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For a half-century, the commercial wild silkworm, Antheraea pernyi, has been protected by coumaphos, which is an internal organophosphorus insecticide used to kill the potential parasitic fly larvae inside. Knowledge about the detoxification genes of A. pernyi as well as the detoxification mechanism for this species remains severely limited. In this study, we identified 281 detoxification genes (32 GSTs, 48 ABCs, 104 CYPs, and 97 COEs) in the genome of this insect, which are unevenly distributed over 46 chromosomes. When compared to the domesticated silkworm, Bombyx mori, a lepidopteran model species, A. pernyi has a similar number of ABCs, but a greater number of GSTs, CYPs, and COEs. By transcriptome-based expression analysis, we found that coumaphos at a safe concentration level significantly changed the pathways related to ATPase complex function and the transporter complex in A. pernyi. KEGG functional enrichment analysis indicated that protein processing in the endoplasmic reticulum was the most affected pathway after coumaphos treatment. Finally, we identified four significantly up-regulated detoxification genes (ABCB1, ABCB3, ABCG11, and ae43) and one significantly down-regulated detoxification gene (CYP6AE9) in response to coumaphos treatment, suggesting that these five genes may contribute to detoxification of coumaphos in A. pernyi. Our study provides the first set of detoxification genes for wild silkworms from Saturniidae and highlights the importance of detoxification gene repertoire in insect pesticide tolerance.

In the present work, reusable magnetic molecularly imprinted polymers have been used for the first time as an adsorbent for the determination of chlorferon. Magnetic molecularly imprinted polymers have been used for the selective separation of chlorferon from the solution and sensitive fluorimetric determination has been performed using the native fluorescence of chlorferon. The developed method is rapid and determination of chlorferon was completed in an hour. Limit of detection (LOD) of the method was found to be 0.0027 mu M and the linearity of the calibration graph was observed within the range of 0.01-0.36 mu M (2.5 - 75 mu g L-1). Adsorption isotherms point out the multilayer adsorption and the heterogeneous distribution of binding sites for imprinted polymer and homogeneous binding site for non-imprinted polymer. The adsorption capacities of imprinted polymer and non-imprinted polymer were calculated as 2.03 mu mol g(-1) and 0.96 mu mol g(-1), respectively and the imprinting factor was found to be 2.11. The interference effects of some organic compounds and characterization studies were also evaluated. The method has been applied to honey and tap water samples and the recoveries were found to be in the range of 91.3 and 101.1 %., 18-EGEMATAL-003, 18-EGEMATAL-003.

The resistance status against deltamethrin, cypermethrin, coumaphos and ivermectin was assessed of Rhipicephalus microplus from five districts of Uttarakhand, through adult immersion test and larval packet test. The field isolates were highly resistant to deltamethrin (median resistance ratio [RR50] = 9.10-29.13-fold) followed by cypermethrin (2.23-3.55). Surprisingly the isolates were susceptible to coumaphos (0.34-3.17). Emerging resistance against ivermectin (1.55-3.27) was also observed in all the isolates. Elevated levels of esterases (enzyme ratio = 2.93-5.84-fold), glutathione S-transferases (5.10-10.06) and monooxygenases (1.68-4.02) in resistant fields isolates were highly correlated (47.4-86.0%) with the resistant factor (RR50) determined by bioassay. All the isolates except Uttarkashi possess mutation at the 190th position in domain II of the sodium channel gene. As a mitigation strategy an Ageratum conyzoides-based characterized natural formulation was tested against all the isolates and was found effective at the concentration of 10.1-11.5%. The possibility of using the natural formulation for the management of multi-acaricide resistant ticks is discussed.

Lactic acid bacteria (LAB) naturally inhabiting the digestive tract of honeybees are known for their ability to detoxify xenobiotics. The effect of chlorpyrifos, coumaphos, and imidacloprid on the growth of LAB strains was tested. All strains showed high resistance to these insecticides. Subsequently, the insecticide binding ability of LAB was investigated. Coumaphos and chlorpyrifos were bound to the greatest extent (up to approx. 64%), and imidacloprid to a much weaker extent (up to approx. 36%). The insecticides were detected in extra- and intracellular extracts of the bacterial cell wall. The ability of selected LAB to reduce the cyto- and genotoxicity of insecticides was tested on two normal (ovarian insect Sf-9 and rat intestinal IEC-6) cell lines and one cancer (human intestinal Caco-2) cell line. All strains exhibited various levels of reduction in the cyto- and genotoxicity of tested insecticides. It seems that coumaphos was detoxified most potently. The detoxification abilities depended on the insecticide, LAB strain, and cell line. The detoxification of insecticides in the organisms of honeybees may reduce the likelihood of the penetration of these toxins into honeybee products consumed by humans and may contribute to the improvement of the condition in apiaries and honeybee health.

Rhipicephalus microplus tick is the ectoparasite causing the greatest economic losses in the livestock industry. Multi-resistance in ticks is increasing, generating the inefficiency of traditional ixodicides, for which biological control has been proposed as an alternative. In this work, we analyze the histomorphological damage caused by the bacterial strain EC-35 on Rhipicephalus microplus. The ixodicidal effect of EC-35 total protein was evaluated on larval or adult ticks comparing with the commercial ixodicide coumaphos 0.02% as a control. Female ticks were processed using the paraffin-embedding technique and stained with hematoxylin-eosin. Also, the pathogenicity of EC-35 was evaluated by capillary feeding and coelom inoculation tests. The identification of the bacterium was performed using the molecular markers 16S RNA and rpoB, by PCR and sequencing technique, and the evolutionary distance was analyzed by Bayesian phylogenetic inference. No differences were observed in the perimeter and area of larvae treated with EC-35 or Coumaphos. The thickness of the integument decreased a 65% with the EC-35 treatment (6.01 ± 0.6 µm) and of 30% in coumaphos (12.04 ± 1.2 µm) in larvae compared with the control group (18.41 ± 2 µm), while no difference was found in adult ticks. The capillary feeding test and coelom inoculation with EC-35 showed an inhibition of reproductive potential of 99.8 ± 7 and an oviposition Inhibition 97 ± 3.02%. The EC-35 strain was genetically related to Serratia marcescens, concluding that these bacteria caused high mortality, oviposition Inhibition, and integument thinning and drastic loss of histoarchitecture in R. microplus tick larvae.

Honey bee populations are declining as occurs with other pollinators. One suggested cause of this decline is the impact of pesticides. To improve bees’ health, pesticides and food sources may be monitored using sentinel hives, given that bees forage in a 2.5 km radius around the hive. We extracted 20 (twenty) bees, as well as samples of wax, honey and pollen from a sentinel hive. Six pesticides were detected in the samples, except for the honey. All detected pesticides in the sentinel hive are pro- hibited in Argentina. Eight different plant families and genera were detected in the honey and pollen samples. Our work suggests that monitoring pesticides with sentinel beehives will be useful to improve agricultural practices in the region.

