Honey Bee Health Research Articles

Discovery of reproductive tissue-associated bacteria and the modes of microbiota acquisition in male honey bees (drones).

Over the last decade, annual honey bee colony loss has increased, resulting in a critical need to determine what factors contribute to honey bee and colony health. Gut microbes have been shown to play important roles in the health of the nonreproductive female honey bee workers, which make up 90% or more of a honey bee colony. However, we currently know very little about the impact of microbes on the health of male honey bees (drones), who only make up a small portion of the colony population but play a very key role in the success of future colonies by mating with virgin queens. Here, we discovered microbes within the reproductive organs of drones and illustrated that social interactions with worker bees are necessary for the proper development of the gut and reproductive tissue microbial communities in drones. Further studies are needed to determine if microbes play an important role in honey bee reproductive health and fitness.

A novel antibody treatment reduces deformed wing virus loads in the western honey bee (Apis mellifera).

Deformed wing virus (DWV) is considered to be a key component of declining honey bee health which threatens global food production. The virus can result in significantly shortened lifespan, deformities in developing bees, and impaired cognition. There is currently no method to directly control the virus. The virus can be indirectly controlled with acaricidal treatments that target a key vector, the parasitic varroa mite (Varroa destructor). But acaricide resistance and a lack of effective alternatives for the control of both Varroa and DWV are major threats to beekeeping and the wider agricultural industry. Our research presents a significant development in the ability to reduce DWV burden in honey bees using IgY antibodies. Moreover, immunoglobulin Y has the potential to be more broadly established as a new treatment modality to combat other pathogens and parasites in A. mellifera.

Proteome of Clothianidin Exposed Honey Bees Reveals a Possible Mechanism Behind Impairment of Sucrose Responsiveness

ABSTRACTNeonicotinoids (NNIs) are the most commonly used insecticides in the world and clothianidin, a type of NNI, is commonly found in honey bee collected pollen. Clothianidin has several negative effects on honey bee health and behaviour, but whether and how it might impact learning and memory remains unclear. Therefore, we exposed honey bee workers to a field‐realistic oral dose of clothianidin for 7 days to assess their sugar responsiveness, and olfactory learning and memory using the proboscis extension response paradigm. Sugar responsiveness impacts important colony‐level traits, such as onset of foraging, that help maintain colony homeostasis as well as impacts learning and memory. We then measured how clothianidin alters protein expression in the brain in an effort to understand the mechanism(s) of clothianidin's effects. Clothianidin‐exposed bees had impaired sugar responsiveness, but no effect was seen on learning and memory. We identified 5069 protein groups and showed that the exposed bees had fewer detectable proteins. The exposed bees also had fewer mitochondrial respiration proteins, as well as reduced levels of nicotinic acetylcholine receptor α7 (nAChRα7) subunit (one of the targets of clothianidin) and reduced levels of acetylcholinesterase and acetylcholinesterase‐2 levels. In the exposed bees, the levels of nAChRα7 subunit positively correlated with sugar responsiveness, providing evidence for a possible mechanical explanation.

The Genetic Characterization of Lake Sinai Virus in Colony Losses Apiaries in Türkiye

