Apis Mellifera Research Articles

Assessing the Electrochemical Performance of a Novel Low‐Cost Screen‐Printed Electrode Based on Apis mellifera Beeswax Modified with Vulcan@Chitosan Used for Acetominophen Detection

AbstractThe present work reports the development of a highly sensitive electrochemical platform, constructed using screen printed electrode based on Apis mellifera beeswax (SPWE) – employed as substrate, and its application for acetaminophen (AC) detection. The sensor was produced using graphite ink and varnish glass solubilized in acetone. The SPWE was modified with a dispersion of chitosan (0.5% m/v) − functionalized Vulcan XC72R carbon black (Vulcan@Chitosan). Electrochemical characterization was performed using cyclic voltammetry (−0.7 to +0.8 V vs. pseudo‐graphite) and chronoamperometry in 0.1 mol L−1 KCl in the presence of 1.0 mmol L−1 [Fe(CN)6]3−/[Fe(CN)6]4−. Through the application of differential pulse voltammetry (DPV) using 0.1 mol L−1 PBS at pH 6.5, the proposed SPWE/Vulcan@Chitosan sensor was found to exhibit significant improvements in current response relative to the AC electrochemical detection. The sensor exhibited linear dynamic range of 0.05 to 4.0 μmol L−1 (n= 3), with R2= 0.992 and theorical LOD of 0.013 μmol L−1 (RSD= 3.80%). The AC detection was conducted in real matrices of river water, synthetic urine and synthetic saliva using the DPV technique yielded a mean recovery rate of 103% (RSD= 3.52%), exhibiting a high degree of accuracy. The SPWE/Vulcan@Chitosan electrochemical platform demonstrated high stability, good reproducibility and satisfactory repeatability; the current responses were not affected by the reutilization of the beeswax substrate for the production of new electrode. The findings of this study show that the proposed modified electrode is a simple, low‐cost sensing device which has proven to be highly efficient when applied for the AC detection in different matrices.

Tolerance and efficient metabolization of extremely high ethanol concentrations by a social wasp

Ethanol, a natural by-product of sugar fermentation, can be found in various fruits and nectar. Although many animals routinely consume ethanol in low concentrations as part of their natural diets, its inherent toxicity can cause severe damage. Even species particularly well adapted to ethanol consumption face detrimental effects when exposed to concentrations above 4%. Here, we investigated the metabolism of ethanol and its impact on survival and behavior in the Oriental hornet (Vespa orientalis), a social wasp that naturally consumes ethanol. We show that chronic ethanol consumption, even at concentrations as high as 80%, had no impact on hornet mortality, construction behavior, or agonistic behavior. Using 13C1 labeled ethanol, we show that hornets efficiently metabolized ingested ethanol and at a much higher rate than honey bees. The presence of multiple copies of the alcohol dehydrogenase (NADP+) gene in the Vespa genera suggests a potential mechanism for ethanol tolerance. These findings support the hypothesis that the mutualistic relationship between ethanol-producing organisms and vespid hosts may be at the origin of their remarkable capacity to utilize and metabolize ethanol.

Distinct pollinator communities persist among co-flowering specialty crops in Indiana

Diverse bee communities pollinate fruits and vegetables, and the composition of these communities has been described for many specialty crops in major production regions. However, pollinator communities in landscapes dominated by agronomic crops may differ in species composition and the contribution of wild bees. With over 4 million ha of maize and soybean, Indiana presents a novel landscape to compare pollinator communities among specialty crops that differ in their use of managed bees and bloom phenology, with potential implications for the composition of wild bee communities and their contributions to flower visitation. We sampled pollinator communities with flower observations and pan traps in spring-blooming apples and blueberries and summer-blooming tomatoes and watermelons, allowing us to compare communities between specialty crops with overlapping and distinct bloom times. Apples, blueberries, and watermelons were stocked with honey bees, and watermelons additionally had managed bumble bees. Across two years of sampling, we observed 1,651 flower visits by 13 taxa, collected 1,967 bees, and identified 84 taxa from collected specimens. Apples and blueberries hosted the richest pollinator communities (34 and 51 taxa, respectively) followed by watermelons (22 taxa), and tomatoes (19 taxa). While 70% of flower visits in apples and blueberries were attributed to honey bees, wild bees accounted for 82% and 99% of flower visits in watermelons and tomatoes, respectively. Our results indicate that distinct pollinator communities persist among co-flowering specialty crops in a landscape dominated by maize and soybean production and underscore the contribution of wild bees for specialty crop pollination, particularly in watermelons and tomatoes.