A survey on concentrations of veterinary drug residues amitraz, coumaphos and thymol in honey, produced in year 2020 by Slovenian beekeepers, was conducted. 100 samples were analysed: 22 from organic and 78 from conventional production, with two analytical methods. In method for determination of coumaphos and thymol samples were extracted with acetone, petroleumether and dichlorometane. In method for determination of amitraz and its degradation products, samples were hydrolisated with HCl and NaOH, extractied with n-hexane and derivatisated with heptafluorobutyric anhydride. Determination in both methods was performed with gas chromatograph coupled with mass spectrometer. Measured concentrations of amitraz, coumaphos and thymol were in the range of 0.01-0.12 mg kg-1, 0.02-0.06 mg kg-1 and 0.08-0.17 mg kg-1, respectively. In 61 % of samples analysed no residues of amitraz, thymol and coumaphos were found. Data obtained were compared with the data from literature. Chronic and acute exposure were calculated for consumers. Maximum chronic exposure for amitraz and thymol was 0.1 % and 0.05 % of acceptable daily intake, respectively. Maximum acute exposure for amitraz and thymol was 4 % and 0.8 % of acute reference dose, respectively

Honey bee queen health is crucial for colony health and productivity, and pesticides have been previously associated with queen loss and premature supersedure. Prior research has investigated the effects of indirect pesticide exposure on queens via workers, as well as direct effects on queens during development. However, as adults, queens are in constant contact with wax as they walk on comb and lay eggs; therefore, direct pesticide contact with adult queens is a relevant but seldom investigated exposure route. Here, we conducted laboratory and field experiments to investigate the impacts of topical pesticide exposure on adult queens. We tested six pesticides commonly found in wax: coumaphos, tau-fluvalinate, atrazine, 2,4-DMPF, chlorpyriphos, chlorothalonil, and a cocktail of all six, each administered at 1, 4, 8, 16, and 32 times the concentrations typically found in wax. We found no effect of any treatment on queen mass, sperm viability, or fat body protein expression. In a field trial testing queen topical exposure of a pesticide cocktail, we found no impact on egg-laying pattern, queen mass, emergence mass of daughter workers, and no proteins in the spermathecal fluid were differentially expressed. These experiments consistently show that pesticides commonly found in wax have no direct impact on queen performance, reproduction, or quality metrics at the doses tested. We suggest that previously reported associations between high levels of pesticide residues in wax and queen failure are most likely driven by indirect effects of worker exposure (either through wax or other hive products) on queen care or queen perception.

Hyalomma dromedarii ticks collected from a camel herd of Hanumangarh district in the Rajasthan State of India were subjected to FAO recommended adult immersion test with discriminating dose (AIT-DD) against amitraz (formamidines), deltamethrin (synthetic pyrethroids) and coumaphos (organophosphate). The country-specific data generated using the IVRI-I line (deltamethrin (59.2 ppm), coumaphos (262 ppm) and amitraz (975.40 ppm)) was taken as discriminating dose for concerned acaricide since the reference susceptible H. dromedarii ticks line is not maintained anywhere in world. Standard FAO protocol for the rapid detection of acaricide resistance was followed. The percent resistance against deltamethrin, coumaphos and amitraz were found to be 26.67%, 46.67% and 0% respectively. Results revealed a high susceptibility of these ticks against amitraz, resulting in 100% mortality at discriminating concentration. Esterase characterization using Native PAGE of Hyalomma dromedarii homogenate reveals a total of 7 esterase bands, viz. Est-1 h-5 h, Est-7 h and Est-8 h in larva and only 5 esterase bands viz. Est-1 h, Est-3 h-4 h, Est-6 h and Est-7 h in adult. Inhibition assay using inhibitors (copper sulphate, p-chloromercuribenzoate, eserine sulphate, phenylmethylsulfonylfluoride) against esterase of H. dromedarii ticks confirms the presence of serine residues in their active site and provide evidence for the presence of Est-7 and Est-8 in larvae as acetylcholinesterases (AChEs) in larvae. It is the first attempt of biochemical characterization of esterases present in H. dromedarii ticks and could be in future related with development of resistance against organophosphates and synthetic pyrethroids compounds. The high susceptibility status to formamidines drugs may be attributed to low selection pressure as they are still preferred next to synthetic pyrethroids in veterinary practices. It also indicates an alternative tick control measure needs to be adopted in camels to extenuate the development of acaricide resistance.

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., A national survey shows that several beekeeping‐associated pesticides remain in recycled beeswax for many years even when the corresponding products are no longer used. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Veterinary biocides used in animal husbandry have the potential to cause human health concerns. Biocidal products for veterinary use, which contain pesticides approved in Korea, comprise 49 active ingredients within 234 products. Within 17 of these products there are 3 ingredients which are highly hazardous pesticides: coumaphos, dichlorvos and methomyl. In this study, the content of the active ingredients of 160 products sold domestically was investigated. Samples were collected for 119 biocidal products for veterinary use. These were analysed by high‐performance liquid chromatography (HPLC) and gas chromatography (GC). Seventeen products were noncompliant (insufficient or excess quantity of active ingredients). The ingredients that were below the stated concentrations were amitraz, chlorpyrifos‐methyl, cypermethrin, cyromazine, dichlorvos, fipronil, muscamone and trichlorfon. The ingredients that exceeded the stated concentrations were abamectin, fluvalinate and pyriproxyfen. The noncompliance rate in biocidal products for veterinary use was 9.19%. The results of this study show that three highly hazardous pesticides (coumaphos, dichlorvos and methomyl) and 10 active ingredients (abamectin, amitraz, chlorpyrifos‐methyl, cypermethrin, cyromazine, fipronil, fluvalinate, muscamone, pyriproxyfen and trichlorfon) deviated from the stated concentrations. Thus, management plans should be established to ensure compliant veterinary drugs by post‐distribution quality control, such as planning for regular inspection., Three highly hazardous pesticides (coumaphos, dichlorvos and methomyl) and 10 active ingredients (abamectin, amitraz, chlorpyrifos‐methyl, cypermethrin, cyromazine, fipronil, fluvalinate, muscamone, pyriproxyfen and trichlorfon) deviated from the stated concentrations. Thus, management plans should be established to distribute compliant veterinary drugs by post‐distribution quality control, such as planning inspection.

Here, a molecular imprinting technique was employed to create an SPR-based nanosensor for the selective and sensitive detection of organophosphate-based coumaphos, a toxic insecticide/veterinary drug often used. To achieve this, UV polymerization was used to create polymeric nanofilms using N-methacryloyl-l-cysteine methyl ester, ethylene glycol dimethacrylate, and 2-hydroxyethyl methacrylate, which are functional monomers, cross-linkers, and hydrophilicity enabling agents, respectively. Several methods, including scanning electron microscopy (SEM), atomic force microscopy (AFM), and contact angle (CA) analyses, were used to characterize the nanofilms. Using coumaphos-imprinted SPR (CIP-SPR) and non-imprinted SPR (NIP-SPR) nanosensor chips, the kinetic evaluations of coumaphos sensing were investigated. The created CIP-SPR nanosensor demonstrated high selectivity to the coumaphos molecule compared to similar competitor molecules, including diazinon, pirimiphos-methyl, pyridaphenthion, phosalone, N-2,4(dimethylphenyl) formamide, 2,4-dimethylaniline, dimethoate, and phosmet. Additionally, there is a magnificent linear relationship for the concentration range of 0.1–250 ppb, with a low limit of detection (LOD) and limit of quantification (LOQ) of 0.001 and 0.003 ppb, respectively, and a high imprinting factor (I.F.4.4) for coumaphos. The Langmuir adsorption model is the best appropriate thermodynamic approach for the nanosensor. Intraday trials were performed three times with five repetitions to statistically evaluate the CIP-SPR nanosensor’s reusability. Reusability investigations for the two weeks of interday analyses also indicated the three-dimensional stability of the CIP-SPR nanosensor. The remarkable reusability and reproducibility of the procedure are indicated by an RSD% result of less than 1.5. Therefore, it has been determined that the generated CIP-SPR nanosensors are highly selective, rapidly responsive, simple to use, reusable, and sensitive for coumaphos detection in an aqueous solution. An amino acid, which was used to detect coumaphos, included a CIP-SPR nanosensor manufactured without complicated coupling methods and labelling processes. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) studies was performed for the validation studies of the SPR.

