Intestinal probiotic bacteria for improving the resistance of honeybees to nitenpyram and use thereof
By inoculating the bee gut with the probiotic strain Gilliamella sp. G0441, the health problems caused by the pesticide acetamiprid in bees were resolved, the bees' resistance and survival ability were improved, and their appetite and cognitive function were restored.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF ZOOLOGY GUANGDONG ACAD OF SCI
- Filing Date
- 2025-01-24
- Publication Date
- 2026-04-24
AI Technical Summary
Honeybees face health threats from exposure to acetamiprid pesticides, including gut microbiota dysbiosis and reduced resistance, leading to decreased survival rates and impaired behavioral abilities.
The probiotic strain Gilliamella sp. G0441 was administered orally to the intestines of bees to enhance their resistance to acetamiprid.
It significantly improves bees' tolerance to acetamiprid, prolongs their lifespan, restores their appetite and cognitive abilities, and reduces the toxic effects of pesticides on bees.
Smart Images
Figure CN119823912B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the agricultural field, specifically relating to a gut probiotic that enhances the resistance of bees to acetamiprid and its application. Background Technology
[0002] Bees provide vital pollination services for natural and agricultural plants, playing a crucial role in both ecology and economy. However, bee populations have declined sharply in recent decades. The overuse of pesticides is a major contributing factor to the serious threat to bee survival. Bees are exposed to a wide variety of pesticides, including herbicides, fungicides, and insecticides, during floristry visits, posing a significant threat to their health. Residual pesticides are carried back by foraging bees and accumulate in the hive; various pesticide residues have been detected in beehives, pollen, honey, and beeswax. High doses of pesticides often lead to mass bee deaths, while sublethal doses can alter bee gene expression, negatively impacting foraging, learning abilities, weight gain, and reproduction. Acetaminophen, a second-generation neonicotinoid insecticide, has been widely used to control pests and diseases in various crops such as rice and corn, achieving satisfactory results in agricultural production. Furthermore, acetamiprid is also widely used to control fleas in dogs and cats. However, due to its broad applicability and high water solubility, it can easily be transferred into the environment. Acetaminophen residues have been found in honey and bee pollen from many countries around the world. Previous studies have found that acetamiprid is highly toxic to honeybees (Apis mellifera). It disrupts the honeybee's gut microbiota and has led to the near-complete disappearance of Gilliamella, a core member of the honeybee family.
[0003] The gut microbiota plays a crucial role in mitigating pesticide toxicity and enhancing host tolerance to these pesticides. In bees, the gut microbiota can also help the host defend against exogenous toxins. For example, bees colonized with *Gilliamella apicola* B1 and *Lactobacillus kunkeei* M1 showed higher survival rates after exposure to deltamethrin compared to germ-free bees. *Pantoea* significantly improved bee survival after thiamethoxam exposure; *Enterobacter* alleviated dysbiosis of the midgut microbiota structure network induced by thiamethoxam. These findings reveal the critical role of the gut microbiota in mitigating pesticide toxicity and enhancing host tolerance to these chemicals.
[0004] The use of pesticides is an indispensable part of modern agricultural practice, used to protect crops from pests and thus ensure yield and quality. However, it is equally urgent to adopt innovative methods to combat the decline in bee populations caused by pesticide toxicity. Based on the protective role of gut bacteria, screening probiotics as supplements to mitigate the harmful effects of pesticides is a promising approach. Oral probiotics may work through a variety of mechanisms: degrading pesticides to reduce their absorption by the host; isolating ingested pesticides to promote their excretion; upregulating the expression of host detoxification enzymes to accelerate pesticide metabolism and ultimately improve host survival. Although the natural gut microbiota has some inherent pesticide detoxification capabilities, their effectiveness is affected by factors such as pesticide type and concentration, exposure duration and seasonal variation
[13] . Studies have found that pesticides such as glyphosate, imidacloprid and coumarin can have a negative impact on the cellular homeostasis of probiotics
[14] . Pesticide exposure can inhibit the growth of beneficial bacteria in the colony, but targeted supplementation with probiotics can overcome this limitation by maintaining bacterial concentrations at effective levels. Summary of the Invention
[0005] The purpose of this invention is to provide an intestinal probiotic that enhances the resistance of bees to acetamiprid and its application.
[0006] The intestinal probiotic that enhances the resistance of bees to acetamiprid is Gilliamella sp. G0441, which was deposited on January 6, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 65723.
