm; Tab. I). The carbamates carbofuran and carbaryl, and the organophosphate parathion methyl have been frequently found at up to 1.4 (Bailey et al., 2005), 94 (cited in Chauzat et al., 2006), and 26 ppm (Rhodes et al., 1979), respectively. High levels of pyrethroids including cyhalothrin and cypermethrin at 1.7 and 1.9 ppm, respectively, have been reported in mustard pollen along with up to 2.2 and 2.1 ppm, respectively, of the cyclodiene endosulfan and the new lipid-synthesis inhibitor insecticide spiromesifen (Choudhary and Sharma, 2008b).

Table I

Maximum pesticide incidence in apiary samples of wax, pollen, bee and honey.

Fungicides often account for most of the pesticide content of pollen. Unprecedented levels (99 ppm) of the widely-used fungicide chlorothalonil were found in honey bee-collected pollen (Tab. I, Mullin et al., 2010). Chorothalonil, a contact and slightly volatile fungicide, was found to be a marker for entombing behavior in honey bee colonies associated with poor health (vanEngelsdorp et al., 2009a). Entombing may be a defensive behavior of honey bees faced with large amounts of potentially toxic food stores. Kubik et al. (1999) noted high residues of the fungicides vinclozolin and iprodione up to 32 and 5.5 ppm, respectively, in beebread.

High residues in the honey bees themselves (Tab. I) are often associated with direct kill from the respective pesticide application, such as with 19.6 ppm of permethrin (LD50 of 1.1 ppm) and 3.1 ppm of fipronil (LD50 0.05 ppm) (Mullin et al., 2010). Anderson and Wojtas (1986) linked dead honey bees to high residues of the insecticides carbaryl (5.8 ppm), chlordane (0.7 ppm), diazinon (0.35 ppm), endosulfan (4.4 ppm), malathion (4.2 ppm), methomyl (3.4 ppm), methyl parathion (3.6 ppm), and fungicide captan (1.7 ppm). Walorczyk and Gnusowski (2009) found exceptional amounts of the organophosphates dimethoate (4.9 ppm), fenitrothion (1 ppm), and omethoate (1.2 ppm), and up to 1.2 ppm of the systemic fungicide tebuconazole in honey bees from other poisoning incidences (Tab. I). Similarly, elevated residues of the organophosphates bromophos methyl (1.7 ppm) and fenitrothion (10.3 ppm) were associated with high honey bee mortality (Ghini et al., 2004). In contrast to systemic fungicides, systemic neonicotinoid residues are generally absent from bee samples, although present in pollen and wax.

Notable honey residues in Europe include 0.65, 0.66 and 4.3 ppm, respectively, of carbofuran, DDT, and lindane (Blasco et al., 2003), and 0.6 ppm of methoxychlor (Fernandez-Muino et al., 1995). A recent broad sampling of US honey following reports of CCD (USDA-PDP, 2008) showed only a few detections of low ppb amounts of external pesticides like dicloran and dicofol, but also revealed more frequent low levels of coumaphos and fluvalinate up to 12 ppb. A standard treatment of synthetic piperonyl butoxide-synergized pyrethrum to kill managed and feral honey bees in a hive (Taylor et al., 2007) can leave high residues in both honey (up to 3, 0.6 ppm, respectively) and wax (470237 ppm).

Very high amounts of the fungicide chlorothalonil (54 ppm) and substantial levels of chlorpyrifos (0.9 ppm), aldicarb (0.7 ppm), deltamethrin (0.6 ppm), and iprodione (0.6 ppm) were found in comb wax (vanEngelsdorp et al., 2009b; Mullin et al., 2010). Elevated levels of the acetylcholinesterase-inhibitors azinphos methyl (0.8 ppm), fenitrothion (0.5 ppm, Chauzat and Faucon, 2007), carbaryl (0.8 ppm), parathion methyl (3.1 ppm, Russell et al., 1998), and malathion (6 ppm, Thrasyvoulou and Pappas, 1988) have been reported. Bogdanov et al., (2004) detected up to 60 ppm of p-dichlorobenzene and Jimenez et al., (2005) up to 0.6 ppm of the miticide tetradifon in beeswax.

3.3. A recent survey found a high diversity of pesticides in beehive samples

The recent phenomenon of CCD triggered a close look at the role of pesticides as a possible contributing factor to honey bee decline in general and CCD specifically. Mullin and Frasier used LC/MS-MS and GC/MS and a modified QuEChERS method to analyze for pesticide residues in honey bees and hive matrices in the USA and Canada to examine colonies exhibiting CCD symptoms (vanEngelsdorp et al., 2009b; Frazier et al., 2008). One hundred twenty-one different pesticides and metabolites were found within 887 wax, pollen, bee and associated hive samples (average of 6.2 detections per sample) from migratory and stationary beekeepers. These included 16 parent pyrethroids, 13 organophosphates, 4 carbamates, 4 neonicotinoids, 4 insect growth regulators, 3 chlorinated cyclodienes, 3 organochlorines, 1 formamidine, 8 miscellaneous miticides/insecticides, 2 synergists, 30 fungicides, and 17 herbicides. Over 40 of the pesticides detected are systemic (Tab. I). Only one of the wax samples, 3 pollen samples and 12 bee samples had no detectable pesticides.

Overall, pyrethroids and organophosphates dominated total wax and bee residues followed by fungicides, systemics, carbamates, and herbicides, whereas fungicides prevailed in pollen followed by organophosphates, systemics, pyrethroids, carbamates, and herbicides. By comparing these residue levels across the matrices, in-hive varroacides were more concentrated in wax than in pollen, whereas externally-derived pesticides were higher or equivalent in pollen compared to wax. This is consistent with chronic use and long-term accumulation of these lipophilic varroacides in the wax as a source to contaminate pollen.

All foundation, beeswax pressed into sheets and used as templates for comb construction, sampled from North America is uniformly contaminated with tau-fluvalinate, coumaphos and lower amounts of other pesticides and metabolites (Mullin et al., 2010). The broad contamination of European foundation, especially with varroacides, has been reviewed previously (Wallner, 1999; Bogdanov, 2004; Lodesani et al., 2008). The uniform presence of these acaricides in foundation is particularly disturbing since replacement of frames is the main avenue currently used to purge a colony of accumulated pesticide contaminants. Fluvalinate residues in beeswax were the best correlative with the French honey bee winter kill of 1999–2000 (Faucon et al., 2002), although disease factors were emphasized in the report.