A novel fluorescent sensor was developed in this study based on glutathione-functionalized graphene quantum dots (GQDs@GSH) to detect organophosphorus pesticide residues in Radix Angelica Sinensis. GQDs@GSH was synthesized by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.9% and its structure was characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. GQDs@GSH exhibited excellent fluorescence property showing strong blue fluorescence under UV irradiation. The fluorescence of GQDs@GSH could be quenched by Fe

Most honey bee (Apis mellifera Linnaeus, 1758) (Hymenoptera: Apidae) colonies in the United States have been exposed to the beekeeper-applied miticides amitraz, coumaphos, and tau-fluvalinate. Colonies are also often exposed to agrochemicals, which bees encounter on foraging trips. These and other lipophilic pesticides bind to the beeswax matrix of comb, exposing developing bees. We explored whether queen-rearing beeswax containing pesticides affects the reproductive health of mated queens. We predicted that queens reared in pesticide-free beeswax would have higher mating frequencies and sperm viability of stored sperm compared with queens reared in wax containing pesticides. Mating frequency and sperm viability are two traditional measurements associated with queen reproductive health. To test these hypotheses, we reared queens in beeswax-coated cups that were pesticide free or contained field-relevant concentrations of 1) amitraz, 2) a combination of tau-fluvalinate and coumaphos, or 3) a combination of the agrochemicals chlorothalonil and chlorpyrifos. We then collected queens once they mated to determine sperm viability, using a dual fluorescent cell counter, and mating frequency, genotyping immature worker offspring at eight polymorphic microsatellite loci. Sperm viability did not differ between control queens and those reared in pesticide-laden wax. However, queens exposed to amitraz during development exhibited higher mating frequency than queens reared in pesticide-free beeswax or beeswax containing the other pesticide combinations. Our results suggest that miticide exposure during development affects queen mating frequency but not sperm viability, at least in newly mated queens. This finding, which has practical implications for commercial queen rearing and overall colony health, calls for further study.

There is mounting evidence that acaricides are among the most prevalent medicinal compounds in honey bee hive matrices worldwide. According to OCDE guideline No. 245 chronic lethal concentration of tau-fluvalinate (at concentrations ranging from 77.5 to 523.18 ppm), coumaphos (59.8 ppm) and dimethoate (0.7 ppm) were determined. The activity of the biomarkers acetylcholinesterase (AChE), carboxylesterase (CbE), glutathione S-transferase (GST), catalase (CAT) and malondialdehyde (MDA) was analysed and as they are implicated in neurotoxicity, biotransformation and antioxidant defences, these values were combined into an integrated biomarker response (IBR). There was enhanced AChE, CAT and GST activity in honey bees exposed to tau-fluvalinate, while dimethoate inhibited AChE activity. Both dimethoate and coumaphos inhibited CbE activity but they enhanced CAT activity and MDA formation. Our results highlight how these biomarkers may serve to reveal honey bee exposure to commonly used acaricides.

11 Pág. Centro de Investigación en Sanidad Animal (CISA), This nationwide monitoring aimed to investigate the prevalence of residues of plant protection products (PPPs) and veterinary medicine products (VMPs) based on random selection of apiaries of Apis mellifera. For a three-year period (2012, 2013 and 2016), this study targeted 306 PPPs, VMPs and other active substances in 442 samples of bee bread honeycomb (BBHC) and 89 samples of honeybees collected from up to 177 apiaries. The results indicate that honeybees were most often exposed to residues of coumaphos, tau-fluvalinate, chlorfenvinphos, and acrinathrin, with a prevalence from a maximum of 98.8% to 49.4% in BBHC samples. Residues of coumaphos, tau-fluvalinate, amitraz (DMF + DMPF), carbendazim and orthophenylphenol were also frequently detected, from a maximum of 55.1% to 13.5% of the honeybee samples. Neonicotinoid residues, namely clothianidin and thiamethoxam, whose outdoor uses in crops are completely banned in EU, were not detected. Imidacloprid was found in 3.4% to 13.3% of samples during 2013 and 2016, respectively. Imidacloprid exceeded its acute toxicity (LD50) value for honey bees in two samples of BBHC. Fipronil was detected in 0.5% of the samples during 2013. The diversity of active substances found (% of different residues analyzed) ranged from 33.9% to 37.2% in BBHC from 2012, 2013 to 2016, and was of 26.5% in honeybees in 2016. In at least 54% of the samples, the total residue load was in the range of 200 to 1500 μg·kg-1. Up to 50% of BBHC samples were positive for one or two residues. No toxic residues for honeybees were detected in up to 88.8% of bee samples. This systematic surveillance of active substances assisted the evaluation of which target pesticides to look for and provided support to the competent authorities in the bee health decision-making., The authors acknowledge the voluntary participation of all the beekeepers involved in the program, the work carried out by the official veterinary services of all the participating Autonomous Communities and their bee inspectors in taking samples. Special mention to the work team of the Ministry of Agriculture, Fisheries and Food in the coordination of the surveillance program integrated by the General Subdirectorate of Animal Health and Hygiene and Traceability, the Department of Bee and Fish Diseases of the Central Veterinary Laboratory of Algete and the Agroalimentary Arbitration Laboratory of Aravaca.

16 Pàg., This work was supported by grants S2013/ABI-2906 (PLATESA), P2018/BAA-4370 (PLATESA2) from Comunidad de Madrid/FEDER and AGL2017-83326-R from Ministerio de Economía y Competitividad and PdC2021-121717-I00 funded by MCIN/AEI /10.13039/501100011033 and by Unión Europea Next Generation EU/ PRTR. AL-V has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovación y Universidades from the Spanish government.

During the 4 years of the project, samples were collected in different parts of Croatia. In the project, samples of honey, olive oil, meat, cheese, soil, and water were collected, while samples of honey, olive oil, meat, cheese and soil were analysed for pesticide residues. For the preparation of samples, the QuEChERS method was utilized for the honey and olive oil samples and a modified version for the soil samples. A gas chromatograph-tandem mass spectrometer (GC-MS/ MS) Shimadzu TQ-8050NX and a liquid chromatograph tandem mass spectrometer (LC-MS/MS) Agilent 1260 Infinity II were used to conduct analyses of pesticide residue. Over 600 active chemicals were examined in a combined 150 honey samples, 200 olive oil samples, and 200 soil samples. It was discovered that certain honey samples contain the unapproved chemical coumaphos while pesticide residues were being monitored. Coumaphos tends to gather in wax, where it stays for a while and then gradually transitions into honey. The oil samples contained a variety of pesticide residues, some of which are illegal to use in the EU (such as chlorpyrifos) and others whose concentrations are over the Maximum Residue Level (MRL). Farmers’ continued usage of outdated supplies is tied to the use of prohibited plant protection products. Farmers are able to buy and use pesticides that have not been approved in Croatia’s neighbouring countries. Pesticide residual levels in all soil samples were below the quantifiable limit (LOQ). The results of this research point to the need for increased monitoring and control of food products for pesticide residues. Acknowledgements: This work was carried out within the project “Food Safety and Quality Center” (KK.01.1.1.02.0004). The project is co-financed by the European Union from the European Regional Development Fund.

Varroosis is the disease caused by the ectoparasitic mite Varroa destructor, one of the most destructive diseases of honeybees. In Spain, there is great concern because there are many therapeutic failures after acaricide treatments intended to control varroosis outbreaks. In some of these cases it is not clear whether such failures are due to the evolution of resistance. Therefore, it is of high interest the development of methodologies to test the level of resistance in mite populations. In this work, a simple bioassay methodology was used to test whether some reports on low efficacy in different regions of Spain were in fact related to reduced Varroa sensitivity to the most used acaricides. This bioassay proved to be very effective in evaluating the presence of mites that survive after being exposed to acaricides. In the samples tested, the mortality caused by coumaphos ranged from 2 to 89%; for tau-fluvalinate, it ranged from 5 to 96%. On the other hand, amitraz caused 100% mortality in all cases. These results suggest the presence of Varroa resistant to coumaphos and fluvalinate in most of the apiaries sampled, even in those where these active ingredients were not used in the last years. The bioassay technique presented here, either alone or in combination with other molecular tools, could be useful in detecting mite populations with different sensitivity to acaricides, which is of vital interest in selecting the best management and/or acaricide strategy to control the parasite in apiaries.

