Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites.

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Sublethal fluvalinate negatively affect the development and flight capacity of honeybee (Apis mellifera L.) workers.

Fluvalinate has been heavily used to control the pest Varroa destructor and residues in honeybee colony causing long-term exposure threat for bees. But, little is known about the lifetime trips and homing ability of worker bees under fluvalinate stresses during the development period. In this study, honeybees from 2-day-old larvae to 7-day-old adults were continuously fed with different concentrations of fluvalinate (0, 0.5, 5 and 50 mg/kg) and the effects of fluvalinate on the development of larvae were examined. And then, all the treated bees were reintroduced into the original source colony and were monitored, and the homing ability of 20 days old bees at 1000 and 2000 m away from the beehive were tested using the radio frequency identification (RFID). We found that fluvalinate significantly activates the superoxide dismutase (SOD) activities of larvae and 5 mg/kg fluvalinate reduced the homing rate of workers at 2000 m away from colony. 50 mg/kg fluvalinate reduced proportion of capped worker cells, activated Cytochrome P450 (CYP450) activity of larvae, affected the foraging times, influenced the homing rate and homing time of one trip at 2000 m away from colony. Our results showed that the larvae can activate the activities of SOD and detoxification enzymes in detoxification of fluvalinate and reduce the influence on honeybees. But, when the concentration is higher than 5 mg/kg fluvalinate, it is difficult for bees to detoxify fluvalinate completely, which affect the homing rate. The results reflect the potential risk for honeybees in the development stage continuously exposed to fluvalinate.

Transcriptomic analysis to elucidate the response of Apis mellifera ligustica brain tissue to fluvalinate exposure.

As a commonly used acaricide in apiculture, fluvalinate is used to kill Varroa mites, while it also damages the nervous system of honeybees. To date, the transcriptomic characteristics associated with fluvalinate-induced neuronal injury in the bee brain have not been reported. Here, we performed transcriptome sequencing on Apis mellifera ligustica (A. mellifera ligustica) brain tissues collected before and after fluvalinate treatment. A total of 546 differentially expressed genes (DEGs) were detected, and these DEGs mainly showed 4 different expression patterns. Further analysis revealed that DEGs with different expression patterns were mainly involved in lipid metabolism, amino acid metabolism, visual transduction, and neural response-related GO terms and KEGG pathways. Moreover, protein-protein interaction network analysis revealed five protein-coding DEGs as key genes, which may play important roles in the resistance to fluvalinate-induced honeybee brain nerve tissue damage. In summary, this study is the first to perform a detailed characterization and functional analysis of genes related to fluvalinate stimulation in honeybee brains.

Molecular basis of selective resistance of the bumblebee BiNav1 sodium channel to tau-fluvalinate.

Insecticides are widely used to control pests in agriculture and insect vectors that transmit human diseases. However, these chemicals can have a negative effect on nontarget, beneficial organisms including bees. Discovery and deployment of selective insecticides is a major mission of modern toxicology and pest management. Pyrethroids exert their toxic action by acting on insect voltage-gated sodium channels. Honeybees and bumblebees are highly sensitive to most pyrethroids, but are resistant to a particular pyrethroid, tau-fluvalinate (τ-FVL). Because of its unique selectivity, τ-FVL is widely used to control not only agricultural pests but also varroa mites, the principal ectoparasite of honeybees. However, the mechanism of bee resistance to τ-FVL largely remains elusive. In this study, we functionally characterized the sodium channel BiNav1-1 from the common eastern bumblebee (Bombus impatiens) in Xenopus oocytes and found that the BiNav1-1 channel is highly sensitive to six commonly used pyrethroids, but resistant to τ-FVL. Phylogenetic and mutational analyses revealed that three residues, which are conserved in sodium channels from 12 bee species, underlie resistance to τ-FVL or sensitivity to the other pyrethroids. Further computer modeling and mutagenesis uncovered four additional residues in the pyrethroid receptor sites that contribute to the unique selectivity of the bumblebee sodium channel to τ-FVL versus other pyrethroids. Our data contribute to understanding a long-standing enigma of selective pyrethroid toxicity in bees and may be used to guide future modification of pyrethroids to achieve highly selective control of pests with minimal effects on nontarget organisms.

Abdominal contact of fluvalinate induces olfactory deficit in Apis mellifera.