Potential Antitumor Mechanism of Propolis Against Skin Squamous Cell Carcinoma A431 Cells Based on Untargeted Metabolomics.

Propolis is a sticky substance produced by honeybees (Apis mellifera) through the collection of plant resins, which they mix with secretions from their palate and wax glands. Propolis can inhibit tumor invasion and metastasis, thereby reducing the proliferation of tumor cells and inducing cell apoptosis. Previous research has shown that propolis has an inhibitory effect on skin squamous cell carcinoma A431 cells. Nevertheless, its inhibitory mechanism is unclear because of many significantly different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the ethanol extract of the propolis (EEP) group and the control group of cells. In this study, the main components of EEP and the antitumor mechanism at an IC50 of 29.04 μg/mL EEP were determined via untargeted metabolomics determined using ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS), respectively. The results revealed 43 polyphenolic components in the EEP and 1052 metabolites, with 160 significantly upregulated and 143 significantly downregulated metabolites between cells treated with EEP and solvent. The KEGG enrichment results revealed that EEP significantly inhibited A431 cell proliferation via the steroid hormone biosynthesis and linoleic acid metabolism pathways. These findings may provide valuable insights for the development of targeted therapies for the treatment of cutaneous squamous cell carcinoma.

Pollen foraging mediates exposure to dichotomous stressor syndromes in honey bees.

Recent declines in the health of honey bee colonies used for crop pollination pose a considerable threat to global food security. Foraging by honey bee workers represents the primary route of exposure to a plethora of toxins and pathogens known to affect bee health, but it remains unclear how foraging preferences impact colony-level patterns of stressor exposure. Resolving this knowledge gap is crucial for enhancing the health of honey bees and the agricultural systems that rely on them for pollination. To address this, we carried out a national-scale experiment encompassing 456 Canadian honey bee colonies to first characterize pollen foraging preferences in relation to major crops and then explore how foraging behavior influences patterns of stressor exposure. We used a metagenetic approach to quantify honey bee dietary breadth and found that bees display distinct foraging preferences that vary substantially relative to crop type and proximity, and the breadth of foraging interactions can be used to predict the abundance and diversity of stressors a colony is exposed to. Foraging on diverse plant communities was associated with increased exposure to pathogens, while the opposite was associated with increased exposure to xenobiotics. Our work provides the first large-scale empirical evidence that pollen foraging behavior plays an influential role in determining exposure to dichotomous stressor syndromes in honey bees.

Pollinator diversity of the food-deceptive orchids in southern Italy.

The orchid family is renowned for its enormous diversity in pollination biology. Many orchid species use deception to attract pollinators, and the main strategy in terrestrial orchids is food deception. Food-deceptive orchids usually show a low number of pollinator visitations, making field observations of pollinators difficult. In this study visual census, pollinator capture and molecular analysis of pollinaria found on caught insects allowed us to obtain information on species composition of orchid pollinators. A total of 321 insects were caught; most specimens were hymenopterans (Apis mellifera, Bombus ruderatus and Eucera rufa) and coleopterans (Tropinota hirta and T. squalida). The identity of species to which pollinaria found on the insect's body belonged was confirmed by molecular analysis. Moreover, some individuals of Billaea lata (Tachinidae, Diptera) were captured and photographed with the pollinaria on their head. Two new and important results emerged clearly in this work: a dipteran, Billaea lata, pollinator of Anacamptis pyramidalis, and two beetles in the genus Tropinota are pollinators of Orchis italica. Our results confirm that generalized food-deceptive orchids of the genera Orchis and Anacamptis show weak pollinator specificity.