High levels of the pyrethroid fluvalinate and the organophosphate coumaphos are co-occuring with lower but significant levels of 119 other insecticides, fungicides and herbicides in hive matrices. Fluvalinate and coumaphos, but not amitraz, are highly persistent in the hive with an estimated half-life in beeswax of 5 years (Bogdanov, 2004). Chronic exposure to high levels of these persistent neurotoxicants elicits both acute and sublethal reductions in honey bee fitness (Stoner et al., 1985; Lodesani and Costa, 2005). The direct association of any one of these varroacides with CCD remains unclear, although higher coumaphos levels may benefit the colony presumably via mite control (vanEngelsdorp et al., 2009b).

Externally-derived, highly toxic pyrethroids were the most frequent and dominant class of insecticides samples (Mullin et al., 2010). Contact pyrethroids, and systemic neonicotinoids and fungicides are often combined as pest control inputs, and many of the latter may synergize the already high toxicity of neonicotinoids and pyrethroids to honey bees (Pilling and Jepson, 1993; Iwasa et al., 2003). Pyrethroids frequently are associated with honey bee kills (Mineau et al., 2008), as has been the case with neonicotinoids (Halm et al., 2006), although the latter with less documentation of acute residues in bees. The effects of toxic chronic exposure to pyrethroids, organophosphates, neonicotinoids, fungicides and other pesticides can range from lethal and/or sub-lethal effects in the larvae and workers to reproductive effects on the queen (Thompson, 2003; Desneux et al., 2007). These chemicals may act alone or in concert, in ways currently unknown, to create a toxic environment for the honey bee.

4. CONCLUSIONS

The widespread planting of transgenic crops appears to be a net benefit for honey bees in the USA, since the pesticidal toxins produced by these plants do not appear to harm honey bees. Additionally, such crops do not require as many applications of traditional pesticides, most of which are known to be toxic to bees.

The systemic nature of the neonicotinoids and phenylpyrazoles present a trade-off from the standpoint of honey bee health. New methods of application help to minimize direct exposure of bees to these compounds during application. The downside is that honey bees may instead be exposed to these pesticides, or their metabolites, in pollen, nectar, and plant exudates over extended periods of time. Further research is needed to assess the true dangers posed by extended low dose exposure to these systemic pesticides.

Beekeepers searching for the primary source of pesticides contaminating bee hives need only to look in a mirror. Unfortunately, the regulatory system governing the veterinary use of pesticides in bee hives in the USA may be perversely contributing to the problem. Two of the handful of pesticides registered for legal use in the USA, coumaphos (CheckMite+®) and tau-fluvalinate (Apistan®), both of which seriously contaminate wax, have become largely useless against the primary pest of honey bees, the Varroa mite. Another effective varroacide used in Europe and Canada, oxalic acid, is not registered in the USA because it is low in cost, readily available, and potential registrants are deterred by the cost of the registration process. There are 3 registered in-hive pesticides that provide effective Varroa control in the USA, fenpyroximate (Hivistan®), formic acid (Miteaway II®) and thymol (ApiGuard® and Api-Life Var®). Other effective pesticides, including amitraz and oxalic acid, are used by some beekeepers in the absence of any regulatory approval. A change in the regulatory system needs to occur to make effective and safe veterinary pesticides available to beekeepers and to spur research into the effects of candidate compounds on honey bee health. Likewise, beekeepers need to realize that honey bee pests and parasites are community problems, as well as individual problems, and that pesticide labels are crafted to protect the sustainability of pesticides. The use of unregistered products is a serious threat to the beekeeping community and should not occur.

Honey bees are being exposed to high levels of in-hive varroacides and agrochemical pesticides. Chronic exposures to neurotoxic insecticides and their combinations with other pesticides, in particular fungicides, are known to elicit reductions in honey bee fitness, but direct association with CCD and declining honey bee health remains to be resolved.

Acknowledgments

We gratefully recognize the major input of Dennis vanEngelsdorp, Pennsylvania Department of Agriculture, and Jeffery S. Pettis, USDA-ARS, for providing samples for the CCD analyses reviewed here; Roger Simonds, USDA-AMS National Science Laboratory, Gastonia, NC for conducting the pesticide analyses; and of Jim Frazier, Sara Ashcraft (PSU), Lizette Peters and Alex Heiden (UNL) for technical support. We thank the National Honey Board, the Florida State Beekeepers, the Tampa Bay Beekeepers, Penn State College of Agriculture Sciences, Preservation Apis mellifera (PAm), The Foundation for the Preservation of Honey Bees, the USDA Critical Issues Program, the USDA CAPS Program, and the EPA for the financial support that made some of the work included in this review possible.