τ-Fluvalinate (fluvalinate) is a highly selective pyrethroid insecticide compound used for controlling ectoparasitic mites that cause major damages in honey bee colonies. Although honey bees have resistance and low toxicity to this xenobiotic chemical, little is known about the effects of this chemical on sensory modulation and behaviors in honey bees. Here we addressed the effect on olfactory cognition at the behavioral, molecular, and neurophysiological levels. First, we found that topical application of fluvalinate to honeybee abdomen elicited somewhat severe toxicity to honey bees. Furthermore, honeybees treated with sublethal doses of fluvalinate showed a significant decrease in olfactory responses. At the molecular level, there was no change in gene expression levels of odorant receptor co-receptor (Orco), which is important for electrical conductivity induced by odorant binding in insects. Rather, small neuropeptide F (sNPF) signaling pathway was involved in olfactory fluctuation after treatment of fluvalinate. This indicates that olfactory deficits by abdominal contact of fluvalinate may stem from various internal molecular pathways in honey bees.

Synthesis of molecularly imprinted dye-silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ-fluvalinate in vodka.

An imprinted fluorescent sensor was fabricated based on SiO2 nanoparticles encapsulated with a molecularly imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core-shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ-fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM, and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operations). The molecularly imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ-fluvalinate in real samples from vodka. The molecularly imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety.

Biomarker responses and lethal dietary doses of tau-fluvalinate and coumaphos in honey bees: Implications for chronic acaricide toxicity.

Evidence suggests that acaricide residues, such as tau-fluvalinate and coumaphos, are very prevalent in honey bee colonies worldwide. However, the endpoints and effects of chronic oral exposure to these compounds remain poorly understood. In this study, we calculated LC50 and LDD50 endpoints for coumaphos and tau-fluvalinate, and then evaluated in vivo and in vitro effects on honey bees using different biomarkers. The LDD50 values for coumaphos were 0.539, and for tau-fluvalinate, they were 12.742 in the spring trial and 8.844 in the autumn trial. Chronic exposure to tau-fluvalinate and coumaphos resulted in significant changes in key biomarkers, indicating potential neurotoxicity, xenobiotic biotransformation, and oxidative stress. The Integrated Biomarker Response was stronger for coumaphos than for tau-fluvalinate, supporting their relative lethality. This study highlights the chronic toxicity of these acaricides and presents the first LDD50 values for tau-fluvalinate and coumaphos in honey bees, providing insights into the risks faced by colonies.

Effect of fluvalinate on the expression profile of circular RNA in brain tissue of Apis mellifera ligustica workers.

Fluvalinate is widely used in apiculture as an acaricide for removing Varroa mites, but there have been growing concerns about the negative effects of fluvalinate on honeybees in recent years. Previous research revealed changes in the miRNA and mRNA expression profiles of Apis mellifera ligustica brain tissues during fluvalinate exposure, as well as key genes and pathways. The role of circRNAs in this process, however, is unknown. The goal of this study was to discover the fluvalinate-induced changes in circular RNA (circRNA) expression profiles of brain tissue of A. mellifera ligustica workers. A total of 10,780 circRNAs were detected in A. mellifera ligustica brain tissue, of which eight were differentially expressed between at least two of the four time periods before and after fluvalinate administration, and six circRNAs were experimentally verified to be structurally correct, and their expression patterns were consistent with transcriptome sequencing results. Furthermore, ceRNA analysis revealed that five differentially expressed circRNAs (DECs) (novel_circ_012139, novel_circ_011690, novel_circ_002628, novel_circ_004765, and novel_circ_010008) were primarily involved in apoptosis-related functions by competitive binding with miRNAs. This study discovered changes in the circRNA expression profile of A. mellifera ligustica brain tissue caused by fluvalinate exposure, and it provides a useful reference for the biological function study of circRNAs in A. mellifera ligustica.

Fluvalinate-Induced Changes in MicroRNA Expression Profile of Apis mellifera ligustica Brain Tissue.

Fluvalinate is a widely used and relatively safe acaricide for honeybees, but it still has a negative impact on honeybee colonies. Such negative effects may be related to fluvalinate-induced brain nerve tissue damage, but the detailed molecular regulatory mechanism of this phenomenon is still poorly understood. In this study, we analyzed the miRNA expression profile changes in the brain tissue of Apis mellifera ligustica by miRNA sequencing after fluvalinate treatment. A total of 1,350 miRNAs were expressed in Apis mellifera ligustica brain tissue, of which only 180 were previously known miRNAs in honeybees. Among all known and novel miRNAs, 15 were differentially expressed between at least two of the four time periods before and after fluvalinate administration. Further analysis revealed five significantly enriched KEGG pathways of the differentially expressed miRNA (DEM) potential target genes, namely, "Hippo signaling pathway-fly," "Phototransduction-fly," "Apoptosis-fly," "Wnt signaling pathway," and "Dorso-ventral axis formation," which indicates that differentially expressed miRNA function may be related to cell apoptosis and memory impairment in the fluvalinate-treated Apis mellifera ligustica brain. Ame-miR-3477-5p, ame-miR-375-3p, and miR-281-x were identified as key miRNAs. Overall, our research provides new insights into the roles of miRNAs in brain tissue during the process of fluvalinate-induced Apis mellifera ligustica poisoning.

