Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray.

In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.

First molecular detection of a viral pathogen in Ugandan honey bees.

Ugandan honey bees (Apis mellifera L.) produce honey, and are key pollinators within commercial crops and natural ecosystems. Real-time RT-PCR was used to screen immature and adult bees collected from 63 beekeeping sites across Uganda for seven viral pathogens. No samples tested positive for Chronic bee paralysis virus, Sacbrood virus, Deformed wing virus, Acute bee paralysis virus, Apis iridescent virus or Israeli acute paralysis virus. However, Black queen cell virus (BQCV) was found in 35.6% of samples. It occurred in adults and larvae, and was most prevalent in the Western highlands, accounting for over 40% of positive results nationally.

Evaluating European Food Safety Authority Protection Goals for Honeybees (Apis mellifera): What Do They Mean for Pollination?

In recent years there has been growing concern regarding the sudden and unexplained failure of honeybee (Apis mellifera) colonies. Several factors have been suggested, including pesticides. In an effort to regulate their impact, guidance published by the European Food Safety Authority (EFSA) has recommended that the magnitude of effects on exposed colonies should not exceed 7% reduction in colony size after 2 brood cycles. However, fears have been raised regarding the practicality of measuring such a loss in the field. It is also unclear how this protection goal relates to maintaining the ecosystem services provided by bees, which we argue should be a primary objective for regulators. Here, we evaluate what these protection goals mean in relation to ecosystems performance using a computational colony model that incorporates mechanisms to simulate both lethal and sublethal pesticide effects. To these simulations, we apply a testing regime similar to that commonly used in field trials to produce standard assessment metrics. By relating these measures to losses in forager activity, we aim to identify which could be used as effective indicators of reduced ecoservice and to quantify acceptable limits below which performance can be maintained. Our findings show that loss of colony size is the best indicator of reduced ecoservice. Metrics that focus on specific colony functions such as increased brood or forager mortality are ineffective indicators for all types of simulated pesticide effects. At the levels of colony loss recommended by EFSA, using our default parameterization, we predict a loss of ecosystems performance of 3% to 4%. However, based on an extensive sensitivity analysis, it is clear that this estimate is subject to substantial uncertainty with losses under alternative parameterizations of up to 14%. Nevertheless, our model provides a valuable framework for assessing protection goals, allowing regulators to test relevant impacts and quantify their magnitude. Integr Environ Assess Manag 2018;14:750-758. © 2018 Crown Copyright and SETAC.

The incidence of honey bee pests and diseases in England and Wales.

The Central Science Laboratory (CSL) National Bee Unit (NBU) has been responsible for maintaining the Integrated Bee Health Programme in England and Wales since the early 1990s. The role of the Bee Health Programme is to protect the honey bee, a major pollinator of agricultural and horticultural crops and wild flora, and to provide up-to-date technical support to beekeepers. The Bee Health Programme is funded in England by the Department for Environment, Food and Rural Affairs (Defra) and in Wales by the Welsh Assembly Government (WAG). The work includes inspection of honey bee colonies, disease and pest diagnosis, development of contingency plans for emerging threats, minimising the risk of introduction of potentially serious exotic pests and diseases through importation by import risk analysis and related extension work and consultancy services to both government and industry. There is also an underpinning programme of research and development.

Modelling long-term effects of IGRs on honey bee colonies.

Systems have been developed to monitor the direct effects of insect growth regulator (IGR) pesticide exposure on honey bee development, but there has been little work on the longer-term impact of exposure on the colony. A honey bee population model provided the opportunity to investigate the effects of short-term mortality of brood and of sublethal changes in behaviour of the surviving adults on honey bee populations. The model showed that brood mortality alone has limited effect on colony size. There were two mechanisms that could have greater influence on productivity. Precocious foraging in affected adult bees, and hence early loss of brood-rearing (nurse) capabilities, had a much larger effect than expected. Increasing mortality rates by 30% to simulate sublethal effects on lifespan, rather than reduced brood-rearing capability, gave a significantly smaller effect. In order to simulate an effect with the 'shortened lifespan' mechanism as large as that with the 'premature ageing' mechanism, the mortality rate of affected adults had to be increased by 500%. A significant finding from the model is that application of IGRs in spring and early summer could have substantial effects on colony size and viability. Sublethal effects such as precocious foraging can have worse effects than massive brood mortality, as it severely reduces the ability to rear the next generation of nurse bees.

Monitoring the effects of thiamethoxam applied as a seed treatment to winter oilseed rape on the development of bumblebee (Bombus terrestris) colonies.

