Apilactobacillus kunkeei Alleviated Toxicity of Acetamiprid in Honeybee

Simple Summary The honeybee is an important pollinator and is key in maintaining ecological balance. Insecticides, especially neonicotinoids, are considered as critical factors in colony collapse disorder. However, the question of how to reduce the toxic effect of pesticides on bees has not been comprehensively answered. Probiotics are an important and valid way to combat stress and have the benefit of maintaining healthy honeybees. Our study found that Apilactobacillus kunkeei, which was isolated from beebread, can reduce the mortality effect of acetamiprid on honeybees. However, the mechanism is not clear, and we attempted to elaborate on it, based on the symbiotic honeybee microbiota. We found that some opportunistic and pathogenic bacteria invaded the intestinal regions of honeybees under acetamiprid exportation. Meanwhile, the community richness and diversity of symbiotic microbiota were decreased, and the community structure of intestinal bacteria was changed and differentiated. However, with the supplementation of A. kunkeei, the community richness and diversity of symbiotic microbiota showed an upward trend, and the community structure was stabilized. These data suggest that A. kunkeei may be beneficial to a stabilized community structure which reduces the toxic effects of acetamiprid on honeybees. Our results offer important insights into the application of probiotics and potential probiotics in beekeeping. Abstract 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.


Introduction
As an important kind of pollinator, the western honeybee (Apis mellifera L.) plays an essential role in maintaining ecological balance and provides huge commercial value. acetamiprid toxicity and mortality, as well as attenuating acetamiprid-induced microbiota dysregulation, in honeybees.

Honeybee Rearing
Brood frames were collected from two healthy colonies in the Beekeeping Research Institute, Anhui Agricultural University, Hefei, China. The Varroa destructor population was controlled using Amitraz one month prior to starting the experiment. Late-stage pupae were removed from the colonies and placed into growth chambers at 33 ± 1 • C in darkness and high humidity (relatively humidity 60%), which simulated colony conditions. The honeybees were fostered using a previously described method [30,31].

Culture Conditions of the Bacterial Strain
The LAB strain used in this study was A. kunkeei BB1 (GenBank accession number: SUB10515234 Apilactobacillus OM755697), which was isolated from beebread. Unless otherwise stated, the routine culturing of this strain was performed under aerobic conditions at 37 ± 1 • C using a constant temperature shaking table (Tianjin, Honour). The culture medium was MRS normal medium supplemented with 10 g/L D-fructose. The final experimental concentration of Lactobacillus cells was 10 4 CFU/mL.

Experimental Design
Acetamiprid (99%, DR) was dissolved in acetone to prepare a stock solution (20,000 mg/L). The acute toxicity of acetamiprid was determined. Based on the results of the pre-experiment, the following concentrations were prepared to determine the LC 50 : 400 mg/L, 300 mg/L, 250 mg/L, 200 mg/L, 150 mg/L, and 0 mg/L. A total of 360 newly emerged honeybees were collected and separated into six groups. They were placed into cages in darkness for four days and fed with freshly prepared mature workers gut homogenate. Then, different concentrations of acetamiprid were added to their diets. The experiment results were recorded at 48 h, and the 48 h LC 50 was calculated. The procedures and methods for the determination of the oral LC 50 were based on previous reports [32]. One-tenth of the LC 50 was selected as the concentration for the following experiment (the results of the acute toxicity of acetamiprid were summarized in the pre-experiment; the LC 50 value was found to be 259.25 mg/L). Meanwhile, our pre-experiment showed that A. kunkeei did not directly depredate acetamiprid in vivo.
Newly emerged honeybees were fed with freshly prepared mature workers gut homogenate in addition to their food (50% sucrose syrup) for four days; the food was replaced every two days [33,34]. This simulated social behavior and ensured that the guts of the newly emerged honeybees established a complete set of symbiotic microorganisms [34,35]. A total of 600 honeybees were collected and separated into 20 cages (into four groups; each group included five experimental replicates). The four groups were designed as shown in Table 1. All honeybees were maintained in an incubator for seven days, and five from each cage were collected on day 11 of the experiment. To study the effects of A. kunkeei and acetamiprid exposure (in sucrose and pollen consumption, and in their gut microbiome) on honeybee survival, data were gathered and analyzed for each group. Acetamiprid was added into sterile sucrose syrup, and A. kunkeei was added to sterile pollen.

