A field realistic model to assess the effects of pesticides residues and adulterants on honey bee gene expression.

While studies on the sublethal effects of chemical residues in beeswax on adult honey bees are increasing, the study protocols assessing the impacts on honey bee brood in realistic conditions still need to be investigated. Moreover, little is known about the residue's effect on gene expression in honey bee brood. This study reports the effects of chlorpyriphos-ethyl, acrinathrin and stearin worker pupae exposure through contaminated or adulterated beeswax on the gene expression of some key health indicators, using a novel in vivo realistic model. Larvae were reared in acrinathrin (12.5, 25, 10 and 100 ppb) and chlorpyriphos-ethyl (5, 10, 500 and 5000 ppb) contaminated or stearin adulterated beeswax (3, 4, 5, 6 and 9%) in newly formed colonies to reduce the influence of external factors. On day 11, mortality rates were assessed. Honey bee pupae were extracted from the comb after 19 days of rearing and were analysed for the gene expression profile of four genes involved in the immune response to pathogens and environmental stress factors (Imd, dorsal, domeless and defensin), and two genes involved in detoxifications mechanisms (CYP6AS14 and CYP9Q3). We found no linear relation between the increase in the pesticide concentrations and the brood mortality rates, unlike stearin where an increase in stearin percentage led to an exponential increase in brood mortality. The immune system of pupae raised in acrinathrin contaminated wax was triggered and the expression of CYP6AS14 was significantly upregulated (exposure to 12.5 and 25 ppb). Almost all expression levels of the tested immune and detoxification genes were down-regulated when pupae were exposed to chlorpyrifos-contaminated wax. The exposure to stearin triggered the immune system and detoxification system of the pupae. The identification of substance-specific response factors might ultimately serve to identify molecules that are safer for bees and the ecosystem's health.

Beekeepers perception of risks affecting colony loss: A pilot survey.

Understanding amateur beekeepers' perception of risks affecting bee health and mortality is essential to analyse the reasons for adopting or rejecting good management practices. A perception survey on how beekeepers perceive and manage factors related to climate change, Varroa infestation, management practices, and pesticide exposure was designed and launched online. This unpreceded sociological survey involved 355 beekeepers spread all over Belgium. A two-sample t test with unequal variances comparing beekeepers with colony loss rates below or exceeding the acceptable level, that is <10% and ≥10%, indicates that beekeepers (N = 213) with colony loss rates <10% generally have greater average levels of perceived risks and the benefits of action that lead to increased motivation to act in better ways. The results of this survey highlight the importance of looking beyond socio-economic determinants in any risk mitigation strategy associated with bee mortality when dealing with amateur beekeepers.

Adulteration of beeswax: A first nationwide survey from Belgium.

Beeswax is intended for use in the beekeeping sector but also in the agro-food, pharmaceutical or cosmetics sectors. The adulteration of beeswax is an emerging issue that was reported lately at several occasions in the scientific literature. This issue tends to become more frequent and global, but its exact extent is not accurately defined. The present study aims to assess the current situation in Belgium through a nationwide survey. Randomized beeswax samples originating from Belgian beekeepers (N = 98) and commercial suppliers (N = 9) were analysed with a Fourier transform infrared spectroscopy (FTIR) coupled with Attenuated Total Reflectance (ATR) accessory (FTIR-ATR spectroscopy) for adulteration. The survey revealed a frequency of 9.2% and 33.3% of adulteration in beekeepers beeswax samples (9 samples out of 98: 2 with paraffin and 7 with stearin/stearic acid) and commercial beeswax samples (3 samples out of 9: all adulterated with stearin/stearic acid), respectively. The analysed samples were adulterated with various percentages of paraffin (12 to 78.8%) and stearin/stearic acid (1.2 to 20.8%). This survey indicates that in the beekeepers samples, beeswax adulteration was more frequent in comb foundation and crude beeswax than in comb wax. With the example of this nationwide survey conducted in Belgium, this study shows the emergence of the issue and the urgent need for action to safeguard the health of both honey bees health and humans, in particular with the setting of a proper regulation legal framework and a specific routine analytical testing of commercial beeswax to ensure beeswax quality.

Risk and protective indicators of beekeeping management practices.

Explaining the reasons for the high honey bee (Apis mellifera) colony loss rate in recent years has become a top global research priority in apicultural and agricultural sciences. Although there are indications of the role played by beekeeping management practices on honey bee health, very little information is currently available. Our study aimed to characterize the beekeeping management practices carried out in Belgium, and to determine the relationship between beekeeping management practices and colony losses. Variables obtained from face-to-face questioning of a representative randomized and stratified sample of Belgian beekeepers (n = 186) were integrated into a logistic regression model (univariate and multivariate) and correlated to the declared colony loss rates to identify risk and protective indicators. We used a classification tree analysis to validate the results. We present evidence of a relationship between poor beekeeping management practices and colony losses. The main factors protecting honey bee colonies are the aptitude of the beekeeper to change his management practices, the hive type, the equipment origin and hygiene, wintering in proper conditions (the use of divider boards, i.e. board blocks or space fillers off part of the hive body), the colony strength estimation before wintering, winter monitoring, and last but not least, appropriate integrated pest management. Proper estimation of the Varroa infestation level should be performed prior to treatment. The consequences of poor beekeeping practices on honey bee health can be addressed by proper training of beekeepers. An online tool was developed and published for beekeepers allowing them to evaluate the effect of their management practices on colony health.