BACKGROUND: The development of bumblebee (Bombus terrestris audax) colonies that had foraged for 5 weeks on flowering winter oilseed rape grown from seed treated with thiamethoxam (as Cruiser OSR) was assessed (two control, one treated field). Colony development was evaluated by monitoring the colony mass, forager activity was assessed, both at the hive and within the crop, and the contribution of oilseed rape to the pollen stored within the colony was analysed. RESULTS: Pollen collected from the treated crop contained residues of 1.0 µg thiamethoxam kg(-1) and 3.0 µg CGA322704 (metabolite likely equivalent to clothiandin) kg(-1) , and nectar contained residues of 1.8 µg thiamethoxam kg(-1) and no metabolite. No residues of thiamethoxam or CGA322704 were detected in samples from the control fields. Up to 93% of bumblebee collected pollen sampled from within the colonies originated from oilseed rape, and B. terrestris were observed actively foraging on all the fields. Colonies on all three fields showed similar rates of mass gain during the exposure phase and comparable production of gynes and drones. CONCLUSIONS: B. terrestris colonies placed adjacent to a field of flowering oilseed rape grown from thiamethoxam-treated seed developed at a comparable rate with colonies placed adjacent to oilseed rape grown from untreated seed. © 2015 Society of Chemical Industry.

Thiamethoxam: Assessing flight activity of honeybees foraging on treated oilseed rape using radio frequency identification technology.

The present study was designed to assess homing behavior of bees foraging on winter oilseed rape grown from seed treated with thiamethoxam (as Cruiser OSR), with 1 field drilled with thiamethoxam-treated seed and 2 control fields drilled with fungicide-only-treated seed. Twelve honeybee colonies were used per treatment group, 4 each located at the field edge (on-field site), at approximately 500 m and 1000 m from the field. A total of nearly 300 newly emerged bees per colony were fitted (tagged) with Mic3 radio frequency identification (RFID) transponders and introduced into each of the 36 study hives. The RFID readers fitted to the entrances of the test colonies were used to monitor the activity of the tagged bees for the duration of the 5-wk flowering period of the crop. These activity data were analyzed to assess any impact on flight activity of bees foraging on the treated compared with untreated crops. Honeybees were seen to be actively foraging within all 3 treatment groups during the exposure period. The data for the more than 3000 RFID-tagged bees and more than 90 000 foraging flights monitored throughout the exposure phase for the study follow the same trends across the treatment and controls and at each of the 3 apiary distances, indicating that there were no effects from foraging on the treated crop. Under the experimental conditions, there was no effect of foraging on thiamethoxam-treated oilseed rape on honeybee flight activity or on their ability to return to the hive.

Neonicotinoids and bumblebees (Bombus terrestris): effects on nectar consumption in individual workers.

BACKGROUND: The objective of this study was to quantify whether the presence of three different neonicotinoid insecticides (imidacloprid, thiamethoxam or clothianidin) in sucrose solution results in antifeedant effects in individual worker bumblebees (Bombus terrestris), and, if so, whether this effect is reversible if bees are subsequently offered untreated feed. RESULTS: Bees exposed to imidacloprid displayed a significant dose-dependent reduction in consumption at 10 and 100 µg L(-1), which was reversed when untreated feed was offered. No consistent avoidance/antifeedant response to nectar substitute with thiamethoxam was detected at the more field-realistic dose rates of 1 and 10 µg L(-1), and exposure to the very high 100 µg L(-1) dose rate was followed by 100% mortality of experimental insects. No reduction in food intake was recorded at 1 µg clothianidin L(-1), reduced consumption was noted at 10 µg clothianidin L(-1) and 100% mortality occurred when bees were exposed to rates of 100 µg clothianidin L(-1). CONCLUSION: This study provides evidence of a direct antifeedant effect of imidacloprid and clothianidin in individual bumblebees but highlights that this may be a compound-specific effect.

Effects of solvent on the toxicity of dimethoate in a honey bee in vitro larval study.

BACKGROUND: There is increasing interest in the toxicity of pesticides to honey bee larvae. The solubility of pesticides in the artificial diet used to dose larvae is a key issue. There has been no full evaluation of the use of solvents to dissolve pesticides in the larval toxicity test system. RESULTS: Data generated for the toxicity of dimethoate to larvae using 5 and 10% acetone solvent in the diet were similar and close to the toxicity data for adults (LD50 0.1-0.3 µg bee(-1)). With only 1.5% acetone as a vehicle, the toxicity (LD50 0.6-1.14 µg larva(-1)) was lower and closer to published validation data of 1.5-3.1 µg larva(-1) (with some reported data as high as 8.8 µg larva(-1)) generated using dimethoate dissolved in water. This suggests that the solubility of the pesticide in the diet is a key factor in determining its toxicity. CONCLUSIONS: Consideration of the solubility of the toxic reference and test items when designing studies is important. The use of higher rates of acetone in the present study more closely represents the true exposure of larvae to pesticides that are not highly soluble in diet. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Investigation into the experimental protocols required to determine maximum residue limits (MRLs) in honey.