Nucleic Acid Extraction and Illumina Sequencing
On day 11 of the experiment, five honeybees from 12 cages were sampled for gut microbiota studies (three replicates were randomly selected out of every five experimental replicates). Each honeybee's whole gut was carefully collected on a clean bench environment and placed in a sterile centrifuge tube (1.5 mL). The gut samples were frozen using liquid nitrogen and stored at −80 • C until use. Microbial community genomic DNA was extracted using the E.Z.N.A. ® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. With the primer pair 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), the hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, NJ, USA). DNA sequencing was performed on an Illumina NovaSeq PE250 platform (Illumina, San Diego, CA, USA) by Shanghai Major Biotechnology.

Quantitative Polymerase Chain Reaction (qPCR)
Universal 16S rRNA gene primers Eub338 (5-ACTCCTACGGGAGGCAGCAG-3) and 355R (5-GGACTACHVGGGTWTCTAAT -3) were used to amplify the total copies of the16S rRNA genes in the samples with a StepOne Plus Real-Time PCR system (Applied Biosystems, USA). The primers (5-AGCAGTAGGGAATCTTCCA -3) and (5-CACCGCTA-CACATGGAG -3) were used to amplify the total copies of A. kunkeei in the samples. The standard curves of total microbial community and A. kunkeei quantification were based on the amplification of the cloned target sequence in the plasmid vector. The quantification of each simple microbial community was estimated based on standard curves and the ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China), according to the manufacturer's instructions.

Statistical Analyses
Operational taxonomic units (OTUs) with 97% similarity cut-off were clustered in UPARSE version 7.1, and chimeric sequences were identified and removed [36,37]. The taxonomy of each OTU representative sequence was analyzed using RDP Classifier version 2.2 against the 16S rRNA database (e.g., Silva v138), applying a confidence threshold of 0.7 [38]. All alpha and beta diversities were calculated using Quantitative Insights Into Microbial Ecology (QIIME2) [39], and the graphs of the analysis results were drawn using the R package, GraphPad Prism 9 (GraphPad Inc., La Jolla, CA, USA), and Adobe Illustrator CS6.

Results
Under the applied culture conditions, the reduction in survival in the control group (96%) represented the natural deaths of honeybees in a colony without acetamiprid stress or supplementation with probiotic A. kunkeei. The treatment experiment (Figure 1a) showed that the survival of the honeybees decreased following acetamiprid exposure (84% compared to the control). Supplementation with probiotic A. kunkeei did not lead to an increase or decrease in the mortalities of the honeybees. However, it significantly increased the survival of the honeybees under acetamiprid exportation, from 84% to 92%. To assess the influence of food consumption under acetamiprid exportation and supplementation with A. kunkeei, the pollen and sucrose consumption of honeybees were recorded and statistically analyzed (Figure 1b). The results showed that the pollen and sucrose consumption of honeybees were not affected under acetamiprid exportation. Following supplementation with A. kunkeei, the sucrose consumption of honeybees did not change, but pollen consumption decreased. The combination with acetamiprid exportation showed similar results. creased the survival of the honeybees under acetamiprid exportation, from 84% to 92%. To assess the influence of food consumption under acetamiprid exportation and supplementation with A. kunkeei, the pollen and sucrose consumption of honeybees were recorded and statistically analyzed (Figure 1b). The results showed that the pollen and sucrose consumption of honeybees were not affected under acetamiprid exportation. Following supplementation with A. kunkeei, the sucrose consumption of honeybees did not change, but pollen consumption decreased. The combination with acetamiprid exportation showed similar results. (c) Impact of acute oral exposure to acetamiprid and of A. kunkeei addition. '*' represents a statistically significant difference between the two groups at p < 0.05. '**' represents a statistically significant difference between two groups at p < 0.01.