International audience; AbstractAcaricides commonly used to control the honey bee parasitic mite, Varroa destructor, may also adversely affect bees. Sublethal LD05 doses of synthetic (tau-fluvalinate, amitraz, and coumaphos) and natural (thymol and formic acid) acaricides did not significantly reduce bee survivorship. However, compared to the ethanol solvent control, hygienic behavior critical for pathogen and parasite control was significantly reduced with coumaphos, whereas both pollen and non-pollen foraging behaviors critical for resource acquisition were reduced with tau-fluvalinate, coumaphos, and formic acid. Thymol significantly reduced non-pollen foraging but also reduced the negative effects of ethanol on hygienic behavior. Amitraz did not affect hygienic and foraging behaviors relative to the solvent. Thymol and amitraz appeared to be the safest acaricides based on these tests.

In this study, the photodegradation of organophosphorus (OPs) pesticides in the honey medium was evaluated under sunlight irradiation. Some of the 22 samples collected at different sites contained OPs pesticides (Methyl parathion, Coumaphos and Fenitrothion) with an average of 8 ng/g. Moreover, three samples were found with pesticide residue levels exceeding the maximum residue limits (MRL ≥ 50 ng/g) imposed by the standard water (WHO). Gas chromatography (GC) combined with a tritium electron capture detector system was used for the analysis of OPs pesticides in honey. Total degradation of the Methyl parathion was obtained in less than 60 min of irradiation. Moreover, the elimination of the other OPs found in the samples was also effective with a rate of 85% for Coumaphos and Fenitrothion after 50 min of sunlight irradiation. The kinetics of the photodegradation reaction of all OPs pesticides studied followed a pseudo-first order model.

Self-poisoning with organophosphorus (OP) insecticides is an important means of global self-harm. The insecticides are formulated with solvents that may also contribute to toxicity. We set up a study to detect changes in osmolal and anion gaps following ingestion of OP insecticides. We recruited consecutive patients admitted to a Teaching Hospital, Sri Lanka, with a history of OP self-poisoning. The osmolal and anion gaps were calculated on admission and at 4, 24 and 72 h post-ingestion together with ethanol concentration. Forty-nine patients were recruited (28 profenofos, 10 diazinon, one coumaphos, one chlorpyrifos, one phenthoate and eight unknown OP). Only modest increases in osmolal and anion gaps were noted. Small rises in osmolal gap above the upper limit of normal were noted in 16/49 (32.7%) of all cases, 9/28 (32.1%) profenofos cases and 4/10 (40.0%) diazinon cases. The anion gap was raised in 24/49 (49.0%) of all cases, 15/28 (53.6%) profenofos cases and 5/10 (50.0%) diazinon cases. We observed a trend for a fall in osmolal gap during the first 24 h, followed by an increase up to 72 h. There was no correlation between the anion gap and serum lactate concentration, indicating that a lactic acidosis was not responsible for the anion gap. Formate, which could have explained the increased gap, was not detected in any of the samples; ketoacids (beta-hydroxybutyrate and acetoacetate) were not measured. This pilot study found that profenofos and diazinon poisoning caused only modest increases in the osmolal and anion gaps in a minority of cases.

Thymol has been considered as an organic varroacide with less toxicity than synthetic chemicals such as amitraz, coumaphos or tau-fluvalinate. The present study evaluates the presence, persistence and distribution of thymol during a five-month sampling period; before, during and after the application of thymol treatment in field conditions. Moreover, its impact on the development of bee brood was also studied at different development stages which consist of larvae, prepupae, pupae and adult honeybee. Samples of beebread (processed pollen), honey, beeswax, adult bees, pupae, prepupae and larvae were analysed by gas chromatography coupled to time of flight mass spectrometry for an accurate identification. Adult bees were the samples with the highest concentration level of thymol during treatment application (Thymovar® strips, 30 gr of thymol per hive) by the beekeeper, however they showed 69% of thymol dissipation three months after treatment. Regarding the bee brood, concentrations of thymol decreased significantly (89-97% of disappearance) in a period of three months, though about 42 µg/kg of thymol persisted in larvae. Thymol was found to accumulate in honey and beeswax at a level of 882 μg/kg and 1305 μg/kg respectively, in the studied period. As the main issue of thymol residues in honey is related to the modification of organoleptic properties, more than toxicology, the levels found in this study indicate that the taste of honey may be affected during and up to three months after the treatment application. In addition, we did not find adverse effects on the evolution of the colonies due to the applied treatment.

Lactic acid bacteria (LAB) naturally inhabiting the digestive tract of honeybees are known for their ability to detoxify xenobiotics. The effect of chlorpyrifos, coumaphos, and imidacloprid on the growth of LAB strains was tested. All strains showed high resistance to these insecticides. Subsequently, the insecticide binding ability of LAB was investigated. Coumaphos and chlorpyrifos were bound to the greatest extent (up to approx. 64%), and imidacloprid to a much weaker extent (up to approx. 36%). The insecticides were detected in extra- and intracellular extracts of the bacterial cell wall. The ability of selected LAB to reduce the cyto- and genotoxicity of insecticides was tested on two normal (ovarian insect Sf-9 and rat intestinal IEC-6) cell lines and one cancer (human intestinal Caco-2) cell line. All strains exhibited various levels of reduction in the cyto- and genotoxicity of tested insecticides. It seems that coumaphos was detoxified most potently. The detoxification abilities depended on the insecticide, LAB strain, and cell line. The detoxification of insecticides in the organisms of honeybees may reduce the likelihood of the penetration of these toxins into honeybee products consumed by humans and may contribute to the improvement of the condition in apiaries and honeybee health.

Beekeepers need new registered products to control the small hive beetle (SHB), Aethina tumida, a significant pest of western honey bee (Apis mellifera) colonies. Few approved chemical controls exist, and there is no standardized system for screening compounds against SHBs. We outline a detailed method for an acute toxicity bioassay that delivers compounds via pollen. We provide a detailed method for a field trial that delivers treated pollen in traps made from modified compact disc cases. We applied both methods in proof-of-concept experiments to assess acetamiprid as a SHB control agent. Using the laboratory bioassay, we found acetamiprid (LC50 = 20.5 µg/g) to be more toxic to SHBs than coumaphos was (LC50 = 1250 µg/g), yet less toxic to SHBs than fipronil was (LC50 = 1.78 µg/g). In our field trial, colonies treated with acetamiprid and fipronil significantly reduced (p < 0.001) live SHB populations over those of control colonies. Traps containing acetamiprid retained significantly higher (p < 0.001) numbers of dead SHBs than did traps containing fipronil. We outline the first detailed methods to assess the toxicity of compounds delivered in pollen for adult SHB control. Our proof-of-concept experiments showed acetamiprid to be a promising control agent for SHBs.