Design, Synthesis, and 3D-QASR of Oxazoline Derivatives Containing an S-S Moiety as Potential Acaricide.

To mitigate the detrimental effects of agricultural pest mites on crop yield, an active substructure splicing strategy was employed to modify etoxazole by introducing an S-S moiety. The target products were obtained efficiently via a bilateral disulfurating reagent (DSMO), which was developed by our group. The leaf dip method was used to evaluate the activities of the designed target compounds against the eggs and larvae of the spider mite (Tetranychus cinnabarinus). Most of the target compounds exhibited good efficacy in controlling the larvae and eggs of T. cinnabarinus. Based on these results, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established to guide the construction of compound 7l. Notably, compound 7l exhibited a better activity against T. cinnabarinus eggs (LC50 = 0.0035 mg/L) compared to etoxazole (LC50 = 0.2990 mg/L). Greenhouse bioassays indicated that compound 7l exhibits excellent acaricidal activity against egg of T. cinnabarinus, which is better than the etoxazole at 1.0 mg/L. Additionally, some of the compounds showed inhibitory effects against Dickeya zeae (D. zeae), Xanthomonas campestris pv campestris (Xcc), Xanthomonas oryzae pvoryza (Xoo), and Xanthomonas oryzae pvoryzicola (Xoc). Furthermore, compounds 7l not only exhibited relatively potent against Plutella xylostella activities (LC50 = 24.0 mg/L) but also had low toxicity (LC50 > 11.0 μg/bee) to Apis mellifera. In conclusion, the current experimental results suggest that oxazoline derivatives containing an S-S moiety have the potential to serve as lead compounds for the development of novel acaricide agents.

The Business Model Canvas Analysis for Honey Business Development at Situak Ni Loba Siarsikarsik Social Forestry Business Group, Lumban Julu District, Toba Regency

Honey has numerous benefits and considerable potential for development within the Social Forestry Business Group (KUPS). In 2020, honey consumption in Indonesia increased to 60 grams per capita per year, leading to growth in the honey bee business. However, more research is needed to be done on developing business strategies for KUPS in North Sumatra Province. Therefore, this research aimed to formulate development strategies for the honey bee business in Situak Ni Loba Siarsikarsik KUPS, Toba Regency, using the Business Model Canvas (BMC) method. This descriptive qualitative research collected data through direct observation, interviews, discussions, and literature research to map the business model through the nine main elements of BMC, namely customer segments, value propositions, channels, customer relationships, revenue flows, main resources, activities, partnerships, and cost structure. The best strategy identified was to increase the value proposition, expand the strategic partner network, and improve market access. The findings highlighted the need to determine the specialty of the honey produced by the KUPS compared to other products, find cooperation partners for investment, and utilize technology and marketplaces to expand market share.

Paenilamicins are context-specific translocation inhibitors of protein synthesis.

The paenilamicins are a group of hybrid nonribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here we determine structures of paenilamicin PamB2-stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site transfer RNAs (tRNAs). In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms used by P. larvae. We further demonstrate that PamB2 interferes with the translocation of messenger RNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a class of context-specific translocation inhibitors.

Autonomous tracking of honey bee behaviors over long-term periods with cooperating robots.

Digital and mechatronic methods, paired with artificial intelligence and machine learning, are transformative technologies in behavioral science and biology. The central element of the most important pollinator species-honey bees-is the colony's queen. Because honey bee self-regulation is complex and studying queens in their natural colony context is difficult, the behavioral strategies of these organisms have not been widely studied. We created an autonomous robotic observation and behavioral analysis system aimed at continuous observation of the queen and her interactions with worker bees and comb cells, generating behavioral datasets of exceptional length and quality. Key behavioral metrics of the queen and her social embedding within the colony were gathered using our robotic system. Data were collected continuously for 24 hours a day over a period of 30 days, demonstrating our system's capability to extract key behavioral metrics at microscopic, mesoscopic, and macroscopic system levels. Additionally, interactions among the queen, worker bees, and brood were observed and quantified. Long-term continuous observations performed by the robot yielded large amounts of high-definition video data that are beyond the observation capabilities of humans or stationary cameras. Our robotic system can enable a deeper understanding of the innermost mechanisms of honey bees' swarm-intelligent self-regulation. Moreover, it offers the possibility to study other social insect colonies, biocoenoses, and ecosystems in an automated manner. Social insects are keystone species in all terrestrial ecosystems; thus, developing a better understanding of their behaviors will be invaluable for the protection and even the restoration of our fragile ecosystems globally.