Compared with the impact of acute oral exposure to acetamiprid supplementation with A. kunkeei (Figure 1c), the LC50 value of the AL group (489 mg/L) was higher than that of the A group (250.3 mg/L). This indicated that the toxicity of acetamiprid was reduced in honeybees. To investigate the influence of intestinal bacteria under acetamiprid exportation and supplementation with A. kunkeei, four groups and 12 samples were collected. Based on 16S rRNA pyrosequencing, information on the intestinal bacteria of honeybees was acquired. As shown in Figure 2, 374 OTUs were common to all groups. Most OTUs were found in the CK group, while the fewest were found in the A group. Following feeding A. kunkeei, the OTUs in the L group were fewer than in the CK group, but there were more in the AL group than in the A group. that of the A group (250.3 mg/L). This indicated that the toxicity of acetamiprid was reduced in honeybees. To investigate the influence of intestinal bacteria under acetamiprid exportation and supplementation with A. kunkeei, four groups and 12 samples were collected. Based on 16S rRNA pyrosequencing, information on the intestinal bacteria of honeybees was acquired. As shown in Figure 2, 374 OTUs were common to all groups. Most OTUs were found in the CK group, while the fewest were found in the A group. Following feeding A. kunkeei, the OTUs in the L group were fewer than in the CK group, but there were more in the AL group than in the A group. As expected, our microbiota composition analysis showed clear differences between the four groups at the genus level ( Figure 3). The core genus (Lactobacillus, Snodgrassella, Frischella, Gilliamella, Bartonella, and Bifidobacterium) in honeybee was detected in all samples and its abundance was over 87%, except for in group A, where it was 58%. The abundance of Lactobacillus was the highest in all treatments (i.e., 45%, 30%, 53%, and 47% in the CK, A, L, and AL groups, respectively). Under acetamiprid exportation, some opportunistic bacteria and pathogens invaded the intestinal regions of honeybees, including Paenibacillus lautus, Paenibacillus sp. The colonization of these bacteria would compress the living area of the core bacteria and reduce the abundance thereof. However, comparing the AL group and the A group, the abundance of core bacteria was higher in the former As expected, our microbiota composition analysis showed clear differences between the four groups at the genus level ( Figure 3). The core genus (Lactobacillus, Snodgrassella, Frischella, Gilliamella, Bartonella, and Bifidobacterium) in honeybee was detected in all samples and its abundance was over 87%, except for in group A, where it was 58%. The abundance of Lactobacillus was the highest in all treatments (i.e., 45%, 30%, 53%, and 47% in the CK, A, L, and AL groups, respectively). Under acetamiprid exportation, some opportunistic bacteria and pathogens invaded the intestinal regions of honeybees, including Paenibacillus lautus, Paenibacillus sp. The colonization of these bacteria would compress the living area of the core bacteria and reduce the abundance thereof. However, comparing the AL group and the A group, the abundance of core bacteria was higher in the former (94%). The presented work estimated the absolute (via qPCR) abundances of intestinal bacterial species in the gut compartments of honeybees, based on a standard curve. The total 16S rRNA gene and A. kunkeei copies of the intestinal bacteria were collected in four groups ( Figure 4). The results showed that the total 16S rRNA gene copies of the intestinal bacteria in the AL, A, and L groups were not significantly differentiated from those of the CK group. The copies of A. kunkeei in the AL and L groups were significantly increased compared to those of the CK group.