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. Graphical abstract

In this case report, we analyze the possible causes of the poor health status of a professional Apis mellifera iberiensis apiary located in Gajanejos (Guadalajara, Spain). Several factors that potentially favor colony collapse were identified, including Nosema ceranae infection, alone or in combination with other factors (e.g., BQCV and DWV infection), and the accumulation of acaricides commonly used to control Varroa destructor in the beebread (coumaphos and tau-fluvalinate). Based on the levels of residues, the average toxic unit estimated for the apiary suggests a possible increase in vulnerability to infection by N. ceranae due to the presence of high levels of acaricides and the unusual climatic conditions of the year of the collapse event. These data highlight the importance of evaluating these factors in future monitoring programs, as well as the need to adopt adequate preventive measures as part of national and international welfare programs aimed at guaranteeing the health and fitness of bees., This research was funded by INIA-FEDER (RTA2017-00004-C02)

The chemical-based tick management method is gradually losing its clutch due to the establishment of resistant ticks. For development of region-specific tick management strategies, the present study was aimed to evaluate the comparative resistance profile of Rhipicephalus microplus isolates collected from seven districts of Uttar Pradesh, a northern state of India. Comparative analysis of the dose–response data using adult immersion test (AIT) against coumaphos, malathion, deltamethrin, ivermectin, and fipronil revealed that all the isolates were resistant to discriminating concentration of deltamethrin having LC50 of 295.12–436.52 ppm with a resistance ratio of 22.02–32.58. An emerging low level of ivermectin resistance (resistance ratio, RR50 = 1.03–2.26) with LC50 in the range of 22.39–48.98 ppm was found across the isolates. The coumaphos was highly effective against all except Amethi (AMT) isolate. Similarly, malathion was efficacious against most of the isolates except Pratapgarh (PRT) and Sultanpur (SUL) isolates showing LC50 of 5128.61 and 5623.41 ppm, respectively. All the isolates were responsive to fipronil. Comparative detoxifying enzymes profiles revealed a significant correlation between the increased activity of esterase and deltamethrin resistance. The GST activity was 51.2% correlated with RR50 of malathion while esterase activity was significantly correlated (68.9%) with RR50 of coumaphos. No correlation between the ivermectin resistance and enzyme activity was established. Multiple sequence analysis of S4-5 linker region of the sodium channel gene of all the isolates revealed a point mutation at 190th position (C190A) which is associated with deltamethrin resistance. The possible tick management strategies in this part of the country are discussed.

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.

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

Honey bee queen health is crucial for colony health and productivity, and pesticides have been previously associated with queen loss and premature supersedure. Prior research has investigated the effects of indirect pesticide exposure on queens via workers, as well as direct effects on queens during development. However, as adults, queens are in constant contact with wax as they walk on comb and lay eggs; therefore, direct pesticide contact with adult queens is a relevant but seldom investigated exposure route. Here, we conducted laboratory and field experiments to investigate the impacts of topical pesticide exposure on adult queens. We tested dose-response relationships of six pesticides commonly found in wax: coumaphos, tau-fluvalinate, atrazine, 2,4-DMPF, chlorpyriphos, chlorothalonil, and a cocktail of all six, each dosed up to 32 times the concentrations typically found in wax. We found no effect of any treatment on queen mass or sperm viability. Furthermore, none of the 1,568 proteins quantified in the queens’ fat bodies (a major site of detoxification enzyme production) were differentially expressed. In a field trial with N = 30 queens exposed to either a handling control, a solvent control, or a pesticide cocktail, we again found no impact on queen egg-laying pattern, mass, or emergence mass of daughter workers. Further, of the 3,127 proteins identified in fluid from the spermatheca (sperm storage organ), none were differentially expressed. These experiments consistently show that at realistic exposure levels, pesticides commonly found in wax have no direct impact on queen performance, reproduction, or quality metrics. We suggest that previously reported associations between high levels of pesticide residues in wax and queen failure are most likely driven by indirect effects of worker exposure (either through wax or other hive products) on queen care or queen perception.

Producción Científica, Pesticides can be found in beehives for several reasons, including contamination from surrounding cultivars; yet one of the most pertinent is related to the fact that beekeepers employ acaricides to control various types of mites, which may accumulate in beeswax due to their lipophilic nature. In the present study, foundation sheets of different origins, collected over a period of three years, were analyzed to detect the residues of seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, alpha-endosulfan, bromopropylate, coumaphos, tau-fluvalinate) by gas chromatography with mass spectrometric detection. An efficient sample treatment (recoveries between 90% and 108%) is proposed, involving solvent extraction with 1% acetic acid in acetonitrile mixture followed by dispersive solid-phase extraction (enhanced matrix removal lipid) and a polishing step. An evaluation was made of the analytical performance of the proposed method. It was shown to be selective, linear from a limit of quantification to 5000 µg/kg, precise (relative standard deviation values were below 6%), and with a goo sensitivity (limit of quantification ranging from 5 to 10 µg/kg). Finally, results showed that a large majority of the sheets analyzed (>90%) contained residues of at least one of these compounds. Coumaphos and tau-fluvalinate residues were the most common, with chlorpyrifos and chlorfenvinphos detected to a lesser extent., Plan Nacional de Investigación Científica y Técnica y Innovación 2013–2016, Instituto Nacional de Investigación y Tecnología Agroalimentaria (INIA) y Fondo Europeo de Desarrollo Regional (FEDER) - (grant RTA2017-00004-C02-01 and 02)

An apiary trial was conducted in 2016 August to October in Szabolcs-Szatmár-Bereg County, Nyírmada to evaluate the influence of queen’s age on the Varroa destructor-burden in the treatment colonies. Sixty colonies of bees belonging to the subspecies Apis mellifera carnica pannonica in Hunor loading hives (with 10 frames in the brood chamber/deep super) were used. The colonies were treated with amitraz and the organophosphate pesticide coumaphos active ingredients. The amitraz treatment includes 6 weeks. The coumaphos treatment with Destructor 3.2% can be used for both diagnosis and treatment of Varroasis. For diagnosis, one treatment is sufficient. For control, two treatments at an interval of seven days are required. The colonies were grouped by the age of the queen: 20 colonies with one-year-old, 20 colonies with two-year-old and 20 colonies with three-year-old queen. The mite mortality of different groups was compared. The number of fallen mites was counted at the white bottom boards. The examination of spring growth of honey bee colonies has become necessary due to the judgement of efficiency of closing treatment. The data was recorded seven times between 16th March 2017 and 19th May 2017. Data on fallen mites were subjected to one-way analysis of variance (ANOVA) and Post-Hoc Tukey-test. Statistical analysis was performed using the software of IBM SPSS (version 21.). During the first two weeks after treatments, the number of fallen mites was significantly higher in the older queen’s colonies (Year 2014). The total mite mortality after amitraz treatment in the younger queen’s colonies was lower (P

A polystyrene-supported molybdate-peroxide polymer (Mo-Y(s)) destroys phosphorothioate pesticides of the form (ArO)P(=S)(OEt)2 in EtOH under mild oxidative (H2O2) conditions and produces a commodity organophosphate. This is the first report of a metal-based system that successfully degrades the “live” pesticides parathion, diazinon and coumaphos. In addition to the operational advantages of heterogeneous reaction chemistry, the Mo-Y(s) support degrades multiple equivalents of the pesticide in H2O2(aq). Of particular importance is the predominant production of diethyl phosphite, a commodity chemical, from diazinon degradation over Mo-Y(s) in EtOH; no toxic oxon is found. Coumaphos and parathion produce the corresponding oxon which have ΔHǂ (kcal/mol) of 15.4 (0.5) and 21.7 (0.8), respectively; these activation parameters are consistent with key observations found in the relative amount of coumoxon and paraoxon produced. Finally, a discrete molybdate-peroxide complex is presented as a possible solution model for this heterogeneous reaction.