Low-Level Fluvalinate Treatment in the Larval Stage Induces Impaired Olfactory Associative Behavior of Honey Bee Workers in the Field.

Fluvalinate is a widely used insecticide for varroa mite control in apiculture. While most beekeepers have ignored the effects of low levels of fluvalinate on bees, this study aims to demonstrate its effects at very low concentrations. We first used fluvalinate doses ranging from 0.4 to 400 ng/larva to monitor the capping, pupation, and emergence rates of larval bees. Second, we used the honey bees' proboscis extension reflex reaction to test the learning ability of adult bees that were exposed to fluvalinate doses from 0.004 to 4 ng/larva in the larval stage. The brood-capped rate of larvae decreased dramatically when the dose was increased to 40 ng/larva. Although no significant effect was observed on brood-capping, pupation, and eclosion rates with a dose of 4 ng/larva, we found that the olfactory associative behavior of adult bees was impaired when they were treated with sublethal doses from 0.004 to 4 ng/larva in the larval stage. These findings suggest that a sublethal dose of fluvalinate given to larvae affects the subsequent associative ability of adult honey bee workers. Thus, a very low dose may affect the survival conditions of the entire colony.

Evaluating the chronic effect of two varroacides using multiple biomarkers and an integrated biological response index.

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.

The adverse effects of synthetic acaricide tau-fluvalinate (tech.) on winter adult honey bees.

Currently several pyrethroids (e.g., flumethrin and tau-fluvalinate) are used in apiculture worldwide as acaricides/miticides. The long half-lives of pyrethroids in synthetic acaricides applied to hive matrices, may adversely affect the health of bee colony. The potentially adverse effects of synthetic acaricide/miticide tau-fluvalinate (tech.) on winter honeybees were assessed in this study (OECD 245 2017). No dose-dependent mortality in in vitro reared winter honeybees was observed after chronic oral 10-day exposure to syrup (50% w/v) spiked with a maximum concentration of 750 µg a.i./kg diet and its 1/10 concentration. The No Observed Effect Concentration is ≥ 750 µg a.i./kg diet. Tau-fluvalinate testing for the sublethal effects on bee immune system showed up-regulated gene expression encoding abaecin, lysozyme, and defensin in both tested groups, however the expression of hymenoptaecin gene was reduced. Moreover, tau-fluvalinate significantly induced levels of DNA damage in exposed bees, which can result in adverse genotoxic effect.

Modification of the existing maximum residue levels for tau-fluvalinate in tomatoes and watermelons.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant ADAMA Agriculture BV on behalf of ADAMA Makhteshim Ltd submitted a request to the competent national authority in Denmark to modify the existing maximum residue levels (MRL) for the active substance tau-fluvalinate in tomatoes and watermelons. The data submitted in support of the request were found to be sufficient to derive an MRL proposal for tomatoes. For watermelons, a change of the MRL recently set in the EU legislation is not required. Adequate analytical methods for enforcement are available to control the residues of tau-fluvalinate in the commodities under consideration. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the intended uses of tau-fluvalinate according to the reported agricultural practices is unlikely to present a risk to consumer health. The risk assessment shall be regarded as indicative.

Residual Tau-Fluvalinate in Honey Bee Colonies Is Coupled with Evidence for Selection for Varroa destructor Resistance to Pyrethroids.

Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy-Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.

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.

A Case Report of Chronic Stress in Honey Bee Colonies Induced by Pathogens and Acaricide Residues.

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.

Efficacy assessment of soft and hard acaricides against Varroa destructor mite infesting honey bee (Apis mellifera) colonies, through sugar roll method.

The parasitic mite Varroa destructor is amongst the most serious problems of honey bees, Apis mellifera (Hymenoptera: Apidae) around the world including Pakistan. The present study estimates the mite density through powdered sugar roll method and evaluates the effectiveness of five miticides (fluvalinate, flumethrin, amitraz, formic acid, and oxalic acid) on A. mellifera colonies in German modified beehives. The results indicated that by treating the bees with one strip and two strips of fluvalinate per colony; the mite population remained below the economic threshold level (ETL) for 14 days and 25 days, respectively. Treatment of flumthrin @1 strip and @ 2 strips per colony resulted in mite population suppressed for 14 days and 39 days, respectively below ETL. Application of Amitraz @ 2 mL per 1.5 L water after every three days interval on sealed brood effectively controlled mites below ETL for 21 days. Formic acid @10 mL per colony applied through plastic applicator proved effective (below 3 mites per bee sample) for 24 days and oxalic acid applied through shop towel method resulted in mite population control for fifteen days. Use of powdered sugar roll method for easy sampling of Varroa mites and application of acaricides on precise economic threshold level during different seasons of the year for integrated management of Varroa mite is hereby advocated by current studies.