[0007] A second objective of this invention is to provide the use of the aforementioned Gilliamella sp. G0441 in the preparation of a drug to improve pesticide resistance in bees.
[0008] Preferably, the pesticide is acetamiprid.
[0009] A third objective of this invention is to provide a drug for improving pesticide resistance in bees, which contains Gilliamella sp. G0441 as an active ingredient.
[0010] The present invention also provides a method for improving pesticide resistance in bees, which involves inoculating Gilliamella sp. G0441 into the intestines of bees.
[0011] Preferably, Gilliamella sp. G0441 is inoculated into the bee's gut by feeding.
[0012] The bees mentioned can be of various types, such as the Chinese honeybee and the Italian honeybee.
[0013] Beneficial effects:
[0014] a) Safety. The chemical probiotic supplement developed in this study—Gilliamella sp. G0441—is a core bacterium in the bee's gut. Therefore, the use of this substance will not pollute bee products or the natural environment, nor will it harm the bees.
[0015] b) High efficiency. Tests have shown that Gilliamella sp. G0441 can significantly reduce the harm of acetamiprid to bees.
[0016] c) Availability. Gilliamella sp. G0441 is a core bacterium in the bee gut, easy to isolate, culture, inexpensive, and widely available.
[0017] Gilliamella sp. G0441 was deposited on January 6, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 65723. Attached image description:
[0018] Figure 1 It is a phylogenetic tree based on the 16S gene sequence.
[0019] Figure 2 It is a phylogenetic tree based on the core genes of the pan-genome.
[0020] Figure 3 It is a phylogenetic tree based on SNPs.
[0021] Figure 4 The effects of oral administration of Gilliamella sp. G0441 and exposure to acetamiprid on bee food consumption.
[0022] Figure 5 The effects of oral administration of Gilliamella sp. G0441 and exposure to acetamiprid on bee weight.
[0023] Figure 6 The effect of different sucrose concentrations on the PER percentage of bees in four treatment groups.
[0024] Figure 7 The effects of oral administration of Gilliamella sp. G0441 and exposure to acetamiprid on the PER score of honeybees.
[0025] Figure 8 These are the survival curves of sterile bees and Gilliamella sp. G0441 colony bees exposed to high concentrations of acetamiprid.
[0026] Figure 9 This is an experimental design flowchart. Detailed Implementation
[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0028] Example 1: Isolation and identification of Gilliamella sp. G0441
[0029] Select healthy, strong colonies from the apiary and randomly collect 10 worker bees into test tubes using soft forceps. Anesthetize the bees by placing them in a 4°C refrigerator for 10–15 minutes. Soak the surface of the worker bees in 75% alcohol for 1 minute, then rinse with sterile water and drain excess water using sterile filter paper. In a laminar flow hood, use sterile dissecting scissors to cut off the heads, then use sterile forceps to grasp the end of the worker bee's tail and gently pull out the entire intestine (including the honey sac, foregut, midgut, and hindgut) into a sterile tube. Collect the intestines of 10 bees into the same tube, add 1 mL of sterile 1×PBS, grind and mix well, and vortex for 1 minute. Dilute the resulting suspension with 1×PBS solution 10 times each. 3 10 4 10 5 Three concentrations were prepared, with 100 μL of each diluted solution plated onto TSA medium (containing 5% defibrinated sheep blood), and each concentration was replicated in triplicate. The plates were sealed with film and incubated in an anaerobic incubator at 35°C and 60% RH for 3 days. Round, smooth, and translucent colonies were picked for purification. Three rounds of purification using the streak plate method were performed to obtain pure strain G0441. Purified colonies were picked from the plates for PCR amplification. The 16S rRNA gene was amplified using universal bacterial primers (27F, 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R, 5'-GGTTACCTTGTTACGACTT-3') for identification. The PCR amplification reaction system was: 25 μL of 2×Taq HiFi PCR mixenzymes, 1 μL of upstream primer, 1 μL of downstream primer, 10 μL of DNA template, and 13 μL of deionized water. The PCR program was as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. The amplification cycle number was 35, with a hold time of 72℃ for 10 min. PCR products were examined by 1% agarose gel electrophoresis. The target band was excised from the gel and recovered for bidirectional sequencing. Sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The obtained nucleic acid sequences were compared and identified in the NCBI database.
