Honey bees for pesticide monitoring in the landscape: which bee matrices should be used?

Among bee species, the western honey bee (Apis mellifera) is preferred in monitoring studies performed in the agricultural landscape, while bee matrices, pollen, and honey are mostly a subject of these studies due to their unique composition. A justified question about the relevance of other bee matrices, like larvae, foragers, beebread, and/or wax, has been raised. The ability of different bee matrices (wax, pollen grains, bee bread, foragers, larvae, nectar, and honey) to absorb pesticide residues is subjected in this study. All samples were collected during a crop flowering season (oilseed rape) on intensively managed agricultural land in Slovakia and Germany. The observed high variability in residue levels, profile, and number of detections among studied matrices from Germany, west, and east Slovakia gave us an assumption of the use of different agricultural practices between these two countries. Fungicides clearly dominated across all samples in all sampling regions. The increased pesticide profile positively correlated with the oilseed rape pollen grains in pollen pellets and/or bee bread. Bee wax, pollen, and bee bread showed a high number of detected active substances and total residue concentrations among matrices, indicating their high ability to absorb pesticide residues in the surrounding hive environment.

Honey bee colonies can buffer short-term stressor effects of pollen restriction and fungicide exposure on colony development and the microbiome.

Honey bees (Apis mellifera) have to withstand various environmental stressors alone or in combination in agriculture settings. Plant protection products are applied to achieve high crop yield, but residues of their active substances are frequently detected in bee matrices and could affect honey bee colonies. In addition, intensified agriculture could lead to resource limitation for honey bees. This study aimed to compare the response of full-sized and nucleus colonies to the combined stressors of fungicide exposure and resource limitation. A large-scale field study was conducted simultaneously at five different locations across Germany, starting in spring 2022 and continuing through spring 2023. The fungicide formulation Pictor® Active (active ingredients boscalid and pyraclostrobin) was applied according to label instructions at the maximum recommended rate on oil seed rape crops. Resource limitation was ensured by pollen restriction using a pollen trap and stressor responses were evaluated by assessing colony development, brood development, and core gut microbiome alterations. Furthermore, effects on the plant nectar microbiome were assessed since nectar inhabiting yeast are beneficial for pollination. We showed, that honey bee colonies were able to compensate for the combined stressor effects within six weeks. Nucleus colonies exposed to the combined stressors showed a short-term response with a less favorable brood to bee ratio and reduced colony development in May. No further impacts were observed in either the nucleus colonies or the full-sized colonies from July until the following spring. In addition, no fungicide-dependent differences were found in core gut and nectar microbiomes, and these differences were not distinguishable from local or environmental effects. Therefore, the provision of sufficient resources is important to increase the resilience of honey bees to a combination of stressors.

Influence of virus abundances in donor colonies and nurse hives on queens of Apis mellifera during the rearing process.

Background: Honeybees are one of the three most important animals for mankind. In order to be safe and increase number of bee colonies for pollination, the breeding of queens is necessary. For several decades, bees were selected on economic and behavioral aspects. With the appearance of the neozootic mite Varroa destructor beekeepers were forced to adapt their methods. Varroa destructor can act as a vector for many different bee pathogenic viruses and by this potentiates its devastating impact. Aim: Methods of rearing queens were not evaluated since the mites' appearance. Besides scientific approaches, viruses received too little attention in regard to the rearing process of honeybee queens. Herein, we present a detailed analysis of virus abundances [Aparavirus, acute bee paralysis virus (ABPV); Triatovirus, black queen cell virus (BQCV); Cripavirus, chronic bee paralysis virus (CBPV); and Iflaviruses, deformed wings virus (DWV), Sacbrood virus (SBV), VDV-1] in breeding hives, donating first instar larvae, hives that are nursing these larvae until the pupa stage, and on queens of Apis mellifera in a breeding apiary. Methods: Nurse and donor colonies of the queen-rearing process were sampled in the year 2020 and analyzed by RT qPCR. Virus quantifications were correlated with queen mortalities and seasonal effects. Results: Virus detections increased in reared queens, however, the elevated virus titers did not increase the mortality of the queens until their exclosure. Moreover, we observed a lower interrelation between virus abundance in queens and their original donor colonies, than between nurse hives and their nursed queens. Conclusion: The bee pathogenic viruses ABPV, BQCV, CBPV, DWV, SBV, and VDV-1 do not influence the mortality of bee queens during the rearing process. Whether respective virus loads result in sublethal or long-term effects remains to be elucidated.

Large-scale study investigating the effects of a tank mixture containing thiacloprid-prochloraz on honey bees (Apis mellifera).