The main objective of this study was to test comparatively the effects of two common insecticides on honey bee Apis mellifera worker’s lifespan, food consumption, mortality, and expression of antioxidant genes. Newly emerged worker bees were exposed to organophosphate insecticide coumaphos, a neonicotinoid imidacloprid, and their mixtures. Toxicity tests were conducted along with bee midgut immunohistological TUNEL analyses. RT-qPCR assessed the regulation of 10 bee antioxidant genes linked to pesticide toxicity. We tested coumaphos at 92,600 ppb concentration, in combination with 5 and 20 ppb imidacloprid. Coumaphos induced significantly higher bee mortality, which was associated with down regulation of catalase compared to coumaphos and imidacloprid (5/20 ppb) mixtures, whereas, both imidacloprid concentrations independently had no effect on bee mortality. Mixture of coumaphos and imidacloprid reduced daily bee consumption of a control food patty to 10 mg from a coumaphos intake of 14.3 mg and 18.4 and 13.7 mg for imidacloprid (5 and 20) ppb, respectively. While coumaphos and imidacloprid mixtures induced down-regulation of antioxidant genes with noticeable midgut tissue damage, imidacloprid induced intensive gene up-regulations with less midgut apoptosis.

Rhipicephalus microplus is posing a serious threat to productive animal husbandry. Excessive use of synthetic chemicals in tick management has led to the development of resistant tick populations. Characterization of resistance to deltamethrin, cypermethrin, coumaphos and ivermectin in ticks is necessary to develop a suitable and sustainable control strategy. Based on adult immersion test and larval packet test, the resistance ratios (RR50) for adults and larvae of R. microplus populations from two Indian states ranged from 3.8 to 19.4 and 1.35-25.0 against deltamethrin, 0.061-26.3 and 0.22-19.2 against cypermethrin, and 0.2-9.5 and 0.01-3.1 against coumaphos, respectively, were recorded. Moreover, the RR50 for adults ranged from 0.212 to 3.87 against ivermectin. The RR50 for different acaricides was significantly (p

The active substances coumaphos, tau-fluvalinate and amitraz are among the most commonly employed synthetic miticides to control varroa infestations in apiculture. These compounds can persist inside the beehive matrices and can be detected long time after their application. The present study describes the application of a new passive sampling methodology to assess the dissipation of these miticides as well as the cross-contamination in neighboring beehives. The APIStrips are a recently developed sampling device based on the sorbent Tenax, which shows a remarkable versatility for the sorption of molecules onto its surface. This avoids the need of actively sampling apicultural matrices such as living bees, wax or reserves (honey and pollen), therefore allowing to obtain representative information of the contamination in the beehive environment in one single matrix. The results show that the amitraz-based treatments have the fastest dissipation rate (half-life of 11-14 days), whereas tau-fluvalinate and coumaphos remain inside the beehive environment for longer time periods, with a half-life up to 39 days. In the present study, tau-fluvalinate originated an intense cross-contamination, as opposed to coumaphos and amitraz. This study also demonstrates the contribution of drifting forager bees in the pesticide cross-contamination phenomena. Moreover, the sampling of adult living bees has been compared to the APIStrip-based sampling, and the experimental results show that the latter is more effective and consistent than traditional active sampling strategies. The active substances included in this study do not migrate to the honey from the treated colonies in significant amounts.

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa. We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [(14)C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4. RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.

Varroa destructor is an ectoparasitic mite causing devastating damages to honey bee colonies around the world. Its impact is considered a major factor contributing to the significant seasonal losses of colonies recorded every year. Beekeepers are usually relying on a reduced set of acaricides to manage the parasite, usually the pyrethroids tau-fluvalinate or flumethrin, the organophosphate coumaphos and the formamidine amitraz. However, the evolution of resistance in the populations is leading to an unsustainable scenario with almost no alternatives to reach an adequate control of the mite.Here we present the results from the first, large-scale and extensive monitoring of the susceptibility to acaricides in the Comunitat Valenciana, one of the most prominent apicultural regions in Spain. Our ultimate goal was to provide beekeepers with timely information to help them decide what would be the best alternative for a long-term control of the mites in their apiaries. Our data show that there is a significant variation in the expected efficacy of coumaphos and pyrethroids across the region, indicating the presence of a different ratio of resistant individuals to these acaricides in each population. On the other hand, the expected efficacy of amitraz was more consistent, although slightly below the expected efficacy according to the label.HIGHLIGHTSVarroa destructor is causing severe damages to honey bee colonies worldwide.There are very few acaricides available to manage the parasite.The evolution of resistance is limiting our capacity to control the mite.We estimated the expected efficacy of the main acaricides in many Spanish apiaries.The information was shared with beekeepers for them to decide the best treatment to control the mite.

Honey is a nutritionally valuable natural product of honey bees which can be contaminated by anthropogenic chemicals from the surrounding environment and beekeeper practices. Good beekeeping practice in conventional and regulations on organic beekeeping aim to reduce harmful residues in honey for human consumption. We compared prominent metal(loid) contaminant (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) and acaricide (pesticides used for treatment of beehives for Varroa mite control) levels in 40 samples of Croatian chestnut honey (Castanea sativa) from Banovina region (Banski med ; 4 organic, 26 conventional) and Ozalj (2 organic, 8 conventional). Metal(loid)s were quantified using the Agilent 7500cx inductively coupled plasma mass spectrometer and acaricides using the Agilent Technology 1260 HPLC system coupled with a Triple Quad LC/MS 6410 mass spectrometer. All honey samples were safe for consumers regarding contaminant residues. Anti-varroa drug residues were not quantified in certified organic honeys (ORG), while amitraz (sum of parent compound and metabolites ; range: 0.007 ̶ 0.035 mg/kg), DMF (N- (2, 4-dimethylphenyl) formamide ; 0.005 ̶ 0.018 mg/kg) and coumaphos (0.005 ̶ 0.031 mg/kg) were detected in 9, 8 and 25 samples of 34 conventional honeys (CONV), respectively. Potentially toxic Cr was the only metal(loid) that showed a statistically significant difference between ORG and CONV (median 5.74 vs. 1.98 µg/kg, respectively ; U = 31, p = 0.009, Mann-Whitney U test) honey groups. On average, this small scale study found organic chestnut honey less burdened with acaricides and more contaminated with Cr residues than conventional honey.

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a serious threat to the honey bee industry, which relies on chemicals for the control of major honey bee pests. We developed a glass vial bioassay for resistance monitoring of adult A. tumida populations in honey bee colonies. We also determined concentrations that could be used to discriminate between susceptible and resistant strains. These include the pyrethroids cypermethrin (25.0 μg per vial) and fluvalinate (25.0 μg per vial) and the organophosphates malathion (10.0 μg per vial), chlorpyrifos (2.5 μg per vial), and coumaphos (25.0 μg per vial). Here, we report that resistance to fluvalinate and coumaphos was widespread in A. tumida populations in Florida in 2019. Aethina tumida populations were still susceptible to cypermethrin, malathion, and chlorpyrifos. The levels of resistance differed between pyrethroid and organophosphate insecticides. Over the last 10 yr, A. tumida populations have developed 43.7-fold resistance to coumaphos and 5.4-fold to fluvalinate. The levels of insecticide resistance were not similar within insecticides in the same class, which suggest that this type of resistance is manageable. Our results demonstrate the usefulness of glass vial bioassays to detect resistance in adult A. tumida and provide the foundation for a resistance management strategy. To the best of our knowledge, this is the first report of insecticide resistance in small hive beetle populations and suggests an urgent need for alternative control strategies for these serious pests of honey bee colonies.

