Does the use of chlorantraniliprole during queen development adversely impact health and viability?

The queen is the sole reproductive individual and the maturing brood replenishes the shorter-lived worker bees. Production of many crops relies on both pesticides and bee pollination to improve crop quantity and quality. Despite the certain knowledge on chemical pesticides caused damage to worker bee physiology and behavior, our understanding of the relationship between honeybee queen development and chemical pesticides remains weak. Here, we comprehensive investigate the effects of the widely used insecticide chlorantraniliprole on the growth, hormone levels, and detoxifying enzyme activity of queen larvae. It has been determined that chlorantraniliprole present a chronic toxic effect on queen larvae and also reduced the fitness of queen, and that these effects are positively correlated with pesticide levels. It has been found that queen larvae began to show reduced capping and emergence rates when exposed to 2 ng/larva of chlorantraniliprole. At 20 ng/larva, queen capping and emergence rates were the lowest, and there were significant reductions in larval hormone level. Chlorantraniliprole have an effect on detoxification enzyme activity and hormone levels in queen larvae. In conclusion, chlorantraniliprole can adversely affect the growth and development of queen larvae. Our findings may guide the scientifically sound use of chemical pesticides to reduce potential risks to queen larvae.

Exposure to sublethal concentrations of thiacloprid insecticide modulated the expression of microRNAs in honeybees (Apis mellifera L.).

This study aimed to investigate the sublethal effects of thiacloprid on microRNA (miRNA) expression in honeybees (Apis mellifera L.) and the role of a specific miRNA, ame-miR-283-5p, in thiacloprid resistance. The high-throughput small RNA-sequencing was used to analyze global miRNA expression profile changes in honeybees orally exposed to sublethal concentrations of thiacloprid (20 mg/L and 4 mg/L) for 72 h. Thiacloprid at 20 mg/L had no observed adverse effects. In addition, bees were fed with miRNA mimics or inhibitors to increase or decrease ame-miR-283-5p expression, and its effects on P450 gene expression (CYP9Q2 and CYP9Q3) were examined. Thiacloprid susceptibility was also detected. The results showed that treatment with thiacloprid at 20 mg/L and 4 mg/L induced 11 and five differentially expressed miRNAs (DEMs), respectively. Bioinformatic analysis suggested that the DEMs are mainly involved in the regulation of growth and development, metabolism, nerve conduction, and behavior. ame-miR-283-5p was downregulated by both concentrations, which was validated using quantitative real-time reverse transcription PCR analysis. Enhancing ame-miR-283-5p expression significantly inhibited CYP9Q2 mRNA and protein expression, and increased thiacloprid toxicity, while reducing ame-miR-283-5p expression significantly promoted CYP9Q2 expression, and decreased thiacloprid susceptibility. Our study revealed a novel role of miRNAs in insecticide resistance in honeybees.

Risk assessment of honeybee larvae exposure to pyrethroid insecticides in beebread and honey.

Honeybee is an essential pollinator to crops, evaluation to the risk assessment of honeybee larvae exposure to pesticides residue in the bee bread and honey is an important strategy to protect the bee colony due to the mixture of these two matrices is main food for 3-day-old honeybee larvae. In this study, a continuous survey to the residue of five pyrethroid insecticides in bee bread and honey between 2018 and 2020 from 17 major cultivation provinces which can be determined as Northeast, Northwest, Eastern, Central, Southwest, and Southern of China, there was at least one type II pyrethroid insecticide was detected in 54.7 % of the bee bread samples and 43.4 % of the honey. Then, we assayed the acute toxicity of type II pyrethroid insecticides based on the detection results, the LD50 value was 0.2201 µg/larva (beta-cyhalothrin), 0.4507 µg/larva (bifenthrin), 2.0840 µg/larva (fenvalerate), 0.0530 µg/larva (deltamethrin), and 0.1640 µg/larva (beta-cypermethrin), respectively. Finally, the hazard quotient was calculated as larval oral ranged from 0.046 × 10-3 to 2.128 × 10-3. Together, these empirical findings provide further insight into the accurate contamination of honey bee colonies caused by chemical pesticides, which can be used as a valuable guidance for the beekeeping industry and pesticide regulation.