Efficacy of Residual Pesticide-Treated Plant Saucers Against Aedes albopictus Larvae Under Semi-Field and Field Conditions.

Plant saucers are ubiquitous, outdoor water-holding receptacles and are one of the most productive domestic mosquito habitats in the urban environment. Two kinds of commonly used plant saucers, clay and plastic, were manually treated with 3 residual insecticides, bifenthrin (Talstar® Professional), lambda-cyhalothrin (Lambda 9.7 CS), and tau-fluvalinate (Mavrik® Perimeter), at their maximum rates to assess their residual efficacy against Aedes albopictus larvae under semi-field and field conditions. Both clay and plastic saucers treated with bifenthrin and lambda-cyhalothrin provided weeks of control of 3rd instars of Ae. albopictus, whereas tau-fluvalinate provided only 1 day of control. Results from this study show that bifenthrin and lambda-cyhalothrin can provide good control of Ae. albopictus larvae for a considerable period of time and have great potential with regard to container mosquito management in the future.

An Examination of Exposure Routes of Fluvalinate to Larval and Adult Honey Bees (Apis mellifera).

Fluvalinate has been extensively used in the United States to combat honey bee colony loss due to Varroa destructor mites. Our objectives were to investigate the extent of fluvalinate contamination in commercially available wax and to define exposure pathways to larval and adult honey bees. All the commercial wax tested contained elevated fluvalinate concentrations, indicating a need for regulation of the sources of wax being rendered for resale. Based on the negative logarithm of the partition coefficient between wax and pollen (-0.54), it is evident that fluvalinate has the potential to actively transfer from the contaminated wax into hive matrices. This was confirmed by adding fluvalinate-dosed wax, fluvalinate-impregnated strips, or a combination of the 2 to hives. Larvae and adult bees were checked for fluvalinate exposure using gas chromatography-mass spectrometry analysis. Larvae had detectable concentrations of fluvalinate in all treatments. Bioaccumulation in adult bees was significantly affected by the interaction between treatment type and application time. In other words, residues from hives that only had fluvalinate-dosed wax were comparable to residues in hives that were actively being treated, suggesting that transfer of fluvalinate from wax into adult bees was an important exposure route. In conclusion, exposure of fluvalinate from contaminated wax and treatment strips to honey bees needs to be considered when the risk for colony loss in hives is being evaluated. Environ Toxicol Chem 2019;38:1356-1363. © 2019 SETAC.

Spatio-temporal dynamics of Varroa destructor resistance to tau-fluvalinate in Czechia, associated with L925V sodium channel point mutation.

BACKGROUND: Extensive application of pyrethroids to control Varroa destructor, an invasive mite devastating bee colonies, has resulted in a global spread of resistant mite populations. In this study, we analyzed the spatio-temporal dynamics of resistant V. destructor populations in Czechia, stemming from the L925V mutation. Mites were collected during 2011-2018 directly or from winter beeswax debris, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and densitometry was used to detect the L925V mutation. RESULTS: Pooled samples of 10 mites were classified, based on their PCR-RFLP patterns, as tau-fluvalinate-sensitive (56%), resistant (9%), or mixed (35%), with the latter including sensitive and resistant homo- and heterozygotes. We identified two zones with higher frequencies of resistance, one in southern Moravia and the other in Bohemia. The mutant populations were evenly distributed throughout the monitored districts, with a few temporal and spatial local fluctuations. The greatest increase in resistance was observed in 2016, following massive losses of bee colonies in the winter of 2015. This event appeared to be closely associated with fluctuations in resistant mite populations and their dispersion. CONCLUSION: Two outbreaks of resistance were detected in Czechia; however, the amount of applied tau-fluvalinate was not correlated with the frequency of resistance in mites. There was no remarkable increase in mite resistance in 2011-2018, although the use of tau-fluvalinate increased 40-fold between 2011 and 2015. PCR-RFLP analysis, performed on mites present in beeswax debris, is a suitable method for monitoring the L925V mutation in V. destructor. © 2018 Society of Chemical Industry.