QUALITY OF ACACIA CARPA HONEY AS A POTENTIAL INGREDIENT FOR CHILDREN’S MOUTHWASH

Dental caries is a dental disease that is most often experienced by people. One way to prevent caries is to gargle with mouthwash. Honey has become one of the natural ingredients that can be used as a mouthwash drug content. Jambi Province is one of the provinces that makes honey a non-timber forest product (NTFP). It is necessary to carry out physical and chemical tests on honey to determine the quality of honey as a basic ingredient for mouthwash. The honey samples examined were honey harvested from honey farms in Danau Lamo, Maro Sebo District, Muaro Jambi Regency, Jambi Province. This type of honey is Acacia Carpa (Acacia crassicarpa) honey from Apis mellifera honey bee farming. The honey tested consisted of honey that had undergone processing and raw honey. The honey testing carried out consists of organoleptic tests, physical tests and chemical tests. Testing was carried out at the Jambi Province Department of Industry and Trade. The inspection results show test results that comply with SNI 8664-2018 such as water content, water-insoluble solids, ash content, HMF, and diastase enzyme levels. The pH of honey is 3.7 and is in accordance with the honey pH standard. Substances that do not comply with SNI 8664-2018 are reducing sugar levels and sucrose levels. Honey with high sucrose content can still be used as a basic ingredient for mouthwash, but it must be based on the results of clinical trials to ensure its effectiveness and safety

Drone Congregation areas of Apis mellifera (hymenoptera: apidae) and Nosema sp. (nosematidae) in Bolivia: first report

Drone Congregation Areas (DCAs) are crucial natural gathering sites for Apis mellifera mating, like male leks in other animals. In these areas, virgin queens and drones from various hives congregate for mating, promoting genetic variability essential for colony adaptation and survival. Identifying drones in DCAs helps to understand the genetic variability within bee populations, which is crucial for promoting colony survival and resilience. Two areas in Villa Montes, Bolivia, were identified as potential DCAs through exploration methods utilizing satellite imagery. Furthermore, drones were captured within these DCAs using an attractive pheromone attached to an unmanned aerial vehicle to assess Nosema infection in the population.

Activity, structure, and diversity of Type II proline-rich antimicrobial peptides from insects.

Apidaecin 1b (Api), the first characterized Type II Proline-rich antimicrobial peptide (PrAMP), is encoded in the honey bee genome. It inhibits bacterial growth by binding in the nascent peptide exit tunnel of the ribosome after the release of the completed protein and trapping the release factors. By genome mining, we have identified 71 PrAMPs encoded in insect genomes as pre-pro-polyproteins. Having chemically synthesized and tested the activity of 26 peptides, we demonstrate that despite significant sequence variation in the N-terminal sequence, the majority of the PrAMPs that retain the conserved C-terminal sequence of Api are able to trap the ribosome at the stop codons and induce stop codon readthrough-all hallmarks of Type II PrAMP mode of action. Some of the characterized PrAMPs exhibit superior antibacterial activity in comparison with Api. The newly solved crystallographic structures of the ribosome complexed with Api andwith the more active peptide Fva1 from the stingless bee demonstrate the universal placement of the PrAMPs' C-terminal pharmacophore in the post-release ribosome despite variations in their N-terminal sequence.

High-resolution outdoor videography of insects using Fast Lock-On tracking.