A Blockchain-Based Authentication and Authorization Scheme for Distributed Mobile Cloud Computing Services.

Authentication and authorization constitute the essential security component, access control, for preventing unauthorized access to cloud services in mobile cloud computing (MCC) environments. Traditional centralized access control models relying on third party trust face a critical challenge due to a high trust cost and single point of failure. Blockchain can achieve the distributed trust for access control designs in a mutual untrustworthy scenario, but it also leads to expensive storage overhead. Considering the above issues, this work constructed an authentication and authorization scheme based on blockchain that can provide a dynamic update of access permissions by utilizing the smart contract. Compared with the conventional authentication scheme, the proposed scheme integrates an extra authorization function without additional computation and communication costs in the authentication phase. To improve the storage efficiency and system scalability, only one transaction is required to be stored in blockchain to record a user's access privileges on different service providers (SPs). In addition, mobile users in the proposed scheme are able to register with an arbitrary SP once and then utilize the same credential to access different SPs with different access levels. The security analysis indicates that the proposed scheme is secure under the random oracle model. The performance analysis clearly shows that the proposed scheme possesses superior computation and communication efficiencies and requires a low blockchain storage capacity for accomplishing user registration and updates.

Community composition, bacterial symbionts, antibacterial and antioxidant activities of honeybee-associated fungi.

BACKGROUND: Fungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica remains elusive. RESULTS: Here, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 µg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 µg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus. CONCLUSIONS: This research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.

Proteome analysis reveals the molecular basis of honeybee brain and midgut response to sulfoxaflor.

Sulfoxaflor is a widely used pesticide in agriculture. However, the molecular effects of sublethal sulfoxaflor on honeybees (Apis mellifera L.) remain elusive. Here, the effects of a sublethal dose of sulfoxaflor (0.05 µg/bee) on the brain and midgut proteome response of the honeybee were investigated. Exposure to sublethal sulfoxaflor doses did not cause significant honeybee death, but it induced significant alterations in the brain and midgut proteomes. After sulfoxaflor challenge, 135 and 28 proteins were differentially regulated in the brain and midgut, respectively. The up-regulated proteins were mainly implicated in energy metabolism, neurotransmitter transport and drug metabolism processes, and included in particular enzymes of the citrate cycle and cellular respiration process, such as ATP citrate synthase, malate dehydrogenase, cytochrome b-c1 complex subunits, and NADH dehydrogenase. These findings suggest that honeybees enhance energy metabolism in the midgut and brain to resist sulfoxaflor challenge. Notably, treatment with sulfoxaflor resulted in a 6.8 times increase in expression levels of the major royal jelly protein 1 (MRJP1) in the brain, and knockdown of MRJP1 mRNA expression using RNA interference significantly decreased the survival rate, indicating that MRJP1 may play an important role in sulfoxaflor tolerance. Our data reveals that sulfoxaflor influences multiple processes related to both metabolism and the nervous system, and provides novel insights into the molecular basis of the honeybee brain and midgut response to sublethal dose of sulfoxaflor.

Annexin A1-derived peptide Ac2-26 facilitates wound healing in diabetic mice.

Impaired wound healing is a common complication of diabetes. In diabetic wounds, macrophages present dysfunctional efferocytosis and abnormal phenotypes, which could result in excessive neutrophil accumulation and prolonged inflammation, thereby eventually hindering wound repair. ANXA1 N-terminal peptide Ac2-26 exhibits a high potential in mitigating inflammation and improving repair; however, its efficacy in diabetic wound repair remains unclear. In this study, a cutaneous excisional wound model was built in genetically diabetic mice. Ac2-26 or a vehicle solution was employed locally in wound sites. Subsequently, wound zones were measured and sampled at different time intervals post-wounding. Using hematoxylin-eosin and Masson's trichrome staining, we observed the histopathological variations and collagen deposition in wound samples. Based on immunohistochemistry and immunofluorescence, the numbers of neutrophils, macrophages, and CD206-positive macrophages in the wound samples were determined. Cytokine expression in wound samples was studied by immunoblot assay. Results showed that Ac2-26 treatment could facilitate diabetic wound closure, down-regulate the number of neutrophils, and improve angiogenesis and collagen deposition. In addition, Ac2-26 application expedited macrophage recruitment and up-regulated the percentage of macrophages expressing CD206, which is a marker for M2 macrophages. Moreover, Ac2-26 inhibited the expressions of TNF-α and IL-6 and up-regulated the expressions of IL-10, TGF-ß, and VEGFA during diabetic wound healing. Hence, based on the aforementioned findings, Ac2-26 application in diabetic wounds could exert anti-inflammatory and pro-repair effects by reducing neutrophil accumulation and facilitating M2 macrophage development.

