Toxicity of Coumaphos Residues in Beeswax Foundation to the Honey Bee Brood.

Coumaphos is one of the most frequently detected pesticides in recycled beeswax. The objective was to assess the maximal level of coumaphos in foundation sheets that could exist without lethal effects on the honey bee larvae. Brood development was followed in cells drawn on foundation squares containing coumaphos ranging from 0 to 132 mg/kg. Furthermore, larval exposure was determined by measuring the coumaphos level in the drawn cells. Coumaphos levels in the initial foundation sheets up to 62 mg/kg did not increase brood mortality because the emergence rates of bees raised on these foundation squares were similar to controls (median of 51%). After a single brood cycle, coumaphos levels in the drawn cells were up to three times lower than the initial levels in foundation sheets. Hence, coumaphos levels of 62 mg/kg in the initial foundation sheets, almost the highest exposures, resulted in levels of 21 mg/kg in drawn cells. A significantly reduced emergence rate (median of 14%) was observed for bees raised on foundation sheets with initial coumaphos levels of 132 mg/kg, indicating increased brood mortality. Such levels resulted in coumaphos concentrations of 51 mg/kg in drawn cells, which is close to the median lethal concentration (LC50) as determined in previous in vitro experiments. In conclusion, brood mortality was increased on wax foundation sheets with initial coumaphos levels of 132 mg/kg, while no elevated mortality was observed for levels up to 62 mg/kg. Environ Toxicol Chem 2023;42:1816-1822. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Lack of evidence for trans-generational immune priming against the honey bee pathogen Melissococcus plutonius.

Trans-generational immune priming involves the transfer of immunological experience, acquired by the parents after exposure to pathogens, to protect their progeny against infections by these pathogens. Such natural mechanisms could be exploited to prevent disease expression in economically important insects, such as the honey bee. This mechanism occurs when honey bee queens are exposed to the pathogenic bacterium Paenibacillus larvae. Here, we tested whether natural or experimental exposure to Melissococcus plutonius-another bacterium triggering a disease in honey bee larvae-reduced the susceptibility of the queen's progeny to infection by this pathogen. Because the immunological response upon pathogen exposure can lead to fitness costs, we also determined whether experimental exposure of the queens affected them or their colony negatively. Neither natural nor experimental exposure induced protection in the honey bee larvae against the deleterious effects of M. plutonius. Our results provided no evidence for the occurrence of trans-generational immune priming upon exposure of the queen to M. plutonius. Whether this lack was due to confounding genetic resistance, to unsuitable exposure procedure or to the absence of trans-generational immune priming against this pathogen in honey bees remains to be determined.

Evaluation of pesticide residues in commercial Swiss beeswax collected in 2019 using ultra-high performance liquid chromatographic analysis.

The aim of this study was to determine residue levels of pesticides in Swiss commercial beeswax. Foundation samples were collected in 2019 from nine commercial manufacturers for analysis of 21 pesticides using ultra-high performance liquid chromatography. Individual samples showed the variability and residue ranges and pooled samples represented the average annual residue values of the Swiss production. In total, 17 pesticides were identified and 13 pesticides were quantified. They included 13 acaricides and/or insecticides, two fungicides as well as a synergist and a repellent. The means calculated from individual samples were similar to the average annual residue values for most tested pesticides. Mean values of 401, 236, 106 and 3 µg·kg-1 were obtained for the beekeeping-associated contaminants coumaphos, tau-fluvalinate, bromopropylate and N-(2,4-Dimethylphenyl)-formamide (DMF; breakdown product of amitraz), respectively. For the other pesticides, the mean values were 203 µg·kg-1 (synergist piperonyl butoxide), 120 µg·kg-1 (repellent N,N-Diethyl-3-methylbenzamide, DEET), 19 µg·kg-1 (chlorfenvinphos) and 4 µg·kg-1 ((E)-fenpyroximate), while the means for acrinathrin, azoxystrobin, bendiocarb, boscalid, chlorpyrifos, flumethrin, permethrin, propoxur and thiacloprid were below the limit of quantification (< LOQ). Individual samples contained from seven to 14 pesticides. The ranges of values for coumaphos and piperonyl butoxide (from 14 to 4270 µg·kg-1; from 6 to 1555 µg·kg-1, respectively) were larger as compared to the ranges of values for DEET and tau-fluvalinate (from < LOQ to 585 µg·kg-1; from 16 to 572 µg·kg-1, respectively). In conclusion, the most prominent contaminants were the pesticides coumaphos and tau-fluvalinate, which are both acaricides with previous authorization for beekeeping in Switzerland, followed by piperonyl butoxide, a synergist to enhance the effect of insecticides.