Insects have important roles globally in ecology, economy, and health, yet our understanding of their behavior remains limited. Bees, for example, use vision and a tiny brain to find flowers and return home, but understanding how they perform these impressive tasks has been hampered by limitations in recording technology. Here, we present Fast Lock-On (FLO) tracking. This method moves an image sensor to remain focused on a retroreflective marker affixed to an insect. Using paraxial infrared illumination, simple image processing can localize the sensor location of the insect in a few milliseconds. When coupled with a feedback system to steer a high-magnification optical system to remain focused on the insect, a high-spatiotemporal resolution trajectory can be gathered over a large region. As the basis for several robotic systems, we show that FLO is a versatile idea that can be used in combination with other components. We demonstrate that the optical path can be split and used for recording high-speed video. Furthermore, by flying an FLO system on a quadcopter drone, we track a flying honey bee and anticipate tracking insects in the wild over kilometer scales. Such systems have the capability to provide higher-resolution information about insects behaving in natural environments and as such will be helpful in revealing the biomechanical and neuroethological mechanisms used by insects in natural settings.

Morphological characterization of spermatozoa in Apis mellifera and the effect of processing and semen storage

The aim of this study was to establish a standardized approach for evaluating sperm morphology in the honey bee and to investigate the impact of processing techniques and semen storage on the incidence of sperm morphoanomalies. The first experiment was designed to characterize the sperm morphoanomalies in unaltered honey bee semen, examining if they are compatible with sperm motility and viability and determining their occurrence in the ejaculates of mature and healthy honey bee drones. The different forms of sperm morphoanomalies were described in detail. In the morphological analysis of fresh drone ejaculates, 7.77% of spermatozoa showed abnormal morphology on average, with 2.20%, 4.75% and 0.81% of head, tail and multiple defects, respectively. The method also allowed us to determine the status of acrosome integrity. The second experiment was designed to assess the impact of smearing and air-drying on the occurrence of sperm morphoanomalies, showing a significant increase in the incidence of both head and tail sperm defects when comparing to wet samples. In the third and fourth experiments, the effect of semen storage at room temperature and semen cryopreservation, respectively, on the occurrence of sperm morphoanomalies were investigated. The preservation of semen at 22 °C led to a significant increase in spermatozoa with coiled tails after day 1, whereas the remaining anomalies did not exhibit significant variations over time. The freezing-thawing process showed a more pronounced effect on the incidence of morphological defects, with an increase in the percentage of spermatozoa with deformed heads and coiled tails.

Reliable molecular detection of small hive beetles

Invasive species require adequate reliable detection methods to mitigate their further spread and impact. However, the reliability of molecular detection methods is often hampered by both false positives (Error type I) and false negatives (Error type II). At present, the reliability of the four published molecular detection methods for small hive beetles (SHB), Aethina tumida, has not been rigorously evaluated considering their extensive genetic diversity. Here, we performed intra- and interlaboratory comparisons of the four available methods using SHB samples representing 78 regions from 27 countries on five continents, beetles from the same genus (Aethina concolor, A. inconspicua, A. flavicollis and A. major), as well as western honey bees, Apis mellifera, and ectoparasitic mites Varroa destructor. The data show that the Idrissou et al. (2018) and Li et al. (2018) methods avoid both false positives and false negatives probably due to lower sensitivity to nucleotide mismatches on the primer and probe’s target sequences. Further, the Li et al. (2018) method can be considered more sensitive because the fluorescent amplification curve crosses the threshold at lower Cq values compared to the Idrissou et al. (2018) one. In light of our data, the Li et al. (2018) method is the most reliable molecular diagnostic tool for SHB. We therefore recommend using this method as it will contribute to management efforts of this invasive species.