Risk assessment of plant protection products (PPPs) will be conducted before authorization for their possible effects on non-target organisms, including honey bees. Tank mixtures are often common practice by farmers, and mostly their effects on honey bees are not routinely assessed. To enable a realistic assessment of laboratory-reported effects of a combination of the insecticide thiacloprid and fungicide prochloraz on honey bees, a large-scale field study with spray application in winter oilseed rape was conducted in four regions in Germany. Several parameters were investigated, including mortality, flight activity, and colony development. Residue analysis of various materials (e.g., dead bees, nectar, and pollen) was conducted to assess exposure level. We observed several intoxication symptoms 2 h after application, including a high number of moribund bees and dead bees on the first day after application (DAA +1) compared to the control. Adverse effects were observed on the number of open brood cells, with a significant reduction of approximately 22% compared to control over the experimental period. High residue concentrations were detected on flowers and dead bees on the day of application, which decreased rapidly within six days. The residue concentrations detected were higher in bee-collected materials than in materials stored in the hive. In conclusion, exposure to a combination containing thiacloprid-prochloraz poses a high risk to honey bees. Thus, the application of such a mixture on flowering crops is restricted in Germany.

Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product.

The increasing use of Bacillus thuringiensis (Bt)-based plant protection products (PPPs) has recently raised some concerns regarding their environmental accumulation and possible chronic exposure of non-target species, including pollinators, to higher than expected doses. The exposure level of such microbial PPPs in bee's matrices under field conditions has not yet been described. Therefore, the current study aims at evaluating the realistic exposure level and comparing the distributions and persistence of Bt spores under field conditions. A field trial with spray application in oilseed rape (Brassica napus) as a representative bee-attractive crop was conducted. During the experimental period, different matrices, including honeybee-collected and -stored matrices as well as bee larvae and dead bees, were collected and analyzed using newly established methods. The concentration of Bt spores in the various matrices was quantified. The results show high levels of Bt spores in honey sac and pollen pellets with reduction over time but no reduction of Bt spores in the stored matrices within the colony, i.e., nectar and bee bread, over time. Our results show for the first time the exposure level of bees to Bt spores under realistic field conditions and are fundamentally important for assessing potential exposure and risks for pollinators.

Rapid identification and genotyping of the honeybee pathogen Paenibacillus larvae by combining culturing and multiplex quantitative PCR.

Background: American Foulbrood (AFB) is a devastating disease of honey bee (Apis mellifera) larvae caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. In most countries, the law requires mandatory reporting of AFB to the veterinary authority. Aim and Methods: To speed up detection and genotyping of P. larvae spores, we compared different culturing protocols on Columbia sheep blood agar and developed a new multiplex quantitative polymerase chain reaction to distinguish between the two relevant P. larvae genotypes enterobacterial repetitive intergenic consensus (ERIC) I and ERIC II. Results and Conclusion: As confirmed by P. larvae reference strains and field isolates, the new identification and genotyping protocol halves the time of current workflows, lessens labor-intension, allows a higher throughput of samples for monitoring, and permits a faster intervention to prevent the spread of AFB.

Feeding deterrence and detrimental effects of pyrrolizidine alkaloids fed to honey bees (Apis mellifera).

Recent studies have shown the occurrence of plant derived pyrrolizidine alkaloids (PAs) in retail honeys and pollen loads, but little is known about how these compounds influence the fitness of foraging honey bees. In feeding experiments, we tested a mix of tertiary PAs and the corresponding N-oxides from Senecio vernalis, pure monocrotaline, and 1,2-dihydromonocrotaline in 50% (w/w) sucrose solutions. The bees were analyzed chemically to correlate the observed effects to the ingested amount of PAs. PA-N-oxides were deterrent at concentrations >0.2%. 1,2-Unsaturated tertiary PAs were toxic at high concentrations. The observed PAs mortality could be linked directly to the presence of the 1,2-double bond, a well established essential feature of PA cytotoxicity. In contrast, feeding experiments with 1,2-dihydromonocrotaline revealed no toxic effects. Levels of less than 50 microg 1,2-unsaturated tertiary PAs per individual adult bee were tolerated without negative effects. PA-N-oxides fed to bees were reduced partially to the corresponding tertiary PAs. Unlike some specialized insects, bees are not able to actively detoxify PAs through N-oxidation. To gain insight into how PAs are transmitted among bees, we tested for horizontal PA transfer (trophallaxis). Under laboratory conditions, up to 15% of an ingested PA diet was exchanged from bee to bee, disclosing a possible route for incorporation into the honey comb. In the absence of alternative nectar and pollen sources, PA-containing plants might exhibit a threat to vulnerable bee larvae, and this might affect the overall colony fitness.