An in vitro model for assessing the toxicity of pesticides in beeswax on honey bee larvae.

While many studies have examined residue levels in beeswax, little is known about the levels that pose a risk for honey bee development. In an in vitro study, we aimed to assess the toxicity of pesticides in wax for worker larvae using coumaphos as a model substance. First, we reared larvae in beeswax with the aim to correlate the larval toxicity to the corresponding levels of coumaphos in beeswax. In a second step, we tested to which extent coumaphos migrates from the beeswax into the larval diet and if such dietary levels are toxic to larvae. We observed dose-related toxicity when larvae were exposed to coumaphos concentrations in beeswax from 30 to 100 mg/kg. The lethal concentration in 50% of the individuals (LC50) was calculated to be 55.9 mg/kg, while the no observed effect concentration (NOEC) for exposure of larvae to coumaphos in wax was 20 mg/kg. Additional test series without larvae allowed to assess the migration of coumaphos from the beeswax into the diet. The resulting dietary coumaphos concentrations were four to five times lower than the initial concentrations in wax. In accordance, the LC50 for chronic exposure of larvae to coumaphos in the diet was 12.5 mg/kg, which was 4.5 times lower than the LC50 obtained for wax exposure. Finally, a coumaphos level of 20 mg/kg in wax led to a dietary concentration of 3.9 mg/kg that was close to the NOEC of 3 mg/kg obtained in the diet. In conclusion, both experimental approaches suggest that coumaphos concentrations of up to 20 mg/kg in wax are non-lethal.

Long-term monitoring of lipophilic acaricide residues in commercial Swiss beeswax.

BACKGROUND: A national survey on pesticides in recycled beeswax originating from beekeeping has been conducted in Switzerland for almost three decades. It allowed obtaining a good overview of the lipophilic products used for beekeeping within the last 30 years. RESULTS: The use of the veterinary drugs containing bromopropylate or tau-fluvalinate two decades ago led to substantial residues in commercial beeswax. These contaminants are still detectable although in Switzerland the corresponding products have been out of use for many years. The level of coumaphos substantially increased in 2015 up to an annual value of 3.25 mg·kg-1 , suggesting that at least a few beekeepers used coumaphos-containing products. Consequently, an information campaign was launched, and the annual value decreased again. Maximal levels of thymol up to an annual value of 87.5 mg·kg-1 were measured in 2009. Since that time, a steady decrease of thymol residues suggests that beekeepers less frequently use thymol-containing products. Twenty-five years ago, 1,4-dichlorobenzene (PDCB) was widely used for the control of the wax moth, resulting in residues in beeswax up to an annual value of 10.9 mg·kg-1 whereas nowadays, PDCB residues are rarely detected in Swiss beeswax. CONCLUSIONS: Our survey illustrates that several beekeeping-associated pesticides persist in recycled beeswax for many years. Most recent analyses show lower residue levels in Swiss beeswax as compared to previous years. Nowadays Swiss beekeepers mostly use hydrophilic substances for treatment against the Varroa destructor that do not accumulate in beeswax, thus reducing exposure of the honey bees to lipophilic contaminants.

Putative determinants of virulence in Melissococcus plutonius, the bacterial agent causing European foulbrood in honey bees.