Next, to determine how acetamiprid and A. kunkeei influence the abundances of intestinal bacteria on the species level, the presented work screened different species based on the 16S rRNA database and analyzed the difference using Student's t-test ( Figure 5). The results showed that the abundances of Lactobacillus helsingborgensis (0.25%) and Lactobacillus sp. (0.13%) in the CK group were higher than the A group (Figure 5a). In the L group, the abundance of Lactobacillus apis (0.15%) was higher than in the CK group, but the abundance of Lactobacillus sp. (0.02%) was lower than in the CK group (Figure 5b). Comparing the A and AL groups, the different species were Bifidobacterium sp. and Snodgrassella alvi (Figure 5c). The abundances thereof in the AL group were higher than in the A group. (94%). The presented work estimated the absolute (via qPCR) abundances of intestinal bacterial species in the gut compartments of honeybees, based on a standard curve. The total 16S rRNA gene and A. kunkeei copies of the intestinal bacteria were collected in four groups ( Figure 4). The results showed that the total 16S rRNA gene copies of the intestinal bacteria in the AL, A, and L groups were not significantly differentiated from those of the CK group. The copies of A. kunkeei in the AL and L groups were significantly increased compared to those of the CK group.   bacterial species in the gut compartments of honeybees, based on a standard curve. The total 16S rRNA gene and A. kunkeei copies of the intestinal bacteria were collected in four groups (Figure 4). The results showed that the total 16S rRNA gene copies of the intestinal bacteria in the AL, A, and L groups were not significantly differentiated from those of the CK group. The copies of A. kunkeei in the AL and L groups were significantly increased compared to those of the CK group.   Next, to determine how acetamiprid and A. kunkeei influence the abundances of intestinal bacteria on the species level, the presented work screened different species based on the 16S rRNA database and analyzed the difference using Student's t-test ( Figure 5). The results showed that the abundances of Lactobacillus helsingborgensis (0.25%) and Lactobacillus sp. (0.13%) in the CK group were higher than the A group (Figure 5a). In the L group, the abundance of Lactobacillus apis (0.15%) was higher than in the CK group, but the abundance of Lactobacillus sp. (0.02%) was lower than in the CK group (Figure 5b). Comparing the A and AL groups, the different species were Bifidobacterium sp. and Snodgrassella alvi (Figure 5c). The abundances thereof in the AL group were higher than in the A group. The biological diversity of intestinal bacteria is an important criterion for assessing intestinal stability and health. The presented work evaluated the influence of intestinal biological diversity under acetamiprid exportation and supplementation with A. kunkeei on the α-diversity ( Figure 6) and β-diversity (Figure 7). Based on statistical data and software, the presented work obtained some diversity indexes (Student's t-test). The observed  The biological diversity of intestinal bacteria is an important criterion for assessing intestinal stability and health. The presented work evaluated the influence of intestinal biological diversity under acetamiprid exportation and supplementation with A. kunkeei on the α-diversity ( Figure 6) and β-diversity (Figure 7). Based on statistical data and software, the presented work obtained some diversity indexes (Student's t-test). The observed species (Sobs) index is regarded as a measurement of community richness in terms of biological diversity. The results of the Sobs index (Figure 6a) showed that the A group had the lowest community richness among all groups. The community richness in the CK and L groups was significantly higher than that of the A group. In the AL group, the community richness was also observed to be higher than in the A group, although this was not statistically significant. The Shannon evenness index (SEI) measures community evenness. The obtained index (Figure 6b) showed that no significant differences existed among groups. The community evenness of the A group fluctuated greatly. The status and queues in the cluster (Qstat) index is usually used to measure community diversity. The obtained Qstat index (Figure 6c) showed that the A group had the lowest community diversity among all groups. The community diversity in the CK and L groups was significantly higher than that of the A group. In the AL group, the community diversity was also observed to be higher than in the A group, although this was not statistically significant. Phylogenetic diversity (PD) was also measured. The results (Figure 6d) showed that the AL group had the lowest community phylogenetic diversity among all of the groups. The community phylogenetic diversity in the CK and L groups was significantly higher than in the AL group. For a more accurate estimation of the biological diversity or the community structure of the intestinal bacteria in all groups, the beta diversity was assessed using principal component analysis (PCA, Figure 7a), principal coordinate analysis (PCoA, Figure 7b), and nonmetric multidimensional scaling (NMDS, Figure 7c). As a common method of dimensionality reduction, PCA visualizes multidimensional data using scatter diagrams. Based on the graphic, we found a variety of intestinal bacteria community structures in the A group. In the AL group, the community structure was clearly stabilized. Similar results were found when complex microbiota information was analyzed using PCoA and NMDS. Overall, compared with the CK group, the community structure of intestinal bacteria had changed, and and the newly differentiated structure had stabilized in AL group compared with the A group. There was little difference between the L groups compared with the CK group regarding the structure of the intestinal bacteria community.