Objective: Honeybees (Apis mellifera) have a unique role in natural pollination and maintaining biodiversity in the ecosystem. The alarming increase in unexpected colony losses, mysterious bee deaths, and the tragic extinction of entire colonies (Colony collapsed disorder- CCD) have sounded a global alarm, demanding immediate attention and collaborative action to address these critical challenges in bee breeding. Diseases, parasites, and pathogens significantly threaten colony health. Türkiye is a significant honey producer, providing an ideal environment for beekeeping due to its unique eco-geographical features. Unexpected colony losses and bee deaths are also questions of concern for beekeepers in Türkiye. Material Method: In this study investigated honey bee viruses in apiaries experiencing sudden bee death losses and CCD-like symptoms between 2021 and 2023 in Türkiye, involving genetic analysis of the LSV RdRp gene region. The honeybee and varroa samples were obtained from 52 colonies in 26 apiaries complaining of unexpected bee deaths and CCD-like symptoms between May 2021 and September 2023. Result: The results showed a high DWV, BQCV, and LSV prevalence, respectively. The sampled apiaries were infested mild-moderate- high grade with Varroa mites. Following PCR results, DWV, BQCV, LSV, IAPV, CBPV, and SBV positivity was detected at 69.2% (n=18), 50% (n=13), 38.46% (n=10), 26.9% (n=7), 19.2% (n=5) and 3.8% (n=1), respectively. High rates of multiple virus coexisting and high varroa infestation were noted in colonies with heavy losses and CCD-like complaints. The RdRp gene from two LSV samples (TrLSV-6474, TrLSV-6517) was sequenced. Turkish LSV samples (TrLSVs) showed a 72.88% homology of each other and clustered LSV4 branches in the phylogenetic tree. Turkish LSV sequences showed a closer similarity rate than reference sequences in GenBank with Asian Korean, Chinese, and Japanese LSV sequences. Conclusion: Further investigation is needed to comprehend the implications of elevated LSV populations on colony losses. The execution of genetic research with a more extensive sample size can significantly enhance the demonstration of species diversity and provide valuable insights into the influence of LSV variants on honeybee health and the management of diseases.

Convergent reductive evolution in bee-associated lactic acid bacteria.

Distantly related organisms may evolve similar traits when exposed to similar environments or engaging in certain lifestyles. Several members of the Lactobacillaceae [lactic acid bacteria (LAB)] family are frequently isolated from the floral niche, mostly from bees and flowers. In some floral LAB species (henceforth referred to as bee-associated LAB), distinctive genomic (e.g., genome reduction) and phenotypic (e.g., preference for fructose over glucose or fructophily) features were recently documented. These features are found across distantly related species, raising the hypothesis that specific genomic and phenotypic traits evolved convergently during adaptation to the floral environment. To test this hypothesis, we examined representative genomes of 369 species of bee-associated and non-bee-associated LAB. Phylogenomic analysis unveiled seven independent ecological shifts toward the bee environment in LAB. In these species, we observed significant reductions of genome size, gene repertoire, and GC content. Using machine leaning, we could distinguish bee-associated from non-bee-associated species with 94% accuracy, based on the absence of genes involved in metabolism, osmotic stress, or DNA repair. Moreover, we found that the most important genes for the machine learning classifier were seemingly lost, independently, in multiple bee-associated lineages. One of these genes, acetaldehyde-alcohol dehydrogenase (adhE), encodes a bifunctional aldehyde-alcohol dehydrogenase which has been associated with the evolution of fructophily, a rare phenotypic trait that is pervasive across bee-associated LAB species. These results suggest that the independent evolution of distinctive phenotypes in bee-associated LAB has been largely driven by independent losses of the same sets of genes.IMPORTANCESeveral LAB species are intimately associated with bees and exhibit unique biochemical properties with potential for food applications and honeybee health. Using a machine learning-based approach, our study shows that adaptation of LAB to the bee environment was accompanied by a distinctive genomic trajectory deeply shaped by gene loss. Several of these gene losses occurred independently in distantly related species and are linked to some of their unique biotechnologically relevant traits, such as the preference for fructose over glucose (fructophily). This study underscores the potential of machine learning in identifying fingerprints of adaptation and detecting instances of convergent evolution. Furthermore, it sheds light onto the genomic and phenotypic particularities of bee-associated bacteria, thereby deepening the understanding of their positive impact on honeybee health.

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.

Monitoring for Amoebic Disease (Malpighamoeba mellificae) in Honey Bee Colonies

Digestive health is an overlooked but vital component of the success of Western honey bee (Apis mellifera) colonies. Viral, bacterial, fungal, and protozoal infections can inhibit nutrient uptake and waste elimination by the honey bee digestive tract. Active management requires monitoring honey bee health on an individual and colony basis, providing adequate nutrition, and controlling for other pests and diseases. One organism that has gained recent attention is Malpighamoeba mellificae. This publication describes how to detect M. mellificae in A. mellifera colonies.

A case for microbial therapeutics to bolster colony health and performance of honey bees

A case for microbial therapeutics to bolster colony health and performance of honey bees

Microplastic ingestion and co-exposure to Nosema ceranae and flupyradifurone reduce the survival of honey bees (Apis mellifera L.)