There is current debate within the EU, and internationally, on how withdrawal periods and maximum residue limits (MRLs) may be set for honey production. Whilst comprehensive EU guidelines exist for calculating the withdrawal times of veterinary medicines in most food-producing species, the analytical variables to be studied for bees/honey are not well defined. The objective of this study was therefore to investigate and understand the factors, for example sampling variability, that is important in the development of a harmonized protocol that can be used to generate the robust scientific data necessary to assist risk assessors in proposing MRLs for honey. Ten bee colonies were treated in the spring with a model compound (ciprofloxacin). One hive was used to study intra-hive variation in residue concentrations and the other nine were used in an inter-hive study over a 41-week sampling period. All samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest mean concentration from nine hives used in the inter-hive study was 4627 µg/kg eight days (D8) after treatment. The concentration of ciprofloxacin declined to an average concentration of 1756 µg/kg at D30 and 733 µg/kg at D283 (over-winter sample). A generalized additive model was used to fit a smooth curve for trend estimation. For some individual hives the concentration of ciprofloxacin increased slightly at the later sampling time-points. Consequently it was not possible to interpolate, with confidence, a finite withdrawal period for ciprofloxacin at theoretical MRLs between 25 and 500 µg/kg. The observed variation in concentration of ciprofloxacin between hives indicates that the validity of the EU guideline for bees/honey, which requires five samples from five hives to calculate a withdrawal period, may require revision.

Study of the depletion of lincomycin residues in honey extracted from treated honeybee (Apis mellifera L.) colonies and the effect of the shook swarm procedure.

Bee colonies were treated with 1.2g lincomycin hydrochloride per hive (single treatment in sucrose solution) and samples of honey were then collected at intervals over a 41-week period. The samples were analysed for lincomycin using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS). The highest mean concentration of lincomycin (pooled analytical results for brood and super honey) was 24 microugg(-1) 3 days after treatment, a mean of 3.5 microgg(-1) after 129 days. The shook swarm procedure was investigated and resulted in a lincomycin concentration of 34 microgg(-1) in honey (pooled results for brood and super honey) 3 days after treatment, declining to 0.38 microgg(-1) 129 days after treatment. Lincomycin was persistent in the hive and detected in all over winter (290 days after dosing) samples of honey collected from both non-shook swarmed and shook swarmed colonies. The results overall indicate that lincomycin parent is a suitable marker compound to detect lincomycin misuse in apiculture.

The effects of four insect growth-regulating (IGR) insecticides on honeybee (Apis mellifera L.) colony development, queen rearing and drone sperm production.

This study assessed the effects of exposure to IGRs on the long-term development of the honeybee colony, viability of queens and sperm production in drones and integrated the data into a honeybee population model. Colonies treated with diflubenzuron resulted in a short-term reduction in the numbers of adult bees and brood. Colonies treated with fenoxycarb declined during the season earlier and started the season slower. The number of queens that successfully mated and laid eggs was affected in the fenoxycarb treatment group but there were no significant differences in the drone sperm counts between the colonies. An existing honeybee population model was modified to include exposure to IGRs. In the model, fenoxycarb reduced the winter size of the colony, with the greatest effects following a June or an August application. Assuming a 'larvae per nurse bee' ratio of 1.5 for brood rearing capability, the reduction in winter size of a colony following a fenoxycarb application was at its worst about 8%. However, even if only those bees reared within 2 weeks of the IGR being applied are subject to premature ageing, this might significantly reduce the size of over-wintering colonies, and increase the chance of the bee population dwindling and dying in late winter or early spring.

Effects of European foulbrood treatment regime on oxytetracycline levels in honey extracted from treated honeybee (Apis mellifera) colonies and toxicity to brood.

This study aimed to assess oxytetracycline (OTC) residue levels in honey up to 12 weeks after treatment of honeybee colonies with two methods of application (in liquid sucrose and in powdered icing sugar). Significantly greater mortality was seen in the all stages of brood development for the treated colonies when compared with the controls. Samples of honey were extracted up to 12 weeks after treatment and analysed by HPLC following metal chelation with a limit of quantitation of 0.05 mg/kg. These data showed that the current method of application of Terramycin in liquid form results in very high residue levels in honey with residues of 3.7 mg/kg eight weeks after application. Further work is required to determine whether the levels can be further reduced by changes in the method of application whilst ensuring efficacy and minimizing the effects on brood.