Viral Diversity in Mixed Tree Fruit Production Systems Determined through Bee-Mediated Pollen Collection

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato necrotic ringspot virus (genus Nepovirus), tobacco necrotic ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

Addressing bee diversity through active learning methodologies enhances knowledge retention in an environmental education project

The Brazilian ecosystems harbor a significant portion of global bee diversity. Despite their magnitude as pollinators, Brazilian native bees are mainly unknown. Considering the importance of scientific literacy in fostering environmental responsibility, we analyzed the BeeDiversity project’s impact on the scientific knowledge of eight to nine-year-old students. This study outlines the challenges of implementing the BeeDiversity project as a curricular component, including live bee observation and active learning methodologies. Students’ knowledge about bees significantly improved after participating in the project. Although the students mastered the concept of the ecological role of bees as pollinators, they were unaware that this ecosystem service is operated mainly by solitary bees. Learning retention was confirmed after five months. Active learning proved more effective than traditional methods. Handling a live hive and learning about solitary bees’ pollination roles generated high enthusiasm and engagement. Therefore, focusing on educational strategies addressing bee diversity beyond the typical honeybee (<i>Apis mellifera</i>) might effectively stimulate a broad environmental consciousness.

Invasive Ant Detection: Evaluating Honeybee Learning and Discrimination Abilities for Detecting Solenopsis invicta Odor.

Invasive red imported fire ants (Solenopsis invicta) create a serious threat to public safety, agriculture, biodiversity, and the local economy, necessitating early detection and surveillance, which are currently time-consuming and dependent on the inspector's expertise. This study marks an initial investigation into the potential of honeybees (Apis mellifera) to detect and discriminate the odor of S. invicta through the olfactory conditioning of proboscis extension responses. Deceased S. invicta were used as conditioned stimuli to ensure relevance to non-infested areas. The results showed that the bees rapidly learned to respond to deceased ant odors, with response levels significantly increasing at higher odor intensities. Bees exhibited generalization across the odors of 25 minor workers, 21 median workers, 1 major worker, and 1 female alate. When conditioned with deceased ant odors, bees effectively recognized live ants, particularly when trained on a single minor worker. Discrimination abilities varied by species and were higher when S. invicta was paired with Polyrhachis dives and Nylanderia yaeyamensis, and lower with S. geminata, Pheidole rabo, and Pheidole fervens. Notably, discrimination improved significantly with the application of latent inhibition. These findings suggest that trained honeybees have the potential to detect S. invicta. Further refinement of this approach could enhance its effectiveness for detection and surveillance.

Chronic oral toxicity protocol for adult solitary bees (Osmia bicornis L.): Reduced survival under long-term exposure to a “bee-safe” insecticide

Pollinators are essential for crop productivity. Yet, in agricultural areas, they may be threatened by pesticide exposure. Current pesticide risk assessments predominantly focus on honey bees, with a lack of standardized protocols for solitary bees. This study addresses this gap by developing a long-term oral exposure protocol tailored for O. bicornis. We conducted initial trials to determine optimal container sizes and feeding methods, ensuring high survival rates and accurate syrup consumption measurements. A validation test involving five laboratories was then conducted with the insecticide Flupyradifurone (FPF). Control mortality thresholds were set at ≤ 15% at 10 days. Three laboratories achieved ≤10%, demonstrating the protocol's effectiveness in maintaining healthy test populations. The seasonal timing of experiments influenced control mortality, underscoring the importance of aligning tests with the natural flight period of the population used. Our findings revealed dose-dependent effects of FPF on syrup consumption, showing stimulatory effects at lower concentrations and inhibitory effects at higher ones. The 10-day median lethal daily dose (LDD50) of FPF for O. bicornis (531.92 ng/bee/day) was 3.4-fold lower than that reported for Apis mellifera (1830 ng/bee/day), indicating Osmia's higher susceptibility. Unlike other insecticides, FPF did not exhibit time-reinforced toxicity. This study introduces a robust protocol for chronic pesticide exposure in solitary bees, addressing a critical gap in current risk assessment. Based on its low risk to honey bees and bumblebees, FPF is approved for application during flowering. However, our results suggest that it may threaten Osmia populations under realistic field conditions. Our findings underscore the need for comparative toxicity studies to ensure comprehensive protection of all pollinators and the importance of accounting for long term exposure scenarios in risk assessment. By enhancing our understanding of chronic pesticide effects in solitary bees, our study should contribute to the development of more effective conservation strategies and sustainable agricultural practices.