Bees are exposed to several threats, including pathogens (i.e. Nosema ceranae), pesticides and environmental contaminants. The new insecticide flupyradifurone, and the microplastics in the environment, have raised significant concerns on bee health. This study evaluated the simultaneous effects of microplastics, flupyradifurone, and N. ceranae on honey bee health, focusing on survival rates, N. ceranae replication, daily food consumption, and bee midgut histological alterations. Results showed a significant decrease in bee longevity across all treatments compared to the control, with the combination of flupyradifurone, microplastics, and N. ceranae having the most severe impact. Microplastics and flupyradifurone exposure also increased N. ceranae proliferation, especially in bees subjected to both stressors. Histological analysis revealed reduced regenerative cell nests in the midgut and changes in the nuclear matrix, indicating stress responses. Overall, the simultaneous presence of both biotic and abiotic stressors in nature can synergistically interact, leading to harmful effects on bees.

Molecular detection and phylogenetic analysis of six bee viruses from Varroa destructor in Korea

In the beekeeping industry, the control of Varroa destructor and viruses is a crucial factor in maintaining the health of the western honey bee Apis mellifera L. Recently, in Korea, beekeepers suffered for high colony losses during the overwintering, and the presence of V. destructor and viruses has been considered an important factor for honey bee health. In this study, we investigated the presence of six honey bee viruses [deformed wing virus (DWV), sacbrood virus (SBV), Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and black queen cell virus (BQCV)] in V. destructor in Korea. V. destructor was collected from 46 apiaries in nine provinces in Korea, and viruses were detected using reverse transcription–polymerase chain reaction. DWV was most predominantly detected at 87% of apiaries, followed by IAPV, SBV, KBV, BQCV, and CBPV that were detected in 65.2%, 17.4%, 13%, 8.7%, and 6.5% of apiaries in Korea. Phylogenetic analysis revealed that five viruses (IAPV, SBV, BQCV, and CBPV) were grouped into clades of viruses originating from China, Russia, and Australia, but KBV showed no distinct clades of countries. This study confirmed the presence of various types of bee viruses in V. destructor in Korea, indicating that domestic honey bees are exposed to multiple viruses via V. destructor.

The IMPACT OF HONEYBEE GUT BACTERIA ON THE SURVIVAL OF HONEYBEES EXPOSED TO INSECTICIDES

The widespread use of insecticides poses a significant threat to the health and sustainability of honeybees (Apis mellifera). This study aimed to isolate, identify, and investigate the potential of honeybee gut bacterial strains in mitigating the detrimental effects of insecticides on honeybees and extending their lifespan. The efficacy of seven honeybee gut bacterial strains in reducing insecticide toxicity was evaluated. Through the identification of honeybee gut bacterial isolates using 16s rRNA, seven strains were identified, namely Priestia endophytica, Bacillus subtilis, Bacillus pumilus, Peribacillus frigoritolerans, B. subtilis, Bacillus pumilus, and Bacillus tequilensis. The experimental results revealed that bees treated with these gut bacteria significantly reduced oxidative stress markers and detoxification enzyme levels compared to untreated bees. Moreover, the treated bees demonstrated enhanced immune responses. The bees treated with deltamethrin + intestinal bacteria showed an increase in bees' lifespan to 6.33 days compared to a lifespan of 4 days with deltamethrin only. Similarly, bees treated with acetamiprid intestinal bacteria had further extended to 10.33 days compared to a lifespan of 7.67 days with acetamiprid. These findings suggest that using honeybee gut bacterial strains may serve as a sustainable strategy to mitigate the harmful impacts of insecticides on honeybees, thereby promoting their overall health and contributing to the preservation of pollination services and apiculture. Further research is warranted to elucidate the underlying mechanisms and optimize the application of these natural products in honeybee management practices.