References

  • Adey M., Walker P., Walker P.T. (1986) Pest Control Safe for Bees, IBRA, London, 224 p. (In the text)
  • Ailouane Y., Lambin M., Armengaud C., El Hassani A., Gary V. (2009) Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior, Environ. Toxicol. Chem. 28, 113–122. [CrossRef] [PubMed] (In the text)
  • Alaux C., Brunet J., Dussaubat C., Mondet F., Tchamitchan S., Cousin M., Brillard J., Baldy A., Belzunces L.P., Le Conte Y. (2010) Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera), Environ. Microbiol., in press, DOI: 10.1111/j.1462-2920.2009.02123.x. (In the text)
  • Albero B., Sanchez-Brunete C., Tadeo J.L. (2004) Analysis of pesticides in honey by solid-phase extraction and gas chromatography – Mass spectrometry, J. Agric. Food Chem. 52, 5828–5835. [CrossRef] [PubMed] (In the text)
  • Alder L., Greulich K., Kempe G., Vieth B. (2006) Residue analysis of 500 high priority pesticides: Better by GC-MS or LC-MS/MS? Mass Spectrometry Reviews 25, 838–865. [CrossRef] [PubMed] (In the text)
  • Aliano N.P., Ellis M.D. (2008) Bee-to bee contact drives oxalic acid distribution in honey bee colonies, Apidologie 39, 481–487. [CrossRef] [EDP Sciences] (In the text)
  • Anderson J.F., Wojtas M.A. (1986) Honey bees (Hymenoptera, Apidae) contaminated with pesticides and polychlorinated biphenyls, J. Econ. Entomol. 79, 1200–1205. (In the text)
  • Arpaia S. (1996) Ecological impact of Bt-transgenic plants: 1. Asessing possible effects of CrtIIIB toxin on honey bee (Apis mellifera) colonies, J. Genet. Breed. 50, 315–319. (In the text)
  • Atkins E.L. (1992) Injury to honey bees by poisoning. In: J.M. Graham (ed.), The Hive and the Honey Bee, Rev. Dadant and Sons, Hamilton, IL. 1324 p. (In the text)
  • Aupinel P., Fortini D., Dufour H., Taséi J.N., Michaud B. (2005) Improvement of artificial feeding in a standard in vitro method for rearing Apis mellifera larvae, Bull. Insectol. 58, 107–111. (In the text)
  • Babendreier D., Joller D., Romeis J., Bigler F., Widmer F. (2007) Bacterial community structures in honeybee intestines and their response to two insecticidal proteins, FEMS Microbiol. Ecol. 59, 600–610. [CrossRef] [PubMed] (In the text)
  • Babendreier D., Kalberer N.M., Romeis J., Fluri P., Mulligan E., Bigler F. (2005) Influence of Bt-transgenic pollen, Bt-toxin and protease inhibitor (SBTI) ingestion on development of the hypopharyngeal glands in honeybees, Apidologie 36, 585–594. [CrossRef] [EDP Sciences] (In the text)
  • Babendreier D., Romeis J., Bigler J., Fluri P. (2006) Neue erkenntnisse, zu möglichen auswirkungen von transgenem Bt-mais auf bienen, Forsch. Agroscope Liebefeld-Posieux ALP, Schweiz.
  • Bailey J., Scott Dupree C., Harris R., Tolman J., Harris B. (2005) Contact and oral toxicity to honey bees in Ontario, Canada, Apidologie 36, 623–633. [CrossRef] [EDP Sciences] (In the text)
  • Balayiannis G., Balayiannis P. (2008) Bee honey as an environmental bioindicator of pesticides’ occurrence in six agricultural areas of Greece, Arch. Environ. Contam. Toxicol. 55, 462–470. [CrossRef] [PubMed] (In the text)
  • Barron A.B., Maleszka R., Vander Meer R.K., Robinson G.E. (2007) Octopamine modulates honey bee dance behavior, Proc. Natl. Acad. Sci. USA 104, 1703–1707. [CrossRef] (In the text)
  • Bernal J.L., Jimenez J.J., del Nozal M.J., Higes M., Llorente J. (2000) Gas chromatographic determination of acrinathrine and 3-phenoxybenzaldehyde residues in honey, J. Chromatogr. A 882, 239–243. [CrossRef] [PubMed] (In the text)
  • Berry J. (2009) Pesticides, bees and wax: an unhealthy, untidy mix, Bee Culture 137, 33–35. (In the text)
  • Blasco C., Fernandez M., Pena A., Lino C., Silveira M.I., Font G., Pico Y. (2003) Assessment of pesticide residues in honey samples from Portugal and Spain, J. Agric. Food Chem. 51, 8132–8138. [CrossRef] [PubMed] (In the text)
  • Blasco C., Font G., Pico Y. (2008) Solid-phase microextraction-liquid chromatography-mass spectrometry applied to the analysis of insecticides in honey, Food Additives and Contaminants 25, 59–69. [CrossRef] [PubMed] (In the text)
  • Boecking O., Genersch E. (2008) Varroosis - the ongoing crisis in bee keeping, Journal Fur Verbraucherschutz Und Lebensmittelsicherheit-Journal of Consumer Protection and Food Safety 3, 221–228. [CrossRef] (In the text)
  • Bogdanov S. (2004) Beeswax: quality issues today, Bee World 85, 46–50. (In the text)
  • Bogdanov S. (2006) Contaminants of bee products, Apidologie 37, 1–18. [CrossRef] [EDP Sciences] (In the text)
  • Bogdanov S., Kilchenmann V., Imdorf A. (1997) Acaricide residues in beeswax and honey, Apiacta 32, 72–80. (In the text)
  • Bogdanov S., Kilchenmann V., Imdorf A. (1998) Acaricide residues in some bee products, J. Apic. Res. 37, 57–67. (In the text)
  • Bogdanov S., Kilchenmann V., Seiler K., Pfefferli H., Frey T., Roux B., Wenk P., Noser J. (2004) Residues of para-dichlorobenzene in honey and beeswax, J. Apic. Res. 43, 14–16. (In the text)
  • Bonmatin J.M., Marchand P.A., Charvet R., Moineau I., Bengsch E.R., Colin M.E. (2005) Quantification of imidacloprid uptake in maize crops, J. Agric. Food Chem. 53, 5336–5341. [CrossRef] [PubMed] (In the text)
  • Burley L., Fell R., Saacke R. (2008) Survival of honey bee (Hymenoptera: Apidae) spermatozoa incubated at room temperature from drones exposed to miticides, J. Econ. Entomol. 101, 1081–1087. [CrossRef] [PubMed] (In the text)
