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.

Quantitation of pesticides in bee bread collected from honey bee colonies in an agricultural environment in Switzerland.

Pesticide contamination of bee products is a widespread phenomenon. Due to its composition, bee bread is affected by both lipophilic and hydrophilic substances. As proof of concept of a monitoring campaign and to better understand the extent of contamination, we developed an analytical method based on a modified QuEChERS extraction, with subsequent separation by liquid chromatography and detection by mass spectrometry. This allowed for the quantitation of 51 agricultural- or beekeeping-associated pesticides in bee bread. The workflow was applied to 60 samples taken biweekly throughout spring to autumn 2022 from five colonies at a Swiss apiary in an agricultural area. In total, 30 pesticides were identified (> LOD), among which 26 pesticides were quantitated. The total number of pesticides detected per colony ranged from 11 to 19. The most prevalent substances (> LOQ) were two neonicotinoid insecticides, acetamiprid and thiacloprid (max. 16 µg/kg and 37 µg/kg, respectively); seven fungicides, azoxystrobin (max. 72 µg/kg), boscalid (max. 50 µg/kg), cyprodinil (max. 1965 µg/kg), difenoconazole (max. 73 µg/kg), mandipropamid (max. 33 µg/kg), pyraclostrobin (max. 8 µg/kg) and trifloxystrobin (max. 38 µg/kg); and two herbicides, prosulfocarb (max. 38 µg/kg) and terbuthylazine (max. 26 µg/kg). The study revealed strong variability in pesticide occurrence and concentrations among colonies sampled at the same site and date. The applied biweekly sampling of bee bread from March to August was shown to be reliable in capturing peak contaminations and revealing the onset of certain pesticides in bee bread. The study provides an adequate practical approach for pesticide monitoring campaigns.

Quantitation of 1,4-Dichlorobenzene and Thymol in Beeswax Using Dynamic Headspace Vacuum Transfer in Trap Extraction Prior to Gas Chromatography-Mass Spectrometry.

A method based on dynamic headspace vacuum transfer in trap extraction, followed by gas chromatography coupled with a mass spectrometer (DHS-VTT-GC-MS), was validated for the fast quantitation of 1,4-dichlorobenzene (p-dichlorobenzene; PDCB) and thymol residues in beeswax. The quantitation limits (LOQ) were 0.05 mg/kg (PDCB) and 0.25 mg/kg (thymol). Recoveries above 80% were obtained for PDCB concentrations between 0.05 and 10 mg/kg and for thymol concentrations between 0.25 and 200 mg/kg. Analysis of beeswax samples showed a good correlation between the results obtained by DHS-VTT-GC-MS analysis and those of a previous method based on SPE extraction followed by gas chromatography and triple- quadrupole mass spectrometry (GC-MS/MS) (R2 = 0.9770 for PDCB and 0.9666 for thymol). However, the sample preparation and chromatography were much shorter using the DHS-VTT-GC-MS method. Forty comb foundations samples produced in Switzerland in 2019 and 2021 were analysed using DHS-VTT-GC-MS. Fourteen samples contained PDCB above the LOQ of 0.05 mg/kg, ranging up to a maximum of 1.53 mg/kg. The mean concentration of the positive samples was 0.22 mg/kg. All samples contained thymol residues ranging between 3.9 and 84.4 mg/kg with mean and median concentrations of 22.7 mg/kg and 17.4 mg/kg. Residue levels of PCDB and thymol in Swiss beeswax were substantially below those measured 20 (PDCB) and 10 (thymol) years ago, respectively.

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.

Antibacterial potential of Swiss honeys and characterisation of their bee-derived bioactive compounds.