New insights into honey bee viral and bacterial seasonal infection patterns using third-generation nanopore sequencing on honey bee haemolymph

Honey bees are rapidly declining, which poses a significant threat to our environment and agriculture industry. These vital insects face a disease complex believed to be caused by a combination of parasites, viruses, pesticides, and nutritional deficiencies. However, the real aetiology is still enigmatic. Due to the conventional analysis methods, we still lack complete insights into the honey bee virome and the presence of pathogenic bacteria. To fill this knowledge gap, we employed third-generation nanopore metagenomic sequencing on honey bee haemolymph to monitor the presence of pathogens over almost a year. This study provides valuable insights into the changes in bacterial and viral loads within honey bee colonies. We identified different pathogens in the honey bee haemolymph, which are not included in honey bee screenings. These pathogens comprise the Apis mellifera filamentous virus, Apis rhabdoviruses, and various bacteria such as Frischella sp. and Arsenophonus sp. Furthermore, a sharp contrast was observed between young and old bees. Our research proposes that transgenerational immune priming may play a role in shaping infection patterns in honey bees. We observed a significant increase in pathogen loads in the spring, followed by a notable decrease in pathogen presence during the summer and autumn months. However, certain pathogens seem to be able to evade this priming effect, making them particularly intriguing as potential factors contributing to mortality. In the future, we aim to expand our research on honey bee transgenerational immune priming and investigate its potential in natural settings. This knowledge will ultimately enhance honey bee health and decrease colony mortality.

Enhancing the Health and Lifespan of Honey Bees (Apis mellifera L.) with an Innovative Soybean-Based Diet Supplemented with Porcine Blood1

Abstract This study investigated the effect of soy-based diets supplemented with porcine blood (PB) on the diet's external morphology, nutritional efficacy, and health impacts on honey bees, Apis mellifera L. (Hymenoptera: Apidae). Scanning electron microscopy revealed that the control diet (maize bee bread) displayed ruptured granules with small particles, whereas the soy-based diets showed varying degrees of protein gelatinization and aggregation depending on the presence of PB. Nutritional analysis indicated that maize bee bread was used as a positive control contained 12.2% protein and provided 312.89 kcal/100 g, whereas the formulated soy-based diets with skim milk or PB showed 13–15% protein, meeting the required needs for optimal honey bee health and development. Survival and longevity assessments showed no significant difference in lifespan between honey bees fed with artificial diets and the control, but all treatment groups outlived the negative control, which fed only on syrup. Moreover, the diameter of the hypopharyngeal gland acini, a key indicator of the nutritional state and health in honey bees, was significantly larger in honey bees fed protein-enriched diets compared with those receiving only syrup. These findings underscore the potential of soy-based artificial protein diets, especially when enhanced with PB, to support honey bee health and longevity, comparable with natural pollen sources.

Synergistic effects between microplastics and glyphosate on honey bee larvae

Microplastic (MPs) pollution has emerged as a global ecological concern, however, the impact of MPs exposure, particularly in conjunction with other pollutants such as glyphosate (GLY) on honey bee remains unknown. This study investigated the effects of exposure to different concentrations of MPs and their combination with GLY on honey bee larvae development, or during the larvae period, regulation of major detoxification, antioxidant and immune genes, and oxidative stress biomarkers. Results revealed that combined exposure to MPs and GLY decreased larvae survivorship and weight, while exposure to MPs alone showed no significant differences. Both MPs and GLY alone downregulated the defensin-1 gene, but only combined exposure with GLY downregulated the hymenoptaecin gene and increased catalase enzyme activity. The data suggest a synergistic effect of MPs and GLY, leading to immunosuppression and reduced larvae survival and weight. These findings highlight potential risks of two prevalent environmental pollutants on honey bee health.

Phytochemical profiles of honey bees (Apis mellifera) and their larvae differ from the composition of their pollen diet.

Pollen and nectar consumed by honey bees contain plant secondary metabolites (PSMs) with vital roles in plant-insect interactions. While PSMs can be toxic to bees, they can also be health-promoting, e.g. by improving pesticide and pathogen tolerances. As xenobiotics, PSMs undergo post-ingestion chemical modifications that can affect their bioactivity and transmission to the brood. Despite the importance of understanding honey bee PSM metabolism and distribution for elucidating bioactivity mechanisms, these aspects remain largely unexplored. In this study, we used HPLC-MS/MS to profile 47 pollen PSMs in honey bees and larvae. Both adult bees and larvae had distinct PSM profiles that differed from their diet. This is likely due to post-ingestion metabolism and compound-dependent variations in PSM transmission to the brood via nurse bee jelly. Phenolic acids and flavonoid aglycones were most abundant in bees and larvae, whereas alkaloids, cyanogenic glycosides and diterpenoids had the lowest abundance despite being consumed in higher concentrations. This study documents larval exposure to a variety of PSMs for the first time, with concentrations increasing from early to late larval instars. Our findings provide novel insights into the post-ingestion fate of PSMs in honey bees, providing a foundation for further exploration of biotransformation pathways and PSM effects on honey bee health.