  • Canadian Honey Council (2005) Conditions of use for oxalic acid dihydrate, HiveLights 18 (Special Suppl.).
  • Cao L.C., Honeyman T.W., Cooney R., Kennington L., Scheid C.R., Jonassen J.A. (2004) Mitochondrial dysfunction is a primary event in renal cell oxalate toxicity, Kidney Int. 66, 1890–1900. [CrossRef] [PubMed] (In the text)
  • CDPR (California department of pesticide regulation) (2006) Top 100 pesticides by acres treated in 2005, CADPR, Sacramento, CA, www.cdpr.gov (accessed 24 August 2009). (In the text)
  • CDPR (California department of pesticide regulation) (2008) Authorization for Section 18 use of Hivastan, http://www.cdpr.ca.gov/docs/registration/sec18/0810.htm (accessed 28 August 2009).
  • Charrière J.D., Imdorf A. (2002) Oxalic acid treatment by trickling against Varroa destructor: recommendations for use in central Europe and under temperate climate conditions, Bee World 82, 51–60. (In the text)
  • Chauzat M.P., Faucon J.P. (2007) Pesticide residues in beeswax samples collected from honey bee colonies (Apis mellifera l.) in France, Pest Manage. Sci. 63, 1100–1106. [CrossRef] [PubMed] (In the text)
  • Chauzat M.P., Carpentier P., Martel A.C., Bougeard S., Cougoule N., Porta P., Lachaize J., Madec F., Aubert M., Faucon J.P. (2009) Influence of pesticide residues on honey bee (Hymenoptera: Apidae) colony health in France, Environ. Entomol. 38, 514–523. [CrossRef] [PubMed] (In the text)
  • Chauzat M.P., Faucon J.P., Martel A.C., Lachaize J., Cougoule N., Aubert M. (2006) A survey of pesticide residues in pollen loads collected by honey bees in France, J. Econ. Entomol. 99, 253–262. [CrossRef] [PubMed] (In the text)
  • Choudhary A., Sharma D.C. (2008a) Pesticide residues in honey samples from Himachal Pradesh (India), Bull. Environ. Contam.Toxicol. 80, 417–422. [CrossRef] (In the text)
  • Choudhary A., Sharma D.C. (2008b) Dynamics of pesticide residues in nectar and pollen of mustard (Brassica juncea (L.) Czern.) grown in Himachal Pradesh (India), Environ. Monit. Assess. 144, 143–150. [CrossRef] [PubMed] (In the text)
  • Claudianos C., Ranson H., Johnson R.M., Biswas S., Schuler M.A., Berenbaum M.R., Feyereisen R., Oakeshott J.G. (2006) A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee, Insect Mol. Biol. 15, 615–636. [CrossRef] [PubMed] (In the text)
  • Colin M.E., Bonmatin J.M., Moineau I., Gaimon C., Brun S., Vermandere J.P. (2004) A method to quantify and analyze the foraging activity of honey bees: relevance to the sublethal effects induced by systemic insecticides, Arch. Environ. Contam. Toxicol. 47, 387–395. [PubMed] (In the text)
  • Collins A.M., Pettis J.S., Wilbanks R., Feldlaufer M.F. (2004) Performance of honey bee (Apis mellifera) queens reared in beeswax cells impregnated with coumaphos, J. Apic. Res. 43, 128–134. (In the text)
  • Crane E., Walker P. (1983) The Impact of Pest Management on Bees and Pollination, IBRA, London, 129 p. (In the text)
  • Croft B.A. (1990) Arthropod Biological Control Agents and Pesticides, New York, Wiley, 723 p. (In the text)
  • Cutler G.C., Scott-Dupree C.D. (2007) Exposure to clothianidin seed-treated canola has no long-term impact on honey bees, J. Econ. Entomol. 100, 765–772. [CrossRef] [PubMed] (In the text)
  • Davies T.G.E., Field L.M., Usherwood P.N.R., Williamson M.S. (2007) DDT, pyrethrins, pyrethroids and insect sodium channels, IUBMB Life 59, 151–162. [CrossRef] [PubMed] (In the text)
  • Decourtye A., Devillers J., Cluzeau S., Charreton M., Pham-Delegue M.H. (2004) Effects of imidacloprid and deltamethrin on associative learning in honeybees under semi-field and laboratory conditions, Ecotoxicol. Environ. Safety 57, 410–419. [CrossRef] [PubMed] (In the text)
  • Decourtye A., Devillers J., Genecque E., Le Menach K., Budzinski H. (2005) Comparative sublethal toxicity of nine pesticides on olfactory learning performances of honey bes, Apis mellifera, Arch. Environ. Toxicol. Chem. 28, 113–122. (In the text)
  • Decourtye A., Mader E., Desneux N. (2010) Landscape enhancement of floral resources for honey bees in agro-ecosystems, Apidologie 41, 264–277. [CrossRef] [EDP Sciences] (In the text)
  • Decourtye A., Pham-Delegue M.H. (2002) The proboscis extension response: assessing the sublethal effects of pesticides on the honey bee. In Honey Bees: Estimaing the Environmental Impact of Chemicals, in: Devillers J., Pham-Delegue M.H. (Eds.), Taylor and Francis, London, 336 p. (In the text)
  • Desneux N., Decourtye A., Delpuech J.M. (2007) The sublethal effects of pesticides on beneficial arthropods, Annu. Rev. Entomol. 52, 81–106. [CrossRef] [PubMed] (In the text)
  • Devillers J., Decourtye A., Budzinski H., Pham-Delègue M.H., Cluzeau S., Maurin C. (2003) Comparative toxicity and hazards of pesticides to Apis and non-Apis bees, A chemometrical study, SAR and QSAR in Environ. Res. 14, 389–403. [CrossRef] (In the text)
  • Duan J.J., Marvier M. Huesing J., Dively G., Huang Z.Y. (2008) A meta-analysis of effects of Bt crops on honey bees (Hymenoptera: Apidae), PLoS ONE 3, e1415. [CrossRef] [PubMed] (In the text)
  • Eischen F.A., Graham R.H. (2008) Feeding overwintering honey bee colonies infected with Nosema ceranae, In Proceedings of the American Bee Research Conference, Am. Bee J. 148, 555. (In the text)