[0030]
[0031] After comparison, this strain showed 99% similarity to *Gilliamella*, a core gut bacterium of bees. Further bacterial genome sequencing was performed by Shanghai Bioengineering Co., Ltd. NCBI Blast was used for genome alignment, and homologous strain information was obtained based on matching scores. Reference representative strains with complete annotations were compiled and collected for subsequent analysis. The 16S rRNA sequences of the strains were collected, multiple sequence alignment was performed, and a phylogenetic tree was constructed. Figure 1 Using Roary, pan-genome analysis and homologous gene cluster analysis were performed based on homologous strain gene information, and a phylogenetic tree was constructed based on homologous genes. Figure 2 Mutation detection was performed using Snippy, and a phylogenetic tree was constructed based on the mutation SNP information. Figure 3 ).
[0032] The strain was named Gilliamella sp. G0441 and deposited on January 6, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 65723.
[0033] Example 2: Analysis of the protective effect of Gilliamella sp. G0441 on bees.
[0034] 1) Methods and steps:
[0035] (1) Preparation of Gilliamella sp. G0441 supplement solution:
[0036] Gilliamella sp. G0441 was isolated from the intestines of *Apis chinensis* using tryptic soy agar (TSA) containing 5% (v / v) defibrinated sheep blood under completely anaerobic conditions at 35°C and 60% RH. The preserved strain was expanded in liquid medium for 48 hours. Bacterial concentration was determined by microscopic counting and diluted with 50% (w / v) sterile sucrose solution to prepare a 10- culture. 6 CFU / mL intestinal flora supplement (prepare and change daily).
[0037] (2) Preparation of acetamiprid test solution:
[0038] Prepare a 1000 mg / L stock solution by mixing acetamiprid (96% purity) with sterile water, and then dilute to the final concentration with 50% (w / v) sterile sucrose solution.
[0039] Three concentrations were designed for this experiment: 1 mg / L, 5 mg / L and 10 mg / L.
[0040] (3) Beekeeping:
[0041] To strictly control variables such as age and growth environment, six combs were selected from three healthy Apis chinensis colonies. Each comb consisted of half brood and half pollen and honey. All worker bees were shaken off the combs, and the combs were transferred to an artificial climate incubator. The incubation conditions were adjusted to darkness, 34°C, and 70% RH. After 24 hours, newly emerged worker bees were collected and marked on their backs with a marker. Approximately 800 newly emerged worker bees were marked. These marked worker bees were returned to the same hive and reared there for 7 days to allow them to establish their gut microbiota in a natural environment.
[0042] Seven days later, tagged bees (7 days old) were collected and randomly assigned to 20 cup cages, each containing 30 bees, with feeders inserted. All were divided into two groups. One group was fed *Gilliamella* sp. G0441 (Group G). The other group was fed a sterile sucrose solution (Group S). Seven days later, half of the bees in Group G (14 days old) were orally exposed to a sublethal concentration of 1 mg / L acetamiprid sucrose solution (Group GN). The other half were fed a 50% sucrose solution as a control (Group GS). The treatment for Group S (14 days old) was the same as above (Groups SN and SS). The experiment consisted of four treatments, with five replicates for each treatment, and pesticide exposure was maintained for 7 days (21 days old). Throughout the experiment, dead bees were recorded and removed every 24 hours. Pollen has been shown to regulate cytochrome P450 (CYP) detoxification enzymes in newly emerging bees. To avoid interference, bees were fed only sucrose solution throughout the experiment.
[0043] (4) Food consumption and weight test
[0044] Calculate the average daily sugar water consumption (A) per bee, A = G / L - (S / 2), where G represents the daily sugar water consumption; L represents the number of surviving bees the previous day; and S represents the number of dead bees each day.
[0045] On day 21, bee weights were recorded. Three cup cages were randomly selected for each treatment, and 10 worker bees from each cup cage were randomly selected from each treatment. Therefore, 30 bees were tested in each group. The bees were weighed after being stunned by the cold to determine their individual weights.
[0046] (5) Proboscis extension reflex (PER) test
[0047] The tests were conducted 7 days after exposure to acetamiprid (21 days old). Three cages were randomly selected from each treatment group, and 15 bees were sampled from each cage, for a total of 180 worker bees used in the study. Sucrose sensitivity in bees was assessed by proboscis response. Proboscis responses were observed by stimulation with 0.1%, 1%, 10%, and 50% (w / vg / ml) sucrose solutions. A glass rod was placed near the bee's antennae, and sucrose solutions of increasing concentrations were applied via dropper. A score of 1 was awarded if the bee's proboscis was fully extended; a score of 0 was awarded if there was no response. Sucrose sensitivity scores for each group were calculated by summing the number of proboscis responses for each bee within that group.