Spermidine supplementation influence on protective enzymes of Apis mellifera (Hymenoptera: Apidae).

Dietary supplementation has been proposed as a sustainable way to improve the health and resilience of honey bees (Apis mellifera, L.), as the decline in their numbers in recent decades has raised scientific, environmental, and economic concerns. Spermidine, a natural polyamine, has been shown to be a promising substance for honey bee supplementation, as its health-promoting effects have been demonstrated in numerous studies and in different organisms. As already shown, supplementation with spermidine at a certain concentration prolonged lifespan, reduced oxidative stress, and increased antioxidative capacity in honey bees. The aim of the present study was to investigate whether spermidine supplementation affects gene expression and/or enzyme activity of antioxidative and detoxification enzymes and immune response markers in honey bee workers. The different gene expression and enzyme activity patterns observed in abdominal and head tissues in response to spermidine supplementation suggest tissue-specific and concentration-dependent effects. In addition, the immune response markers suggest that spermidine has the ability to boost honey bee immunity. The observed changes make a valuable contribution to understanding the molecular mechanisms by which spermidine may exert its beneficial effects on the bee's health and lifespan. These results support the idea of the use of spermidine supplementation to promote bee health and resilience to environmental stressors, emphasizing that the dose must be carefully chosen to achieve a balance between the pro- and antioxidant effects of spermidine.

Acaricidal properties of Corsican Humulus lupulus L. (Cannabaceae) essential oils against Varroa destructor: a honeybee health perspective

ABSTRACT Six Corsican essential oils (EOs) from Humulus lupulus were studied for their activity against Varroa destructor adults and Apis mellifera pupae. The GC/MS analysis of EOs showed that the major compounds of all accessions were α-Humulene, Myrcene, α-Selinene, β-Selinene, (E)-β-Farnesene, and (E)-β-Caryophyllene. Bioassays showed that EOs from each accession acted as toxins and repellents against V. destructor adults, with lower toxicity towards A. mellifera pupae. In terms of efficacy against V. destructor adults, Smaragd 2020 oil displayed the highest performance, with LC50 and LC90 values of 2.33 and 7.83 µL/L air, respectively. Meanwhile, Smaragd 2019 and Tettnanger EOs showed moderate toxicity, with LC50 values of 3.101 and 3.66 µL/L air, along with LC90 values of 11.67 and 19.89µL/L air, respectively. The oils from Biguglia and Ajaccio were strongly repellent to Varroa adults. At the lowest concentration (2 µL/mL), these EOs exhibited percentage of repellence greater than 70% after 2h exposure, with RC50 values of 1.66 and 1.26 µL/L air, respectively. Concerning their insecticidal effects, EOs demonstrate reduced toxicity towards bee pupae when compared to Varroa mites. Notably, Ajaccio stands out for inducing lower mortality among bee pupae, with a selectivity ratio below 2 and LC50 of 11.25 µL/L air.

Exploring bee venom and silver nanoparticles for controlling foulbrood pathogen and enhancing lifespan of honeybees