Metabolomic analysis of honey bee, Apis mellifera L. response to thiacloprid.

The cyano-substituted neonicotinoid insecticide, thiacloprid, is nowadays widely used in agriculture for controlling insect pests. However, it also simultaneously has adverse effects on the health of important pollinators, such as honey bees. Previous studies have reported that sublethal doses of neonicotinoids impaired immunocompetence, learning and memory performance, and homing behaviour in honey bees. In the present study, using LC-MS-based combined with GC-MS-based metabolomic approaches, we profiled the metabolic changes that occur in the head of honey bee after subchronic exposure to 2 mg/L thiacloprid over 3 days. The estimated total dose of thiacloprid fed to each bee was 0.12 µg. The results showed that there were 115 metabolites significantly affected in thiacloprid-treated bees compared to control. The metabolites with high level of abundance enriched to wide range pathways associated with oxidative stress and detoxification suggest that the honey bees have activated their detoxification system to resistant toxicity of thiacloprid. While, the reduction of serotonin suggest thiacloprid may hinder the brain activity implicated in learning and behaviour development. Our study expand the understanding of the molecular basis of the complex interactions between neonicotinoids and honey bees.

Effects of Field-Realistic Concentrations of Carbendazim on Survival and Physiology in Forager Honey Bees (Hymenoptera: Apidae).

Carbendazim is nowadays widely used to control fungus in various nectariferous crops. Little is known about how honey bees, Apis mellifera L. (Hymenoptera: Apidae), respond to carbendazim exposure. In this study, the effects of field-realistic concentrations of carbendazim (4.516, 0.4516, and 0.04516 ppm) on the survival, biomarker enzyme activity (AChE, GST, CarE, and P450), and four antimicrobial peptide gene expression (hymenoptaecin, defensin, apidaecin, and abaecin) in forager honey bees were evaluated. The forager bees were fed with the pesticides for 10 d. The results showed that the field-realistic concentrations of carbendazim did not affect survival; activities of AChE, GST, and CarE; and expression levels of defensin and abaecin in forager bees. However, 4.516, 0.4516, and 0.04516 ppm of carbendazim all significantly inhibited the expression of hymenoptaecin and apidaecin (P < 0.01), while P450 (7-ethoxycoumarin-O-deethylase) activity was downregulated by 4.516 ppm of carbendazim (P < 0.05). Our results indicate that the field-realistic concentrations of carbendazim may alter the immune response and P450-mediated detoxification of honey bees. Thus, carbendazim should be discreetly used on nectariferous crops during florescence.

Evaluation of Highly Detectable Pesticides Sprayed in Brassica napus L.: Degradation Behavior and Risk Assessment for Honeybees.

Honeybees are major pollinators of agricultural crops and many other plants in natural ecosystems alike. In recent years, managed honeybee colonies have decreased rapidly. The application of pesticides is hypothesized to be an important route leading to colony loss. Herein, a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was used to determine eight highly detectable pesticides (carbendazim, prochloraz, pyrimethanil, fenpropathrin, chlorpyrifos, imidacloprid, thiamethoxam, and acetamiprid) in rape flowers. A field experiment was conducted at the recommended dose to evaluate the contact exposure risk posed to honeybees for 0⁻14 days after treatment. The initial residue deposits of neonicotinoids and fungicides among these compounds were 0.4⁻1.3 mg/kg and 11.7⁻32.3 mg/kg, respectively, and 6.4 mg/kg for fenpropathrin and 4.2 mg/kg for chlorpyrifos. The risk was quantified using the flower hazard quotient (FHQ) value. According to the data, we considered imidacloprid, thiamethoxam, chlorpyrifos, fenpropathrin, and prochloraz to pose an unacceptable risk to honeybees after spraying in fields, while fungicides (carbendazim and pyrimethanil) and acetamiprid posed moderate or acceptable risks to honeybees. Therefore, acetamiprid can be used instead of imidacloprid and thiamethoxam to protect rape from some insects in agriculture, and the application of prochloraz should be reduced.

Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10 days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10 days post-injury, which were >1.8 at 10 days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

Occurrence of erythromycin and its degradation products residues in honey. Validation of an analytical method.

Erythromycin A, the main component of erythromycin, is widely used to treat and control foulbrood diseases in honey bees. In this study, we developed a fast and sensitive method to simultaneously determine erythromycin A and its degradation products in honey. The analytical methodology was based on dispersive liquid-liquid microextraction and liquid chromatography coupled with tandem mass spectrometry with advanced i-Funnel technology. The liquid-liquid microextraction and liquid chromatography coupled with tandem mass spectrometry parameters were optimized. The recoveries of erythromycin A and its degradation products from spiked honey samples were 76.1-102.1%, with reproducibility rates of 7.1-13.1% and correlation coefficients  >0.99. The decision limit and detection capability were 0.02-0.07 and 0.03-0.10 ng/g, respectively. The proposed method was validated and successfully applied to the determination of the target analytes in commercial honey samples. It was efficient and sensitive, and it lays the foundation for further research on honey safety.

Influence of the Neonicotinoid Insecticide Thiamethoxam on miRNA Expression in the Honey Bee (Hymenoptera: Apidae).

MicroRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs regulating gene expression in eukaryotes. They play important roles in regulating caste differentiation, behavior development, and immune defences in the honey bee, Apis mellifera (Linnaeus) (Hymenoptera: Apidae). In this study, we explored the effect of the neonicotinoid insecticide, thiamethoxam, on miRNA expression in this species using deep small RNA sequencing. The results showed that seven miRNAs were significantly differentially expressed (q-value <0.01 and |log2(fold-change)| >1) upon exposure to 10 ppb thiamethoxam over 10 d. Some candidate target genes were related to behavior, immunity, and neural function. Several miRNAs, including ame-miR-124, ame-miR-981, ame-miR-3791, and ame-miR-6038, were selected and further validated using real-time quantitative PCR analysis. The findings expand our understanding of the effects of neonicotinoid insecticides on honey bees at the molecular level.

Genetic structure of Mount Huang honey bee (Apis cerana) populations: evidence from microsatellite polymorphism.

BACKGROUND: The Mount Huang eastern honey bees (Apis cerana) are an endemic population, which is well adapted to the local agricultural and ecological environment. In this study, the genetic structure of seven eastern honey bees (A. cerana) populations from Mount Huang in China were analyzed by SSR (simple sequence repeat) markers. RESULTS: The results revealed that 16 pairs of primers used amplified a total of 143 alleles. The number of alleles per locus ranged from 6 to 13, with a mean value of 8.94 alleles per locus. Observed and expected heterozygosities showed mean values of 0.446 and 0.831 respectively. UPGMA cluster analysis grouped seven eastern honey bees in three groups. CONCLUSION: The results obtained show a high genetic diversity in the honey bee populations studied in Mount Huang, and high differentiation among all the populations, suggesting that scarce exchange of honey bee species happened in Mount Huang. Our study demonstrated that the Mount Huang honey bee populations still have a natural genome worth being protected for conservation.

Multi-Residue Analysis of Pesticide Residues in Crude Pollens by UPLC-MS/MS.

A multi-residue method for the determination of 54 pesticide residues in pollens has been developed and validated. The proposed method was applied to the analysis of 48 crude pollen samples collected from eight provinces of China. The recovery of analytes ranged from 60% to 136% with relative standard deviations (RSDs) below 30%. Of the 54 targeted compounds, 19 pesticides were detected. The major detection rates of each compound were 77.1% for carbendazim, 58.3% for fenpropathrin, 56.3% for chlorpyrifos, 50.0% for fluvalinate, 31.3% for chlorbenzuron, and 29.2% for triadimefon in crude pollen samples. The maximum values of each pesticide were 4516 ng/g for carbendazim, 162.8 ng/g for fenpropathrin, 176.6 ng/g for chlorpyrifos, 316.2 ng/g for fluvalinate, 437.2 ng/g for chlorbenzuron, 79.00 ng/g for triadimefon, and so on. This study provides basis for the research on the risks to honeybee health.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Tumor-Derived Membrane Vesicles Restrain Migration in Gliomas By Altering Collective Polarization.