Insects 2022, 13, 1167 9 of 15 method of dimensionality reduction, PCA visualizes multidimensional data using scatter diagrams. Based on the graphic, we found a variety of intestinal bacteria community structures in the A group. In the AL group, the community structure was clearly stabilized. Similar results were found when complex microbiota information was analyzed using PCoA and NMDS. Overall, compared with the CK group, the community structure of intestinal bacteria had changed, and and the newly differentiated structure had stabilized in AL group compared with the A group. There was little difference between the L groups compared with the CK group regarding the structure of the intestinal bacteria community. ; the status and queues in the cluster index (c); and the phylogenetic diversity index (d). '*' represents a statistically significant difference between two groups (independent-sample t test, p < 0.05). '**' represents a statistically extremely significant difference between two groups (independent-sample t test, p < 0.01). ; the status and queues in the cluster index (c); and the phylogenetic diversity index (d). '*' represents a statistically significant difference between two groups (independent-sample t test, p < 0.05). '**' represents a statistically extremely significant difference between two groups (independent-sample t test, p < 0.01).

Discussion
Neonicotinoids and acetamiprid are widely applied to control pests in agricultural and in domestic and public health activities. In agriculture, these compounds have been used to control aphids, hemipterans, lepidopterans, and other pests via foliar sprays, soil drenches, trunk injections, and seed soaks [40,41]. Following its application, residual levels of acetamiprid may occur in pollen, nectar, and water, with sublethal effects, and even mortality, on honeybees. As a widely applied insecticide, acetamiprid is considered to be slightly toxic or practically non-toxic to mammals compared to organophosphorus and carbamate insecticides; however, it has a very distinct toxicity toward pollinating insects (such as honeybees). In our study, we found that sublethal doses of acetamiprid exhibit toxicity on honeybees, significantly decreasing survival rate. This phenomenon was also observed under semi-field conditions [42]. Acetamiprid is a neurotoxin which also reduces sucrose sensitivity, interferes with memory function, and impacts on foraging and learning abilities [43]. Exposure to sublethal levels of acetamiprid also leads to a decrease in the honeybee's weight and affects its development from larva to adult [44]. Continuous exposure to can lead to death. Therefore, acetamiprid can have adverse effects on honeybee health and colony productivity. In the present study, during exposure to sublethal levels of acetamiprid, sucrose and pollen consumption by honeybees were not significantly different when compared to the control treatments, suggesting that food consumption by honeybees is not influenced by acetamiprid exposure. A previous study showed that sucrose consumption by honeybees decreased significantly following exposure to very high concentrations of thiacloprid, but that it did not change significantly following exposure to low concentrations [45]. We speculated that the characteristics of acetamiprid are similar to those of thiacloprid; both are cyano-substituted neonicotinoids which are moderately toxic to honeybees [43]. Exposure to a high concentration of acetamiprid could induce escape behavior responses of honeybees.
LAB has been widely administered as a probiotic to improve the health of humans and animals. As a common LAB, A. kunkeei is widely distributed in the colony environment, including bee pollen, beebread, and combs [46]. Honeybees acquire it through trophallaxis with companions or through the consumption of bee pollen and beebread. A. kunkeei was also isolated from the honey crops of nine Apis species and three stingless bee species [47]. This suggests that A. kunkeei is safe for hosts and is globally present in bees. A previous study showed that the microbiota associated with maternally provisioned host pollen perform critical functions in bee larval nutrition and survival [48]. Meanwhile, metabolic pathways with phenolic acids allow A. kunkeei to contribute to the degradation of secondary plant metabolite phenolic acids in pollen, to reduce the toxicity of phenolic acids in pollen [49]. It has both technological and functional beneficial attributes. This suggests that A. kunkeei may play an important role in the development and growth of honeybees. It has great probiotic potential. In our research, supplementation with A. kunkeei as a potential probiotic in pollen did not induce the escape behavior response, and food consumption did not decrease during the experiment. It also did not increase the mortality of honeybees when compared to the control treatment. Moreover, pollen consumption by honeybees decreased when A. kunkeei was added, in comparison with the acetamiprid-exposed and control groups. The reason for this may be that the quality and nutrition of pollen increased after supplementation with A. kunkeei, i.e., the litter pollen could provide enough nutrients for development and growth. The decrease in the pollen consumption of the colony following A. kunkeei treatment manifested in a significant increase in the quantity of reserve pollen in the colony [50]. As a probiotic, A. kunkeei has been applied for the treatment of major honeybee microbial infections, such as P. larvae [13], Melissococcus plutonius [47], and N. ceranae [12]. However, few studies have been conducted to explore the relationship between honeybees, probiotics, and pesticides. Our study shows that supplementation with A. kunkeei significantly improved the survival of honeybees following acetamiprid exposure. In order to clarify the mechanism for this, the presented work obtained and analyzed information about the intestinal microorganisms in sample honeybees.