Quality and safety of food, including honey, is one of the leading priorities regarding residues of anthropogenic chemicals with proven adverse health effects. In total, 61 honey samples of known botanical origin were collected in period 2018–2019 from Croatian registered organic and conventional beekeepers (N = 16 organic and N = 45 conventional honey samples). Eleven trace metal(loid)s (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn; quantitated by ICP-MS), 24 antibiotics (LC-MS/MS, ELISA, microbiological inhibition test), six indicator PCBs (GC-MS/MS) and 121 pesticides (LC-MS/MS, GC-MS/MS) originating from environment and/or beekeeping practice were measured to assess possible differences in contaminant residues between organic and conventional honeys. All honey samples had contaminant residues below the legal maximum levels and are considered safe for consumers. However, 2/16 organic and 34/45 conventional honeys contained one or two synthetic acaricides (most frequently coumaphos), while other pesticides, antibiotics and PCBs were not quantified. Also, organic honey contained lower levels of coumaphos, amitraz and amitraz metabolite N-(2,4-dimethylphenyl) formamide than conventional honeys, on average. Higher levels of Cr (p = 0.006) were detected in organic compared to conventional chestnut honeys. This study pinpointed beehive disease control treatment as prominent pesticide residue source, which was to some extent reduced in organic honeys. Quantified pesticide and metal(loid) levels were within range or lower than reported in recent literature regarding honey of the same botanical origin.

The aim of this study was to investigate reported cases of honeybee mortality incidents and the potential association to pesticide exposure and to their metabolites. The same honeybee samples were also assessed for Varroa mites, and Nosema microsporidia provoked infections to provide an integrated picture of all observable stressors that may impact bees' survival. Thus, honeybee samples from different areas of Greece (2014–2018) were analyzed for the presence of pesticide residues and metabolites. In this context, an existing LC-ESI-QqQ-MS multiresidue method of analytes of different chemical classes such as neonicotinoids, organophosphates, triazoles, carbamates, was enriched with additional active substances, developed and validated. A complementary GC-EI-QqQ-MS method was also exploited for the same scope covering pyrethroid compounds. Both methods monitored more than 150 active substances and metabolites and presented acceptable linearity over the ranges assayed. The calculated recoveries ranged from 65 to 120% for the three concentration levels, while the precision (RSD%) values ranged between 4 and 15%. Therefore, this approach proved sufficient to act as a monitoring tool for the determination of pesticide residues in cases of suspected honeybee poisoning incidents. From the analysis of 320 samples, the presence of 70 active substances and metabolites was confirmed with concentrations varying from 1.4 ng/g to 166 μg/g. Predominant detections were the acaricide coumaphos, several neonicotinoids exemplified by clothianidin, organophosporous compounds dimethoate and chlorpyrifos, and some pyrethroids. Metabolites of imidacloprid, chlorpyrifos, coumaphos, acetamiprid, fenthion and amitraz were also identified. Concerning Nosema and Varroa they were identified in 27 and 22% of samples examined, respectively, verifying their prevalence and coexistence with pesticides and their metabolites in honeybees.

Pollinator diversity and abundance in North America have been at a steep decline over the last two decades due to the combinatorial effects of several environmental and anthropogenic stressors. In particular, managed honey bees (Apis mellifera) face multiple health risks including nutritional stress, exposure to pests and pathogens, poor queen quality, and pesticide contamination, which cause problems at the individual and colony levels. One of the gravest problems faced by honey bees is parasitization by the mite Varroa destructor, which is typically controlled through the application of miticides such as tau-fluvalinate, coumaphos, and amitraz. In addition to miticides, colonies are also exposed to pesticides brought by foragers from agricultural settings, including the fungicide chlorothalonil and the insecticide chlorpyrifos. Here, we explored whether exposure of wax to combinations of these pesticides during development affects honey bee queen physiology and worker behavior. To do this, we reared queens in plastic cups coated with molten beeswax that was either pesticide-free or containing field-relevant concentrations of tau-fluvalinate and coumaphos, amitraz, or chlorothalonil and chlorpyrifos. Once queens mated naturally, we placed them in observation hives to measure egg-laying rate and worker retinue size. We then dissected the queens and used the contents of their mandibular glands to measure worker attractiveness in caged bioassays and to analyze their chemical components using GC-MS. Exposure of wax to field-relevant concentrations of the tested pesticides during queen development significantly lowered the adult queens’ egg-laying rate and worker retinue size. Miticide exposure during development also lowered the attractiveness of queen mandibular gland contents to workers and affected the relative amounts of the glands’ chemical components. Our results support the ideas that mandibular gland pheromones act as honest indicators of queen reproductive fitness and that pesticide exposure of wax during bee development is an important and concerning factor impairing honey bee health. Pollinator health is currently being threatened by multiple environmental factors, including poor nutrition, habitat fragmentation, and pesticide contamination. In particular, honey bee health is facing challenges caused by combinations of these factors, leading to an annual mortality of over 30% of all managed colonies in the USA. The conditions in which honey bee queens are reared can cause severe physiological and behavioral effects in adults, which in turn can affect the behavior of workers that tend to those queens, thus affecting colony productivity, growth, and survival. In this study, we show that queens reared in beeswax contaminated with miticides or agrochemicals lay fewer eggs over time and exhibit mandibular gland pheromone profiles that differ from those of queens reared in pesticide-free wax. Workers discern these differences, preferentially investigating the gland contents and forming larger retinues (thus performing more tending behaviors) for queens reared in pesticide-free wax. These results indicate that pesticide contamination of the queen-rearing wax directly affects queen physiology and behavior and further affirm that queen mandibular pheromones seem to act as honest indicators to workers of a queen’s reproductive capacity and overall health.

We studied the distribution of coumaphos in beeswax after a single application of CheckMite® to 15 bee colonies in the fall according to the manufacturer’s instruction. Immediately after the treatment, residue levels were especially elevated in the wax that came into contact with the CheckMite® strips. During the following spring season, coumaphos levels ranged from 36 to 159 mg/kg in the wax of the brood frames next to the CheckMite® strips, and residue levels were about 10 times lower in the wax of frames that did not come into contact with the strips. Beeswax that was newly constructed 5 months after removing the treatment strips contained up to 7.3 mg/kg of coumaphos, and up to 0.5 and 2.3 mg/kg was detected in the honeycomb and capping wax, respectively. These results suggest that beeswax exposed to CheckMite® should not be recycled in order to prevent elevated concentrations of coumaphos in new foundations and hence to prevent honeybee larvae from being exposed to detrimental residue levels.

Zoonoses are major causes of morbidity and mortality worldwide. Among them, Brazilian Spotted Fever (BSF) is an important one that occurs in some regions of South America and can be transmitted by the “star tick” Amblyomma sculptum. Application of acaricides against the larval stage is important as strategy of population control. However, there is still a deficiency of studies on chemical control of A. sculptum and the present work aims to evaluate the in vitro acaricidal activity of cypermethrin, flumethrin, deltamethrin, fipronil, coumaphos and chlorpyrifos against A. sculptum larvae. Bioassays were performed using the larval immersion test method. A discriminatory analysis between the antiparasitic classes most used for tick control was carried out, which made it possible to determine the classes with higher potential for controlling A. sculptum larvae. Our results showed that A. sculptum larvae present highest sensitivity to the synthetic pyrethroid group, followed by the phenylpyrazole, organophosphate and macrocyclic lactone groups. These findings may support studies on improvement of tick control as in animals as in the environment. Resumo As zoonoses são a maior causa de morbidade de mortalidade no mundo. A Febre Maculosa Brasileira (FMB) é uma importante zoonose que ocorre em algumas regiões da América do Sul e pode ser transmitida pelo “carrapato-estrela” Amblyomma sculptum. A aplicação de acaricidas, frente ao estágio larval, é importante como estratégia no controle da população. No entanto, ainda há uma deficiência de estudos para o controle químico de A. sculptum. Devido à necessidade de mais informações sobre o controle de A. sculptum, o presente trabalho tem como objetivo avaliar a atividade acaricida in vitro de cipermetrina, flumetrina, deltametrina, fipronil, coumafós e clorpirifós frente a larvas de A. sculptum. Os bioensaios foram realizados pelo método Teste de Imersão de Larva. Foi realizada uma análise discriminatória entre as classes antiparasitárias mais utilizadas para controle de carrapatos, possibilitando determinar classes com maior potencial para o controle de larvas de A. sculptum. Os resultados deste trabalho mostraram que as larvas de A. sculptum apresentam maior sensibilidade ao grupo dos piretroides sintéticos, seguido pelos grupos fenilpirazóis, organofosforados e lactonas macrocíclicas. Esses achados poderiam apoiar estudos visando ao controle do carrapato tanto em animais quanto no meio ambiente.