  • Ellis M.D., Baxendale F.P. (1997) Toxicity of seven monoterpenoids to tracheal mites (Acari: Tarsonemidae) and their honey bee (Hymenoptera: Apidae) hosts when applied as fumigants, J. Econ. Entomol. 90, 1087–1091. (In the text)
  • Ellis M.D., Baxendale F.P., Keith D.L. (1998) Protecting bees when using insecticides, NebGuide G98-1347, University of Nebraska Cooperative Extension. (In the text)
  • Ellis M.D., Nelson R., Simonds C. (1988) . A comparison of the fluvalinate and ether roll methods of sampling for Varroa mites in honey bee colonies, Am. Bee J. 128, 262–263. (In the text)
  • Elzen P.J., Westervelt D. (2002) Detection of coumaphos resistance in Varroa destructor in Florida, Am. Bee J. 142, 291–292. (In the text)
  • Elzen P.J., Baxter J.R., Spivak M., Wilson W.T. (2000) Control of Varroa jacobsoni Oud. resistant to fluvalinate and amitraz using coumaphos, Apidologie 31, 437–441. [CrossRef] [EDP Sciences] (In the text)
  • Elzen P.J., Eischen F.A., Baxter J.B., Elzen G.W., Wilson W.T. (1999). Detection of resistance in US Varroa jacobsoni Oud. (Mesostigmata: Varroidae) to the acaricide fluvalinate, Apidologie 30, 13–17. [CrossRef] [EDP Sciences] (In the text)
  • Enan E. (2001) Insecticidal activity of essential oils: octopaminergic sites of action, Comparative Biochemistry and Physiology Part C, Toxicol. Pharmacol. 130, 325–337. (In the text)
  • EPA (environmental protection agency) (2009a) Federal insecticide fungicide and rodenticide act, http://www.epa.gov/pesticides/regulating/laws.htm (accessed 24 August 2009). (In the text)
  • EPA (environmental protection agency) (2009b) Pesticide registration review, http://www.epa.gov/oppsrrd1/registration_review/highlights.htm (accessed 24 August 2009). (In the text)
  • Estep C.B., Menon G.N., Williams H.E., Cole A.C. (1977) Chlorinated hydrocarbon insecticide residues in Tennessee honey and beeswax, Bull. Environ. Contam. Toxicol. 17, 168–174. [CrossRef] [PubMed] (In the text)
  • Evans P.D., Gee J.D. (1980) Action of formamidine pesticides on octopamine receptors, Nature 287, 60–62. [CrossRef] [PubMed] (In the text)
  • Faucon J.P., Mathieu L., Ribiere M., Martel A.C., Drajnudel P., Zeggane S., Aurieres C., Aubert M.F.A. (2002) Honey bee winter mortality in France in 1999 and 2000, Bee World 83, 14–23. (In the text)
  • Federal Register (2000) Coumaphos: pesticide tolerance for emergency action, U.S. Environmental Protection Agency, Washington, DC.
  • Fernandez-Muino M.A., Sancho M.T., Muniategui S., Huidobro J.F., Simallozano J. (1995) Nonacaricide pesticide residues in honey – Analytical methods and levels found, J. Food Protect. 58, 1271–1274. (In the text)
  • Fernandez M., Pico Y., Manes J. (2002) Analytical methods for pesticide residue determination in bee products, J. Food Protect. 65, 1502–1511. (In the text)
  • Floris I., Satta A., Cabras P., Garau V.L., Angioni A. (2004) Comparison between two thymol formulations in the control of Varroa destructor: effectiveness, persistence, and residues, J. Econ. Entomol. 97, 187–191. [CrossRef] [PubMed] (In the text)
  • Frazier M., Mullin C., Frazier J., Ashcraft S. (2008) What have pesticides got to do with it? Am. Bee J. 148, 521–523. (In the text)
  • Fries I. (1991) Treatment of sealed honey bee brood with formic acid for control of Varroa jacobsoni, Am. Bee J. 131, 313–314. (In the text)
  • Ghini S., Fernandez M., Pico Y., Marin R., Fini F., Manes J., Girotti S. (2004) Occurrence and distribution of pesticides in the province of Bologna, Italy, using honeybees as bioindicators, Arch. Environ. Contam. Toxicol. 47, 479–488. [CrossRef] [PubMed] (In the text)
  • Girolami V., Greatti M., Di Bernardo A., Tapparo A., Giorio C., Squartini A., Mazzon L., Mazaro M., Mori N. (2009) Translocation of neonicotinoid insecticides from coated seeds to seedling guttation drops: a novel way of intoxication for bees, J. Econ. Entomol. 102, 1808–1815. [CrossRef] [PubMed] (In the text)
  • Gregorc A., Bowen I. (2000) Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, amitraz and oxytetracycline, Cell Biol. Int. 24, 319–324. [CrossRef] [PubMed] (In the text)
  • Gregorc A., Smodisskerl M.I. (2007) Toxicological and immunohistochemical testing of honeybees after oxalic acid and rotenone treatments, Apidologie 38, 296–305. [CrossRef] [EDP Sciences] (In the text)
  • Gunasekara A., Truong T., Goh K., Spurlock F., Tjeerdema R. (2007) Environmental fate and toxicology of fipronil, J. Pestic. Sci. 32, 189–199. [CrossRef] (In the text)
  • Haarmann T., Spivak M., Weaver D., Weaver B., Glenn T. (2002) Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations, J. Econ. Entomol. 95, 28–35. [CrossRef] [PubMed] (In the text)
  • Halm M.P., Rortais A., Arnold G., Taséi J.N., Rault S. (2006) New risk assessment approach for systemic insecticides: The case of honey bees and imidacloprid (Gaucho), Environ. Sci. Technol. 40, 2448–2454. [CrossRef] [PubMed] (In the text)
  • Higes M., Meana A., Suarez M., Llorente J. (1999) Negative long-term effects on bee colonies treated with oxalic acid against Varroa jacobsoni Oud., Apidologie 30, 289–292. [CrossRef] [EDP Sciences] (In the text)
  • ISB (inf. syst. biotechnol.) (2007) Field test release applications in the US (Database provided by APHIS biotechnology regulatory services, http://www.isb.vt.edu/CFDOCS/fieldtests1.cfm (accessed 24 August 2009). (In the text)