Intestinal microorganisms play an important role in the development of insects based on their secondary metabolites. Intestinal microorganisms positively impact the growth, development, and reproduction of insects via the advanced metabolism of the host [51]. Stable intestinal symbiotic bacteria are beneficial to host health. Meanwhile, intestinal microorganisms enhance the ability of the host to cope with stress from the environment. Our previous research showed that honeybees maintained a stable intestinal bacteria environment during overwintering, and that an abundance of Lactobacillus may help the host to successfully survive the extreme coldness of winter [52]. As an intestinal microorganism, A. kunkeei supplementation for honeybees did not have an influence on the diversity and ecological structure of the intestinal bacteria of the host. However, it likely decreased the abundance of Lactobacillus sp. and increased the abundance of L. apis. This was similar to previous reported results [50]. This may be contrary to the supplementation with A. kunkeei, where sublethal acetamiprid exposure seriously changed the diversity and ecological structure of intestinal bacteria. This was, again, consistent with previous studies [53]. However, the difference in terms of the gut microbiome richness was that the alpha diversity decreased after acetamiprid treatment compared to that of the control. The apparent differences could be partially caused by different doses and lab environments. Other neonicotinoid pesticides, such as thiacloprid [45], thiamethoxam [54], and imidacloprid [54], also cause imbalances in honeybee gut microbiomes. However, there were no significant differences in the diversity and richness of intestinal bacteria after exposure to 1.0 mg/L amitraz [55]. The apparent differences could be partially caused by differences in the pesticide type, i.e., acetamiprid and amitraz. Amitraz has been used globally to control Varroa mites in beekeeping due to its reduced toxicity to honeybees compared to acetamiprid. Some opportunistic and pathogenic bacteria were detected following sublethal acetamiprid exposure. Odoribacter has been proven to be an important pathogenic bacteria in humans and cattle [56]. Although the pathogenicity of Odoribacter on honeybees has not been determined, it is also a potential pathogen and may impact the health of honeybees. Paenibacillus is an important pathogen, causing American foulbrood of honeybee larva; therefore, it is a major hazard to beekeeping. Interestingly, honeybees that were damaged by acetamiprid were shown to significantly improve in health after supplementation with A. kunkeei. This suggests that A. kunkeei could enhance the pesticide resistance of the host through the maintenance a stable ecosystem, or by increasing the diversity of the intestinal bacteria [57]. As a core genus, the abundance of Bifidobacterium was increased; this was beneficial to the host. Bifidobacterium was mainly involved in the degradation of the toxic substance trehalose and in the absorption of some carbohydrates [15]. When dealing with the stress of toxicity, the host needs energy and nutrients. The increase of Bifidobacterium could effectively enhance the energy intake of the host and assist in efficiently responding to acetamiprid stress. As for other core bacteria, S. alvi distributed in the ileum can form an oxygen pressure environment through respiration. The increase in S. alvi could inhibit the passage of opportunistic and pathogenic bacteria. This bacterium enhances the stability and persistence of the characteristic honeybee intestinal bacteria. Meanwhile, the redundant fermentation products of Bifidobacterium may be taken up and oxidized by S. alvi for energy and carbon [23]. A previous study demonstrated that the gut microbiota may influence detoxification gene expression in the honeybee digestive tract [58]. Overall, A. kunkeei can reduce the acetamiprid toxicity of honeybees by increasing microbial diversity and maintaining the ecological structure of intestinal bacteria. These findings were reported in a previous study [59].  Data Availability Statement: All data generated or analysed during this study are included in this published article. The datasets of microbial community genomic for this study can be found in the SRA database (BioProject ID: PRJNA820132).