The beekeeping industry plays a crucial role in local economies, contributing significantly to their growth. However, bee colonies often face the threat of American foulbrood (AFB), a dangerous disease caused by the Gram-positive bacterium Paenibacillus larvae (P. l.). While the antibiotic Tylosin has been suggested as a treatment, its bacterial resistance necessitates the search for more effective alternatives. This investigation focused on evaluating the potential of bee venom (BV) and silver nanoparticles (Ag NPs) as antibacterial agents against AFB. In vitro treatments were conducted using isolated AFB bacterial samples, with various concentrations of BV and Ag NPs (average size: 25nm) applied individually and in combination. The treatments were administered under both light and dark conditions. The viability of the treatments was assessed by monitoring the lifespans of treated bees and evaluating the treatment's efficiency within bee populations. Promising results were obtained with the use of Ag NPs, which effectively inhibited the progression of AFB. Moreover, the combination of BV and Ag NPs, known as bee venom/silver nanocomposites (BV/Ag NCs), significantly extended the natural lifespan of bees from 27 to 40 days. Notably, oral administration of BV in varying concentrations (1.53, 3.12, and 6.25 mg/mL) through sugary syrup doubled the bees' lifespan compared to the control group. The study established a significant correlation between the concentration of each treatment and the extent of bacterial inhibition. BV/Ag NCs demonstrated 1.4 times greater bactericidal efficiency under photo-stimulation with visible light compared to darkness, suggesting that light exposure enhances the effectiveness of BV/Ag NCs. The combination of BV and Ag NPs demonstrated enhanced antibacterial efficacy and prolonged honeybee lifespan. These results offer insights that can contribute to the development of safer and more efficient antibacterial agents for maintaining honeybee health.

From molecules to organisms: A multi-level approach shows negative effects of trace elements from sewage sludge used as soil improver on honeybees

The use of sewage sludge as a soil improver has been promoted in agroecosystems. However, sludges can contain toxic trace elements because of suboptimal wastewater treatment. Nonetheless, field studies investigating the negative effects of these practices on pollinators are lacking. We collected honeybees from an area where sewage sludge use is widespread, and one where it is precluded. Trace elements in soils and bees were quantified. Cadmium, chromium, lead, mercury, and nickel were investigated because they were the least correlated elements to each other and are known to be toxic. Their levels were related to oxidative stress and energy biomarkers, midgut epithelial health, body size and wing asymmetry of honeybees. We found increased carbohydrate content in sites with higher cadmium levels, increased histological damage to the midgut epithelium in the sewage sludge area, and the presence of dark spherites in the epithelium of bees collected from the sites with the highest lead levels. Finally, we found that honeybees with the highest lead content were smaller, and that wing fluctuating asymmetry increased in sites with increasing levels of mercury. To the best of our knowledge, this is the first comprehensive study of the concentration and effects on honeybees of trace elements potentially deriving from soil amendment practices.

Assessment of Lambda-Cyhalothrin and Spinetoram Toxicity and Their Effects on the Activities of Antioxidant Enzymes and Acetylcholinesterase in Honey Bee (Apis mellifera) Larvae.

Honeybees play a crucial role as agricultural pollinators and are frequently exposed to various pollutants, including pesticides. In this study, we aimed to evaluate the toxicity of lambda-cyhalothrin (LCY) and spinetoram (SPI) in honey bee larvae reared in vitro through single (acute) and repeated (chronic) exposure. The acute LD50 values for LCY and SPI were 0.058 (0.051-0.066) and 0.026 (0.01-0.045) μg a.i./larva, respectively. In chronic exposure, the LD50 values of LCY and SPI were 0.040 (0.033-0.046) and 0.017 (0.014-0.019) μg a.i./larva, respectively. The chronic no-observed-effect dose of LCY and SPI was 0.0125 μg a.i./larva. Adult deformation rates exceeded 30% in all LCY treatment groups, showing statistically significant differences compared to the solvent control group (SCG). Similarly, SPI-treated bees exhibited significantly more deformities than SCG. Furthermore, we examined the activities of several enzymes, namely, acetylcholinesterase (AChE), glutathione-S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD), in larvae, pupae, and newly emerged bees after chronic exposure at the larval stage (honey bee larval chronic LD50, LD50/10 (1/10th of LD50), and LD50/20 (1/20th of LD50)). LCY and SPI induced significant changes in detoxification (GST), antioxidative (SOD and CAT), and signaling enzymes (AChE) during the developmental stages (larvae, pupae, and adults) of honey bees at sublethal and residue levels. Our results indicate that LCY and SPI may affect the development of honey bees and alter the activity of enzymes associated with oxidative stress, detoxification, and neurotransmission. These results highlight the potential risks that LCY and SPI may pose to the health and normal development of honey bees.