[0048] (6) Test of sterile (microbiota-free, MF) bee model
[0049] Sterile bees were produced in the laboratory. Late-stage pupae were removed from the comb using sterile tweezers and placed in sterile plastic boxes. The pupae emerged in an incubator at 35°C and 50% humidity. Newly emerged sterile bees (0 days old) were placed in sterile cup cages for 24 hours, with 15 bees per cup cage, and supplemented with sterile sucrose syrup (50%, w / v). Then, the sterile bees (1 day old) were divided into two groups, one group was fed with Gilliamella sp. G0441 supplement, and the other group continued to be fed with sterile sucrose syrup. After 7 days, both groups were treated with the following 3 methods (each treatment included 3 cup cages): (1) CV: continued feeding with sterile sucrose syrup; (2) feeding with 5 mg / L acetamiprid sucrose solution; (3) feeding with 10 mg / L acetamiprid sucrose solution. Dead bees were cleaned and recorded daily.
[0050] (7) Raw data collection and processing
[0051] 2) Test Results
[0052] like Figure 4 As shown, the average food consumption per bee over 14 days was 0.22 mg for SN, 0.29 mg for SS, 0.25 mg for GN, and 0.29 mg for GS. Compared with the control SS, acetamiprid exposure (SN) significantly reduced food consumption in bees (P = 0.0092) and negatively affected their appetite. Food consumption in GN was higher than in SN but lower than in GS. Although this trend was not significant (P = 0.1759, P = 0.1907), it suggests to some extent that supplementation with *Gilliamella sp.* G0441 may alleviate anorexia induced by acetamiprid exposure.
[0053] like Figure 5As shown: Comparison of SN and SS revealed that bees exposed to acetamiprid had significantly lower body weight than healthy bees (P < 0.0001). Comparison of GS and SS, and GN and SN, showed that supplementation with Gilliamella sp. G0441 significantly promoted host weight gain (P < 0.0001).
[0054] like Figure 6 As shown: SN's PER response to all four sucrose concentrations was significantly lower than that of the control SS (P<0.0001). At a 1% concentration, Gilliamella sp. G0441 supplement (GS) significantly improved sucrose sensitivity compared to the control SS. An increasing trend was observed at 10% and 0.1% concentrations, but it was not significant (P=0.0860, P=0.9265). At high concentrations (50%), the sucrose responses of GS and SS were comparable. Except at the 1% concentration, GN showed higher sucrose sensitivity than SN. No significant difference was observed between GN and SS after stimulation with a 50% sucrose solution (P=0.8158), indicating that GN's PER response to this sucrose concentration was almost as strong as that of healthy bees.
[0055] like Figure 7 As shown: To fully evaluate the effects of acetamiprid and Gilliamella sp. G0441 on the recognition and learning abilities of bees, the sucrose sensitivity of each group of bees was scored. The mean sucrose sensitivity scores for SN, SS, GN, and GS were 1.15, 2.73, 2.02, and 3.07, respectively. This indicates that acetamiprid exposure leads to impaired cognitive abilities in bees, and that Gilliamella sp. G0441 supplementation can effectively mitigate these negative effects.
[0056] like Figure 8 As shown, when acetamiprid was administered orally at a concentration of 10 mg / L, the median survival was 1 day in the MF group and 2.5 days in the Gilliamella sp. G0441 group. When acetamiprid was administered orally at a concentration of 5 mg / L, the median survival was 2 days in the MF group and 3 days in the Gilliamella sp. G0441 group. These results indicate that Gilliamella sp. G0441 can enhance host resistance to acetamiprid and significantly prolong the host's median survival.
Claims
1. A strain that enhances pesticide resistance in bees Gilliamella sp. G0441, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC NO: 65723.
2. The claim 1 Gilliamella Application of sp. G0441 in the preparation of a drug to improve pesticide resistance in bees, wherein the pesticide is acetamiprid and the bee is the Chinese honeybee.
3. The application according to claim 2, characterized in that, It is the same as that described in claim 1 Gilliamella sp.G0441 was inoculated into the bee's gut.
4. The application according to claim 3, characterized in that, is By feeding Gilliamella sp.G0441 was inoculated into the bee's gut.
5. A drug for improving pesticide resistance in bees, characterized in that, Containing the contents of claim 1 Gilliamella sp. G0441 is the active ingredient, and the pesticide in question is acetamiprid.