MELISSOCOCCUS PLUTONIUS: is a bacterial pathogen that causes epidemic outbreaks of European foulbrood (EFB) in honey bee populations. The pathogenicity of a bacterium depends on its virulence, and understanding the mechanisms influencing virulence may allow for improved disease control and containment. Using a standardized in vitro assay, we demonstrate that virulence varies greatly among sixteen M. plutonius isolates from five European countries. Additionally, we explore the causes of this variation. In this study, virulence was independent of the multilocus sequence type of the tested pathogen, and was not affected by experimental co-infection with Paenibacillus alvei, a bacterium often associated with EFB outbreaks. Virulence in vitro was correlated with the growth dynamics of M. plutonius isolates in artificial medium, and with the presence of a plasmid carrying a gene coding for the putative toxin melissotoxin A. Our results suggest that some M. plutonius strains showed an increased virulence due to the acquisition of a toxin-carrying mobile genetic element. We discuss whether strains with increased virulence play a role in recent EFB outbreaks.

Pyrrolizidine Alkaloids: The Botanical Origin of Pollen Collected during the Flowering Period of Echium vulgare and the Stability of Pyrrolizidine Alkaloids in Bee Bread.

Previous studies have shown that pollen products sold as nutritional supplements and used in apitherapy may contain toxic pyrrolizidine alkaloids (PAs) if bees collect pollen from PA-containing plants, such as Echium vulgare. In this study, the botanical origin of pollen from two observation sites was studied. Despite a high PA content in pollen samples that bees collected during E. vulgare's flowering period, bees were found to collect relatively few Echium pollen loads. Thus, the monitoring of pollen loads collected at the apiaries is unviable to estimate the risk of PA contamination in pollen or bee bread. In a second step, the stability of PAs in bee bread samples containing PAs at concentrations of 2538 ng/g and 98 ng/g was assessed over a period of five or six months, respectively. No significant PA reduction was observed in bee bread stored at 15 °C, but there were overall PA reductions of 39% and 33% in bee bread stored at 30 °C, reflecting hive conditions. While PA N-oxides decreased over time, other types of PAs remained relatively stable. Monitoring PAs in pollen products remains important to ensure consumer safety and should include echivulgarine (and its N-oxide), the major PA type found in pollen from E. vulgare.

Nursing protects honeybee larvae from secondary metabolites of pollen.

The pollen of many plants contains toxic secondary compounds, sometimes in concentrations higher than those found in the flowers or leaves. The ecological significance of these compounds remains unclear, and their impact on bees is largely unexplored. Here, we studied the impact of pyrrolizidine alkaloids (PAs) found in the pollen of Echium vulgare on honeybee adults and larvae. Echimidine, a PA present in E. vulgare pollen, was isolated and added to the honeybee diets in order to perform toxicity bioassays. While adult bees showed relatively high tolerance to PAs, larvae were much more sensitive. In contrast to other bees, the honeybee larval diet typically contains only traces of pollen and consists predominantly of hypopharyngeal and mandibular secretions produced by nurse bees, which feed on large quantities of pollen-containing bee bread. We quantified the transfer of PAs to nursing secretions produced by bees that had previously consumed bee bread supplemented with PAs. The PA concentration in these secretions was reduced by three orders of magnitude as compared to the PA content in the nurse diet and was well below the toxicity threshold for larvae. Our results suggest that larval nursing protects honeybee larvae from the toxic effect of secondary metabolites of pollen.

Chemical fingerprinting identifies Echium vulgare, Eupatorium cannabinum and Senecio spp. as plant species mainly responsible for pyrrolizidine alkaloids in bee-collected pollen.