Mechanobiology is a cornerstone in physiology. However, its role in biomedical applications remains considerably undermined. In this study, we employed cell membrane vesicles (CMVs), which are currently being used as nanodrug carriers, as tactile cues for mechano-regulation of collective cell behaviors. Gliomas, which are among the most resilient brain tumors and have a low patient survival rate, were used as the cell model. We observed that mechanical responses due to the application of glioma- or microglia-derived CMVs resulted in the doubling of the traction stress of glioma cell collectives with a 10-fold increase in the CMV concentration. Glioma-CMVs constrained cell protrusions and hindered their collective migration, with the migration speed of such cells declining by almost 40% compared to the untreated cells. We speculated that the alteration of collective polarization leads to migration speed changes, and this phenomenon was elucidated using the cellular Potts model. In addition to intracellular force modulation and cytoskeletal reorganization, glioma-CMVs altered drug diffusion within glioma spheroids by downregulating the mechano-signaling protein YAP-1 while also marginally enhancing the associated apoptotic events. Our results suggest that glioma-CMVs can be applied as an adjuvant to current treatment approaches to restrict tumor invasion and enhance the penetration of reagents within tumors. Considering the broad impact of mechano-transduction on cell functions, the regulation of cell mechanics through CMVs can provide a foundation for alternative therapeutic strategies.

Digestion dynamics of acetamiprid during royal jelly formation and exposure risk assessment to honeybee larva based on processing factor.

Previous studies to the exposure effects of acetamiprid on honeybees were based on the analysis of bee pollen and honey sacs from field trials or of beebread and honey in the hive, which overestimate or underestimate the risk of exposure to pesticide residues. It was believed that the processing factor (PF) is an important variable to determine the final pesticide residue during royal jelly formation and the actual risk to honeybee larva. Hence, a QuEChERS method to determine acetamiprid contents in honeybee samples was established in this study. Then, the PFs for acetamiprid in beebread fermentation, honey brewing, and royal jelly formation were determined to be 0.85, 0.76, and 0.16, respectively. The PF for royal jelly formation was 0.04 when acetamiprid was detected in beebread alone, and it was 0.12 when acetamiprid was only detected in honey. Finally, the predicted exposure concentration of acetamiprid in royal jelly was calculated to be 2.05 µg/kg using the PF without significant difference with the 90th percentile value (3.64 µg/kg) in the actual sample. However, the value was 16.62 µg/kg without considering the PF. This study establishes a methodology for the correct evaluation of the risk to bee larva of acetamiprid residues in bee pollen and honey sac contents and the residual levels in royal jelly.

Apilactobacillus kunkeei Alleviated Toxicity of Acetamiprid in Honeybee.

Nowadays, colony collapse disorder extensively affects honeybees. Insecticides, including acetamiprid, are considered as critical factors. As prevalent probiotics, we speculated that supplementation with lactic acid bacteria (LAB) could alleviate acetamiprid-induced health injuries in honeybees. Apilactobacillus kunkeei was isolated from beebread; it significantly increased the survival of honeybees under acetamiprid exportation (from 84% to 92%). Based on 16S rRNA pyrosequencing, information on the intestinal bacteria of honeybees was acquired. The results showed that supplementation with A. kunkeei significantly increased survival and decreased pollen consumption by honeybees under acetamiprid exportation. Under acetamiprid exportation, some opportunistic and pathogenic bacteria invaded the intestinal regions. Subsequently, the community richness and diversity of symbiotic microbiota were decreased. The community structure of intestinal bacteria was changed and differentiated. However, with the supplementation of A. kunkeei, the community richness and community diversity of symbiotic microbiota showed an upward trend, and the community structure was stabilized. Our results showed that A. kunkeei alleviated acetamiprid-induced symbiotic microbiota dysregulation and mortality in honeybees. This demonstrates the importance of symbiotic microbiota in honeybees and supports the application of Apilactobacillus kunkeei as probiotics in beekeeping.