Pesticide and veterinary drug residues in Belgian beeswax: Occurrence, toxicity, and risk to honey bees.

Pesticide and veterinary drug residues are one of the stress factors affecting bee health and mortality. To investigate the occurrence, the concentration and the toxicity risk to bees of pesticide residues in four different types of beeswax (brood comb wax, recycled comb wax, honey comb wax, and cappings wax), 182 samples were collected from apiaries located all over the Belgian territories, during spring 2016 and analysed by LC-MS/MS and GC-MS/MS for the presence of 294 chemical residues. The toxicity risk to bees expressed as the Hazard Quotient (HQ) was calculated for each wax sample, according to two scenarios with different tau-fluvalinate LD50 values. Residues showing the highest prevalence were correlated to bee mortality in a multivariate logistic regression model and a risk-based model was used to predict colony bee mortality. Altogether, 54 different pesticide and veterinary drug residues were found in the four types of beeswax. The residues with a higher likelihood to be retained in beeswax are applied in-hive or with a high lipophilic nature. The multivariate logistic regression model showed a statistically significant influence of chlorfenvinphos on bee mortality. All our results indicated that cappings wax was substantially less contaminated. This national survey on beeswax contamination provides guidelines on the re-use of beeswax by beekeepers and shows the necessity to introduce maximum residue levels for global trade in beeswax. An online tool was developed to enable beekeepers and wax traders to estimate the risk to honey bee health associated with contaminated wax.

Honeybee and consumer's exposure and risk characterisation to glyphosate-based herbicide (GBH) and its degradation product (AMPA): Residues in beebread, wax, and honey.

In order to assess bee and human exposure to residues of glyphosate-based herbicide (GBH) and its main degradation products aminomethylphosphonic acid (AMPA) and to characterise the risk posed by these substances, we analysed 3 different bee matrices; beebread (N = 81), wax (N = 100) and 10-paired samples of wax/honey collected in 2016/2017 from 379 Belgian apiaries. A high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS-MS) was used as analytical method. Limit of quantification and detection (LOQ and LOD) for GBH residues and AMPA in the 3 matrices was respectively of 10 ng g-1 and 1 ng g-1. In beebread, 81.5% of the samples showed a residue concentration > LOQ and 9.9% of the samples a residue concentration < LOQ (detection without quantification); no significant difference in detection rate was found between the north and the south of the country. Glyphosate was detected in beeswax less frequently than in beebread (i.e. 26% >LOQ versus 81.5% >LOQ). The maximum GBH residues and AMPA concentration found in beebread (respectively 700 ng g-1 and 250 ng g-1) led to sub-lethal exposure to bees. The Hazard Quotient (HQ) for beebread and beeswax (7 and 3.2, respectively) were far below the "safety" oral and contact thresholds for bees. For human health, the highest exposure to GBH residues in pollen corresponded to 0.312% and 0.187% of the ADI and of the ARfD respectively and, to 0.002% and to 0.001% for beeswax. No transfer of glyphosate from wax to honey was detected. Considering our results and the available regulatory data on the glyphosate molecule considered solely, not including the adjuvants in GBH formulation, the consumption of these three contaminated matrices would not be a food safety issue. Nonetheless, caution should be taken in the interpretation of the results as new studies indicate possible glyphosate/GBH residues toxicity below regulatory limits and at chronic sub-lethal doses.

Belgian case study on flumethrin residues in beeswax: Possible impact on honeybee and prediction of the maximum daily intake for consumers.

To assess the health risk posed by flumethrin residues in beeswax to honeybees and honey consumers, 124 wax samples randomly distributed in Belgium were analysed for flumethrin residues using liquid chromatography/tandem mass spectrometry. The risk posed by flumethrin residues in beeswax to honeybee health was assessed through the calculation of a non-pondered and a pondered Hazard Quotient by the prevalence rate of flumethrin considering an oral or topical exposure. No statistical difference was found when comparing both the average flumethrin residues concentrations and contact and oral pondered hazard quotients between apiaries with lower and equal or higher than 10% of colony loss. Flumethrin residues estimated daily intake by Belgian consumers through honey and wax ingestion was estimated via a deterministic (worst-case scenario) and a probabilistic approach. The probabilistic approach was not possible for beeswax consumption due to the lack of individual consumption data. The highest estimated exposure was <0.1% of the theoretical maximum daily intake for both approaches, meaning no risk for human health.

Moku virus detection in honey bees, Belgium, 2018.

We report the detection of Moku virus in honey bees (Apis mellifera) collected in 2017 from hives with a history of attacks by invasive Asian hornets (Vespa velutina nigrithorax) in Belgium. End 2016, Moku virus was reported in Asian hornets from the same area. In addition, the Moku virus was already present in historical samples of bees collected in 2013, that is, 2 years after the official first detection of Asian hornets in the same area of Belgium. This study suggests a spread of Moku virus to honey bees with possible consequences.

Moku Virus in Invasive Asian Hornets, Belgium, 2016.

We report the detection of Moku virus in invasive Asian hornets (Vespa velutina nigrithorax) in Belgium. This constitutes an unexpected report of this iflavirus outside Hawaii, USA, where it was recently described in social wasps. Although virulence of Moku virus is unknown, its potential spread raises concern for European honeybee populations.