BACKGROUND: Antibacterial activity of honey is not only crucial characteristic in selection of honey for medical usage but also an important honey quality marker. The aim of the study was to characterise the antibacterial potential of 29 honey samples representing the main types of multi-floral blossom and honeydew honeys produced in Switzerland. Antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa was expressed as a minimum inhibitory and bactericidal concentrations (MIC and MBC). Furthermore, the content of bee-derived glucose oxidase (GOX) and its enzymatic product, H2 O2 , were also evaluated. RESULTS: All honey samples successfully met basic defined criteria (moisture and hydroxymethylfurfural (HMF)) tested in this study. Honeydew honeys were the most effective honey samples and generated the highest levels of H2 O2 . A strong significant correlation was found between the overall antibacterial activity and the level of H2 O2 among all honey samples. Interestingly, the content of GOX in honey samples did not correlate with their antibacterial activity as well as H2 O2 production capacity. A weak antibacterial activity was determined in five floral honeys, most likely due to increased enzymatic activity of pollen-derived catalase. CONCLUSION: This study showed that antibacterial effect of Swiss honey samples is associated mainly with H2 O2 . © 2019 Society of Chemical Industry.

Acetyl-CoA carboxylase 1-dependent lipogenesis promotes autophagy downstream of AMPK.

Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.

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%.

Evaluation of baker's yeast in honey using a real-time PCR assay.

Occasionally, melissopalynological analysis reveals the presence of baker's yeast (Saccharomyces cerevisiae) in honey sediments. A field experiment reproducing a common spring bee feeding practice, using sugar paste containing baker's yeast, was performed to understand how S. cerevisiae are introduced into honey. Apart from classical microscopy, a real-time quantitative PCR (qPCR) system specific for S. cerevisiae was established for quantification of S. cerevisiae in honeys. Results showed that S. cerevisiae cells are stored in the honey of the brood combs and are also transferred into honey in the supers. The concentrations of S. cerevisiae were highest in honey of the brood frames immediately after the feeding and decreased over time to low concentrations at the end of the year. A high content of S. cerevisiae cells were also found in the honey from supers of the spring harvest. Observed S. cerevisiae cells were not able to multiply in a high-sugar environment, such as honey, and their viability decreased rapidly after addition to the honey. The screening of 200 Swiss honeys revealed the presence of S. cerevisiae in 4.5% of the samples, as determined by microscopy and qPCR. Finally, the method described here may indicate an unwanted sucrose addition to honey through bee-feeding.

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.

The ERK/MAPK pathway regulates the activity of the human tissue factor pathway inhibitor-2 promoter.

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5'-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions -89 and -384, whereas the minimal inducible promoter activity is between -89 and -222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position -156 (inducible activity) and a Sp1 site at position -134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2.

Chronic left heart catheterization for microvascular blood flow determination in the rabbit: a minimally invasive technique using specially designed port devices.

BACKGROUND: This study describes a modified catheterization technique with subcutaneously implanted port catheters to be inserted in a retrograde manner across the aortic valve into the left heart ventricle through the right carotid artery to measure organ perfusion. MATERIALS AND METHODS: The specially designed arterial port catheters were implanted in New Zealand rabbits (n = 11, 3.7 +/- 0.1 kg [mean +/- SEM]) under iv anesthesia (medetomidine/ketamine) and single-shot perioperative antibiotic therapy. Hemodynamics were registered continuously during the operation via an ear artery catheter. RESULTS: Implantation of ports was performed in all animals (11/11) without major complications (mean operation time: 70 +/- 3 min). We did not observe catheter-associated arrhythmia, fall in mean arterial pressure (MAP before and post OP: 70 +/- 2 and 68 +/- 2 Torr, respectively), or change in arterial oxygen saturation (SaO2 before and post OP: 89 +/- 3 and 95 +/- 2%, respectively). With a specifically modified microsurgical insertion technique, cerebral blood supply was effectively preserved as evidenced from postmortem histological examinations, cerebral blood flow determination with fluorescent microspheres, and measurement of S-100b protein serum concentrations, a specific marker of neuronal damage. The positioning of the catheter tip in the left ventricle was found to be correct in 10/11 animals. CONCLUSIONS: Repeated and atraumatic microsphere injections into the left ventricle have become feasible by transcutaneous puncture of subcutaneous port systems over several weeks under light sedation. Hence, this new approach (i) avoids the necessity of repeated intracardiac injections and port insertions via thoracotomy, thus reducing the perioperative stress for the animals, and (ii) allows for the first time minimally invasive repetitive and chronic measurements of regional organ blood flow under various experimental settings.