Various studies have shown that bee-collected pollen sold as nutritional supplements may contain toxic pyrrolizidine alkaloids (PAs) and, thus, pose a potential health risk for consumers. The level of contamination may vary according to its geographical and botanical origin. Here, the PA content of pollen produced in Switzerland was studied and 32 commercially available bee-collected pollen supplements produced between 2010 and 2014 were analysed. In addition, at what time period bees collect PA-containing pollen was investigated. Hence, this study looked into the occurrence of PAs in pollen samples collected daily during two-to-three consecutive seasons. Furthermore, the PA spectrum in pollen was compared to the spectrum found in flower heads of PA-plants to unambiguously identify plants responsible for PA contamination of pollen. The PA concentration of commercial and daily collected pollen was determined by target analysis using an HPLC-MS/MS system, allowing the detection of 18 different PAs and PA N-oxides found in the genera Echium, Eupatorium and Senecio, while the comparison of the PA spectrum in pollen and flower heads was performed by LC-HR-MS, allowing the detection of all PA types in a sample, including saturated, non-carcinogenic PAs. Of the commercially available pollen, 31% contained PAs with a mean concentration of 319 ng/g, mainly Echium- and Eupatorium-type PAs, while the PA concentrations were below the limit of quantitation (LOQ) in 69% of the pollen samples. Bees collected pollen containing Echium-type PAs mainly in June and July, while they gathered pollen containing Eupatorium-type PAs from mid-July to August. Senecio-type PAs appeared from June to September. Comparison of the PA array in pollen and plants identified E. vulgare and E. cannabinum as the main plants responsible for PA contamination of Swiss bee-collected pollen, and to a lesser extent also identified plants belonging to the genus Senecio.

Impact of the Asian Chestnut Gall Wasp, Dryocosmus kuriphilus (Hymenoptera, Cynipidae), on the Chestnut Component of Honey in the Southern Swiss Alps.

The Asian chestnut gall wasp (ACGW; Dryocosmus kuriphilus Yasumatsu, Hymenoptera, Cynipidae) is considered as one of the most dangerous pests of the genus Castanea. In southern Switzerland, repeated heavy ACGW attacks prevented chestnut trees from vegetating normally for years before the arrival and spread of the biological control agent Torymus sinensis (Kamijo, Hymenoptera, Torymidae). This resulted in a greatly reduced green biomass and flower production. In this paper, we analyze the impact of such an ecosystem alteration of the environment on the composition of produced honey. Six beekeepers were chosen from sites with different densities of chestnut trees, each of which providing series of honey samples from 2010 to 2016. We determined the chestnut component in the honeys via a combined chemical and sensory approach, and correlated the obtained results with the degree of yearly ACGW-induced crown damage and weather conditions during the period in question in the surrounding chestnut stands. The chestnut component in the analyzed honey sample series showed a strong correlation with the degree of ACGW-induced crown damage, whereas meteorological conditions of the corresponding year had a very marginal effect. Decreases in the chestnut component of the honey were statistically significant starting from a ACGW infestation level of 30%.

Pyrrolizidine Alkaloids from Echium vulgare in Honey Originate Primarily from Floral Nectar.

Pyrrolizidine alkaloids (PAs) in honey can be a potential human health risk. So far, it has remained unclear whether PAs in honey originate from pollen or floral nectar. We obtained honey, nectar, and plant pollen from two observation sites where Echium vulgare L. was naturally abundant. The PA concentration of honey was determined by targeted analysis using a high pressure liquid chromatography-mass spectrometry system (HPLC-MS/MS), allowing the quantification of six different PAs and PA-N-oxides present in E. vulgare. Echium-type PAs were detected up to 0.153 µg/g in honey. Nectar and plant pollen were analyzed by nontargeted analysis using ultrahigh pressure liquid chromatography-high resolution-mass spectrometry (UHPLC-HR-MS), allowing the detection of 10 alkaloids in small size samples. Echium-type PAs were detected between 0.3-95.1 µg/g in nectar and 500-35000 µg/g in plant pollen. The PA composition in nectar and plant pollen was compared to the composition in honey. Echimidine (+N-oxide) was the main alkaloid detected in honey and nectar samples, while echivulgarine (+N-oxide) was the main PA found in plant pollen. These results suggest that nectar contributes more significantly to PA contamination in honey than plant pollen.