Purpose. To collect information on available on the Ukrainian market veterinary medicinal products to treat honey bee varroosis caused by Varroa destructor Anderson and Trueman, 2000; to systematize the information and compile the list of the veterinary medicinal products taking into account the content of active substances, dosage form and producer. Methods. Analysis of the information obtained in 2015–2020 by surveying beekeepers, direct communication with them, browsing Internet resources, visiting veterinary pharmacies; creation of an information database of anti-Varroa medicinal products. Results. As for September 2020, at least 136 items of veterinary medicinal products to treat varroosis are available on the market in Ukraine, of which 79 are imported. They contain ten active substances (amitraz – in 37 products, fluvalinate – 30, flumethrin – 12, bromopropylate – 5, coumaphos – 2, acrinatrin – 1, thymol – 14, formic acid – 6, lactic acid – 6, oxalic acid – 9), vegetable essential oils and other substances. However, as for February 2020, only 17 anti-Varroa products have been included into the State Register of Veterinary Medicinal Products Permitted for Use in Ukraine. Analogs with the same concentration of the same active substance from different producers have been identified; drugs with the same name but different origins, differing both in the concentration of the active substance and in the dosage form; numerous technical errors in the indication of initial concentrations on the sites of online stores, as well as other risks associated with the dishonesty of sellers. Conclusions. The obtained results stress the need to strengthen control over the market of veterinary medicaments to treat honey bee varroosis in Ukraine.

Apart from the efficiency of coumaphos against Varroa mites, its impact on the oxidative status and survival of the honey bee (Apis mellifera) was assessed. The research was conducted on hives from the same apiary, equalised regarding the number of bees, brood area and food storage. Based on Varroa infestation the hives were allotted to two groups: Non-infested (N) and infested (I). Both groups were either treated (T)-NT and IT, or untreated (U)-NU and IU. The treatment of infested bees was controlled with a follow-up treatment with amitraz. The efficiency of coumaphos was 96-97%. This organophosphate had a negligible effect on bee survival, but it significantly affected their oxidative status: Superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities, and the concentrations of malonyl dialdehyde (MDA). Coumaphos significantly (pË 0.0001) decreased SOD activity in non-infested bees, but increased it in those infested. By contrast, both CAT and GST activities, as well as MDA concentrations significantly increased (from pË 0.05 to pË 0.0001) after treatment in all groups, with the exception of IT, where it declined. Coumaphos in non-infested hives caused oxidative stress per se, not unlike varroa in infested colonies. However, in infested colonies it decreased oxidative stress, owing to its efficacy against Varroa mites and contributed to the recovery of bee colonies. In spite of its certain downsides, coumaphos remains an effective anti-varroa substance, but should be used with precaution, not to add to the effects of environmental factors which may cause red-ox misbalance. Procenjivana je efikasnost kumafosa protiv varoe, kao i njegov uticaj na oksidativni status i preživljavanje medonosne pčele (Apis mellifera). Ispitivanje je obavljeno na košnicama iz istog pčelinjaka, ujednačenim po broju pčela, površini legla i količini uskladištene hrane. Na osnovu infestacije varoom, košnice su podeljene u dve grupe: neinfestirane (N) i infestirane (I). Obe grupe su bile ili tretirane (T) – NT i IT, ili netretirane (U) – NU i IU. Tretman infestiranih društava je kontrolisan naknadnom primenom amitraza. Efikasnost kumafosa iznosila je 96-97%. Ovaj organofosfat imao Zikic et al.: Anti-varroa efficiency of coumaphos and its influence on oxidative stress and survival of honey bees 373 je zanemarljiv efekat na preživljavanje pčela, ali je značajno uticao na njihov oksidativni status: aktivnosti superoksid-dismutaze (SOD), katalaze (CAT) i glutation Stransferaze (GST), i koncentracije malonil-dialdehida (MDA). Kumafos je značajno (p˂0,0001) smanjio aktivnost SOD kod neinfestiranih pčela, ali ju je povećao kod infestiranih. Za razliku od toga, aktivnosti CAT i GST, kao i koncentracije MDA značajno su se povećale (od p˂0,05 do p˂0,0001) posle tretmana u svim grupama, sa izuzetkom IT, u kojoj su opale. Kumafos je sam po sebi u neinfestiranim društvima prouzrokovao oksidativni stres kod pčela, slično kao Varroa u infestiranim. Međutim, u infestiranim košnicama on je smanjio oksidativni stress zahvaljujući efikasnosti protiv varoe, čime je doprineo oporavku društava. Uprkos određenim nedostacima, kumafos ostaje efikasna supstanca u borbi protiv varoe, ali ga treba koristiti sa oprezom, da bi se izbeglo dodatno opterećenje pčela prouzrokovano faktorima sredine koji mogu da izazovu red-oks neravnotežu.

작은벌집딱정벌레(Small hive beetle(SHB), Aethina tumida(Coleoptera: Nitidulidae)는 아프리카 대륙 사하라 사막 이남 지역이 원산지이다. 벌통내 양봉산물과 꿀벌에 직·간접적인 피해를 준다. 국내에서는 밀양지역에서 2016년 9월 첫 피해 보고되었다. 현재 발생 지역은 밀양과 그 주변지역인 창녕, 양산, 기장에서 발생하고 있다. 이에 국내 작은벌집딱정벌레 확산 방지 및 방제를 위해 본 연구를 실시하였다. 본 연구는 유인제 선호성, 방제 약제 선발, 꿀벌안전성 평가, 선발 약제를 이용한 현장 적용 평가로 나눌 수 있다. 유인제 선호성은 유충과 성충을 대상으로 이루어졌으며, 유충은 대용화분을 가장 선호하였으며, 성충은 분쇄화분+설탕물을 가장 선호하였다. 이에 유충과 성충은 기본적으로 화분성분을 선호하여, 방제 약제 개발시 약제와 화분을 혼합하여 사용할 수 있게 개발하였다. 방제 약제 선발은 국외 문헌 조사를 통해 일차적으로 coumaphos, fluvalinate, cymiazole hydrochlorid을 후보 목록으로 선정하였다. 이에 대해 실내 독성 평가를 통해 적합 약제를 선발하였다. 선발된 약제는 coumaphos이며, 방제 효율이 높으며, 꿀벌에 안전한 것으로 나타났다. coumaphos를 이용하여 대용화분과 혼합하여 섭식을 통해 방제하였으며, 유충에 대한 방제 농도는 1,000ppm, 성충은 10,000ppm으로 설정되었으며, 꿀벌 안전성 평가를 위해 실내·외 평가에서도 coumaphos 10,000ppm 혼합 대용화분은 안전한 것으로 나타났다. 이를 이용하여 CD형 케이스에 coumaphos 10,000ppm 혼합 대용화분과 Apitor, 오일 트랩을 이용하여 작은벌집딱정벌레 발생 현장에서 실험한 결과 높은 방제 효율이 나타났다. 이에 coumaphos 1,000ppm(유충), 10,000ppm(성충)을 대용화분과 혼합하여CD형 케이스에 넣어 사용한다면 발생현장에서 작은벌집딱정벌레 방제에 적합한 것으로 나타났다.