  • Isman M.B. (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world, Annu. Rev. Entomol. 51, 45–66. [CrossRef] [PubMed] (In the text)
  • Iwasa T., Motoyama N., Ambrose J.T., Roe R.M. (2003) Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera, Crop Prot. 23, 371–378. [CrossRef] (In the text)
  • Jeschke P., Nauen R. (2008) Neonicotinoids - from zero to hero in insecticide chemistry, Pest Manage. Sci. 64,1084–1098. [CrossRef] (In the text)
  • Jimenez J.J., Bernal J.L., del Nozal M.J., Martin M.T. (2005) Residues of organic contaminants in beeswax. Eur. J. Lipid Sci. Technol. 107, 896–902. [CrossRef] (In the text)
  • Johansen C.A. (1977) Pesticides and pollinators, Annu. Rev. Entomol. 22, 177–192. [CrossRef] (In the text)
  • Johansen C.A., Mayer D.F. (1990) Pollinator Protection: a Bee and Pesticide Handbook, Wicwas Press, Cheshire, CT, 212 p. (In the text)
  • Johnson R.M., Pollock H.S., Berenbaum M.R. (2009) Synergistic interactions between in-hive miticides in Apis mellifera, J. Econ. Entomol. 102, 474–479. [CrossRef] [PubMed] (In the text)
  • Johnson R.M., Wen Z., Schuler M.A., Berenbaum M.R. (2006) Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases, J. Econ. Entomol. 99, 1046–1050. [CrossRef] [PubMed] (In the text)
  • Keyhani J., Keyhani E. (1980) EPR study of the effect of formate on cytochrome oxidase, Biochem. Biophys. Res. Comm. 92, 327–333. [CrossRef] (In the text)
  • Kim Y.J., Lee S.H., Lee S.W, Ahn Y.J. (2004) Fenpyroximate resistance in Tetranychus urticae (Acari: Tetranychidae): cross-resistance and biochemical resistance mechanisms, Pest Manage. Sci. 60, 1001–1006. [CrossRef] (In the text)
  • Kubik M., Nowacki J., Pidek A., Warakomska Z., Michalczuk L., Goszczynski W. (1999) Pesticide residues in bee products collected from cherry trees protected during blooming period with contact and systemic fungicides, Apidologie 30, 521–532. [CrossRef] [EDP Sciences] (In the text)
  • Kubik M., Nowacki J., Pidek A., Warakomska Z., Michalczuk L., Goszczynski W., Dwuznik B. (2000) Residues of captan (contact) and difenoconazole (systemic) fungicides in bee products from an apple orchard, Apidologie 31, 531–541. [CrossRef] [EDP Sciences] (In the text)
  • Laurent F.M., Rathahao E. (2003) Distribution of (14)C imidacloprid in sunflowers (Helianthus annuus L.) following seed treatment, J. Agric. Food Chem. 51, 8005–8010. [CrossRef] [PubMed] (In the text)
  • Lemaux P.G. (2008) Genetically engineered plants and foods: a scientist’s analysis of the issues, Annu. Rev. Plant Biol. 59, 771–812. [CrossRef] [PubMed] (In the text)
  • Li A.Y., Davey R.B., Miller R.J., George J.E. (2004) Detection and characterization of amitraz resistance in the southern cattle tick, Boophilus microplus (Acari: Ixodidae), J. Med. Entomol. 41, 193–200. [CrossRef] [PubMed] (In the text)
  • Li A.Y., Pruett J.H., Davey R.B., George J.E. (2005) Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae), Pestic. Biochem. Physiol. 81, 145–153. [CrossRef] (In the text)
  • Lodesani M., Costa C. (2005) Limits of chemotherapy in beekeeping: development of resistance and the problem of residues, Bee World 86, 102–109. (In the text)
  • Lodesani M., Colombo M., Spreafico M. (1995) Ineffectiveness of Apistan(R) treatment against the mite Varroa jacobsoni Oud. in several districts of Lombardy (Italy), Apidologie 26, 67–72. [CrossRef] [EDP Sciences] (In the text)
  • Lodesani M., Costa C., Serra G., Colombo R., Sabatini A.G. (2008) Acaricide residues in beeswax after conversion to organic beekeeping method, Apidologie 39, 324–333. [CrossRef] [EDP Sciences] (In the text)
  • Lodesani M., Pellacani A., Bergomi S., Carpana E., Rabitti T., Lasagni P. (1992) Residue determination for some products used against Varroa infestation in bees, Apidologie 23, 257–272. [CrossRef] [EDP Sciences] (In the text)
  • Macedo P.A., Ellis M.D., Siegfried B.D. (2002) Detection and quantification of fluvalinate resistance in Varroa mites in Nebraska, Am. Bee J. 147, 523–526. (In the text)
  • Marchetti S., Barbattini R. (1984) Comparative effectiveness of treatments used to control Varroa jacobsoni Oud., Apidologie 15, 363–378. [CrossRef] [EDP Sciences] (In the text)
  • Martel A.C., Zeggane S., Aurieres C., Drajnudel P., Faucon J.P., Aubert M. (2007) Acaricide residues in honey and wax after treatment of honey bee colonies with Apivar (R) or Asuntol (R) 50, Apidologie 38, 534–544. [CrossRef] [EDP Sciences] (In the text)
  • Meimaridou E., Jacobson J., Seddon A.M., Noronha-Dutra A.A, Robertson W.G., Hothersal J.S. (2005) Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes, Free Radical Biology and Medicine 38, 1553–1564. [CrossRef] (In the text)
  • Mineau P., Harding K.M., Whiteside M., Fletcher M.R., Garthwaite D., Knopper L.D. (2008) Using reports of bee mortality in the field to calibrate laboratory-derived pesticide risk indices, Environ. Entomol. 37, 546–554. [CrossRef] [PubMed] (In the text)
  • Motoba K., Nishizawa H., Suzuki T., Hamaguchi H., Uchida M., Funayama S. (2000) Species-specific detoxification metabolism of fenpyroximate, a potent acaricide, Pestic. Biochem. Physiol. 67, 73–84. [CrossRef] (In the text)
  • Motoba K., Suzuki T., Uchida M. (1992) Effect of a new acaricide, fenpyroximate, on energy metabolism and mitochondrial morphology in adult female Tetranychus urticae (two-spotted spider mite), Pestic. Biochem. Physiol, 43, 37–44. [CrossRef] (In the text)
  • Mullin C.A., Frazier M., Frazier J.L., Ashcraft S., Simonds R., vanEngelsdorp D., Pettis J.S. (2010) High levels of miticides and agrochemicals in North American apiaries: Implications for honey bee health, PLoS One, accessed 19 March 2010, http://dx.plos.org/10.1371/journal.pone.0009754. (In the text)
  • NAS (national academy of sciences) (2007) Status of pollinators in North America, National Academy Press, Washington, DC. (In the text)
  • Nguyen B.K., Saegerman C., Pirard C., Mignon J., Widart J., Tuirionet B., Verheggen F.J., Berkvens D., De Pauw E., Haubruge E. (2009) Does imidacloprid seed-treated maize have an impact on honey bee mortality? J. Econ. Entomol. 102, 616–623. [CrossRef] [PubMed] (In the text)
  • NOD Apiary Products, Directions for use, www.miteaway.com/MAII_flyer.pdf (accessed 20 August 2009). (In the text)
  • OEPP/EPPO (1992) Guideline on test methods for evaluating the side effects of plant protection products on honeybees, Bull. OEPP/EPPO 22, 201–215. (In the text)
  • PAN (pesticide action network) Accessed 12 August 2009, http://www.pesticideinfo.org. (In the text)
  • Pettis J.S., Collins A.M., Wilbanks R., Feldlaufer M.F. (2004) Effects of coumaphos on queen rearing in the honey bee, Apis mellifera, Apidologie 35, 605–610. [CrossRef] [EDP Sciences] (In the text)
  • Pilling E.D., Jepson P.C. (1993) Synergism between EBI fungicides and A pyrethroid insecticide in the honeybee (Apis mellifera), Pestic. Sci. 39, 293–297. [CrossRef] (In the text)
  • Pilling E.D., Bromley-Challenor K.A.C., Walker C.H., Jepson P.C. (1995) Mechanism of synergism between the pyrethroid insecticide λ-cyhalothrin and the imidazole fungicide orochloraz, in the honeybee (Apis mellifera L.), Pestic. Biochem. Physiol. 51, 1–11. [CrossRef] (In the text)
  • Plapp F.W. Jr. (1979) Synergism of pyrethroid insecticides by formamidines against Heliothis pests of cotton, J. Econ. Entomol. 72, 667–670. (In the text)
  • PMRA – Canada’s pest management regulatory agency (2009) PMRA approves emergency use of Apivar in Canada, http://cba.stonehavenlife.com/2009/07/apivarvarroaemergencyuseincanada/ (accessed 10 November 2009). (In the text)
  • Priestley C.M., Williamson E.M., Wafford K.A., Sattelle D.B. (2003) Thymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABA(A) receptors and a homo-oligomeric GABA receptor from Drosophila melanogaster, British J. Pharmacol. 140, 1363–1372. [CrossRef] (In the text)
  • Quarles W. (1996) EPA exempts least-toxic pesticides, IPM Practitioner 18, 16–17. (In the text)
  • Quarles W. (2008) Pesticides and honey bee colony collapse disorder, IPM Practitioner 30, 1–10. (In the text)
  • Rademacher E., Harz M. (2006) Oxalic acid for the control of varroosis in honey bee colonies – a review, Apidologie 37, 98–120. [CrossRef] [EDP Sciences] (In the text)
  • Ramirez-Romero R., Chaufaux J., Pham-Delegue M.H. (2005) Effects of Cru1Ab protoxin, deltamethrin and imidacloprid on the foraging activity and the learning performances of the honeybee, Apis mellifera, a comparative approach, Apidologie 36, 601–611. [CrossRef] [EDP Sciences] (In the text)
  • Rhodes H.A., Wilson W.T., Sonnet P.E., Stoner A. (1979) Exposure of Apis mellifera (Hymenoptera, Apidae) to bee-collected pollen containing residues of microencapsulated methyl parathion, Environ. Entomol. 8, 944–948. (In the text)
  • Rinderer T.E., De Guzman L.I., Lancaster V.A., Delatte G.T., Stelzer J.A. (1999) Varroa in the mating yard: I. The effects of Varroa jacobsoni and Apistan (R) on drone honey bees, Am.. Bee J. 139, 134–139. (In the text)
  • Rissato S.R., Galhiane M.S., de Almeida M.V., Gerenutti M., Apon B.M. (2007) Multiresidue determination of pesticides in honey samples by gas chromatography-mass spectrometry and application in environmental contamination, Food Chem. 101, 1719–1726. [CrossRef] (In the text)
  • Rissato S.R., Galhiane M.S., Knoll F.R.N., Apon B.M. (2004) Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection, J. Chromatogr. A 1048, 153–159. [PubMed] (In the text)
  • Robertson J.L., Russell R.M., Preisler H.K., Savin N.E. (2007) Bioassays with Arthropods, CRC Press, Boca Raton. (In the text)
  • Rortais A., Arnold G., Halm M.P., Touffet-Briens F. (2005) Modes of honeybees exposure to systemic insecticides: estimated amounts of contaminated pollen and nectar consumed by different categories of bees, Apidologie 36, 71–83. [CrossRef] [EDP Sciences] (In the text)
  • Russell D., Meyer R., Bukowski J. (1998) Potential impact of microencapsulated pesticides on New Jersey apiaries, Am.. Bee J. 138, 207–210. (In the text)
  • Sammataro D., Untalan P., Guerrero F., Finley J. (2005) The resistance of Varroa mites (Acari: Varroidae) to acaricides and the presence of esterase, Int. J. Acarol. 31, 67–74. [CrossRef] (In the text)
  • Schmidt L.S., Schmidt J.O., Rao H., Wang W., Ligen X. (1995) Feeding preferences and survival of young worker honey bees (Hymenoptera:Apidae) fed rape, sesame and sunflower pollen, J. Econ. Entomol. 88, 1591–1595. (In the text)
  • Schulz D.J., Robinson G.E. (2001) Octopamine influences division of labor in honey bee colonies, J. Compar. Physiol. A 187, 53–61. [CrossRef] (In the text)
  • Sherer T.B., Richardson J.R., Testa C.M., Seo B.B., Panov A.V., Yagi T., Matsuno-Magi A., Miller G.W., Greenamyre J.T. (2007) Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease, J. Neurochem. 100, 1469–1479. [PubMed] (In the text)
  • Smith R.K., Wilcox M.M. (1990) Chemical residues in bees, honey and beeswax, Am. Bee J. 130, 188–192.
  • Soberon M., Gill S., Bravo A. (2009) Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells? Cell. Mol. Life Sci. 66, 1337–1349. [CrossRef] [PubMed] (In the text)
  • Song C., Scharf M.E. (2008) Formic acid: A neurologically active, hydrolyzed metabolite of insecticidal formate esters, Pestic. Biochem. Physiol. 92, 77–82. [CrossRef] (In the text)
  • Stoner A., Wilson W.T., Harvey J. (1985) Honey bee exposure to beeswax foundation impregnated with fenvalerate or carbaryl, Am. Bee J. 125, 513–516. (In the text)
  • Suchail S., Guez D., Belzunces L.P. (2000) Characteristics of imidacloprid toxicity in two Apis mellifera subspecies, Environ. Toxicol. Chem. 19, 1901–1905. (In the text)
  • Suchail S., Guez D., Belzunces L.P. (2001) Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera, Environ. Toxicol. Chem. 20, 2482–2486. [PubMed] (In the text)
  • Sylvester H.A., Watts R.P., De Guzman L.I., Stelzer J.A., Rinderer T.E. (1999) Varroa in the mating yard: II. The effects of Varroa and fluvalinate on drone mating competitiveness, Am. Bee J. 139, 225–227. (In the text)
  • Taséi J.N. (2003) Impact of agrochemicals on non-Apis bees, In Devillers J. and Pham-Delegue M.H., eds., Honey bees: Estimating the environmental impact of chemicals, Taylor and Francis, London, UK, pp. 101–131. (In the text)
  • Taylor M.A., Goodwin R.M., McBrydie H.M., Cox H.M. (2007) Destroying managed and feral honey bee (Apis mellifera) colonies to eradicate honey bee pests, N. Z. J. Crop Hort. Sci. 35, 313–323. [CrossRef] (In the text)
  • Thompson H.M. (2003) Behavioral effects of pesticides in bees: their potential for use in risk assessment, Exotoxicol. 12, 317–330. [CrossRef] (In the text)
  • Thrasyvoulou A.T., Pappas N. (1988) Contamination of honey and wax with malathion and coumaphos used against the Varroa mite, J. Apic. Res. 27, 55–61. (In the text)
  • Trapp S., Pussemier L. (1991) Model calculations and measurements of uptake and translocation of carbamates by bean plants, Chemosphere 22, 327–339. [CrossRef] (In the text)
  • Tremolada P., Bernardinelli I., Colombo M., Spreafico M., Vighi M. (2004) Coumaphos distribution in the hive ecosystem: Case study for modeling applications, Ecotoxicology 13, 589–601. [CrossRef] [PubMed] (In the text)
  • Underwood R., Currie R. (2003) The effects of temperature and dose of formic acid on treatment efficacy against Varroa destructor (Acari: Varroidae), a parasite of Apis mellifera (Hymenoptera: Apidae), Exp. Appl. Acarol. 29, 303–313. [CrossRef] [PubMed] (In the text)
  • USDA-AMS (2009) Pesticide data program, annual summary, calendar year 2008, http://www.ams.usda.gov/pdp (accessed 20 Jan. 2010). (In the text)
  • USDA-Biotech Crop Data (2009) Adoption of genetically engineered crops in the U.S. http://www.ers.usda.gov/Data/BiotechCrops/#200971 (accessed 20 Aug. 2009). (In the text)
  • USDA-PDP (Pesticide Data Program) (2008) Pesticide testing history 1991–2008, http://www.ams.usda.gov/AMSv1.0/pdp (accessed 20 Aug. 2009). (In the text)
  • vanEngelsdorp D., Evans J.D., Donovall L., Mullin C., Frazier M., Frazier J., Tarpy D.R., Hayes J., Pettis J.S. (2009a) ``Entombed Pollen'': A new condition in honey bee colonies associated with increased risk of colony mortality, J. Invertebr. Pathol. 101, 147–149. [CrossRef] [PubMed] (In the text)
  • vanEngelsdorp D., Evans J.D., Saegerman C., Mullin C., Haubruge E., Nguyen B.K., Frazier M., Frazier J., Cox-Foster D., Chen Y., Underwood R., Tarpy D.R., Pettis J.S. (2009b) Colony Collapse Disorder: A descriptive study, PLoS ONE 4, 1–17. [CrossRef] [PubMed] (In the text)
  • Varrox Vaporizer (2007) The Varrox Vaporizer, HiveLights 20, 16. (In the text)
  • Vita Europe Ltd., ApiGuard, http://www.vitaeurope.com/ (accessed 15 August 2009). (In the text)
  • Wallner K. (1995) The use of varroacides and their influence on the qualify of bee products, Am. Bee J. 135, 817–821. (In the text)
  • Wallner K. (1999) Varroacides and their residues in bee products, Apidologie 30, 235–238. [CrossRef] [EDP Sciences] (In the text)
  • Walorczyk S., Gnusowski B. (2009) Development and validation of a multi-residue method for the determination of pesticides in honeybees using acetonitrile-based extraction and gas chromatography-tandem quadrupole mass spectrometry, J. Chromatogr. A 1216, 6522–6531. [CrossRef] [PubMed] (In the text)
  • Wang R.W., Liu Z.Q., Dong K., Elzen P.J., Pettis J., Huang Z.Y. (2002) Association of novel mutations in a sodium channel gene with fluvalinate resistance in the mite, Varroa destructor, J. Apic. Res. 41, 17–25. (In the text)
  • Wellmark, Hivistan product information, http://www.centralapiary.com/products.htm#hivastan (accessed August 30, 2009). (In the text)
  • Whittington R., Winston M.L., Melathopoulos A.P., Higo H.A. (2000) Evaluation of the botanical oils neem, thymol, and canola sprayed to control Varroa jacobsoni Oud. (Acari: Varroidae) and Acarapsis woodi (Acari: Tarsonemidae) in colonies of honey bees (Apis mellifera L., Hymenoptera: Apidae), Am. Bee J. 140, 565–572. (In the text)

All Tables

Table I

Maximum pesticide incidence in apiary samples of wax, pollen, bee and honey.