Intestinal probiotics for improving nitenpyram resistance of bees and application of intestinal probiotics

Abstract

The invention discloses intestinal probiotics for improving nitenpyram resistance of bees and application of the intestinal probiotics. The preservation number of the Gilliamella sp.G0441 is GDMCC (China General Microbiological Culture Collection Center) NO: 65723. The developed chemical substance probiotic supplement, namely Gilliamella sp.G0441, is a core bacterium of intestinal tracts of bees, so that the substance does not cause pollution to bee products and the natural environment and does not cause harm to the bees. The efficiency is high. Tests prove that the Gilliamela sp.G0441 can be used for remarkably reducing the harm of the nitenpyram to the bees.

Description

Technical Field

[0001] The invention belongs to the field of agriculture, and particularly relates to an intestinal probiotic for improving the resistance of honey bees to nitenpyram and an application thereof. Background Art

[0002] Bees provide important pollination services for natural and agricultural plants and are of great ecological and economic significance. However, in recent decades, the number of bees has declined sharply. The abuse of pesticides is an important reason why the survival of bees is facing serious threats. When bees visit flowers, they are exposed to a variety of pesticides, including herbicides, fungicides and insecticides, which are an important threat to the health of bees. Residual pesticides are brought back by foraging bees and accumulated in the hives. A variety of pesticide residues have been detected in beehives, pollen, honey and beeswax. High doses of pesticides usually cause a large number of bee deaths, while sublethal doses of pesticides can also change the gene expression of bees, causing negative effects on bee foraging, learning ability, weight gain and reproduction. Nitenpyram is a second-generation neonicotinoid insecticide that has been widely used to control pests and diseases of a variety of crops such as rice and corn, and has achieved satisfactory results in agricultural production. In addition, nitenpyram can also be used to control fleas on dogs and cats, and is currently widely used. However, due to its wide range of application and high water solubility, it can be easily transferred to the environment. Nitenpyram residues have been found in honey and bee pollen in many countries around the world. Previous studies have found that nitenpyram is highly toxic to honey bees (Apis mellifera). It disrupts the bee gut microbiota and has led to the almost complete disappearance of Gilliamella, one of the core members of the bee species.

[0003] The gut microbiota plays a key role in mitigating the toxicity of pesticides and enhancing host tolerance to these pesticides. In honey bees, gut microbes can also help the host defend against exogenous toxicants. For example, honey bees colonized with Gilliamella apicola B1 and Lactobacillus kunkeei M1 showed higher survival rates after exposure to deltamethrin compared with sterile bees. Pantoea can significantly improve the survival rate of honey bees after clothianidin exposure; Enterobacter alleviates the imbalance of the microbiota of the honey bee midgut structure network induced by clothianidin. These findings reveal the key role of the gut microbiota in mitigating the toxicity of pesticides to the host and enhancing the host tolerance to these chemicals.

[0004] The use of pesticides is an integral part of modern agricultural practices, used to protect crops from pests and thus ensure yield and quality. However, innovative approaches are equally urgent to combat the decline in bee populations due to pesticide toxicity. Screening for probiotics as supplements to mitigate the harmful effects of pesticides based on the protective effects of gut bacteria is a promising approach. Oral probiotics may work through a variety of mechanisms: degradation of pesticides to reduce host uptake; sequestration of ingested pesticides to promote their excretion; and upregulation of host detoxification enzymes to accelerate pesticide metabolism and ultimately improve host survival. Although natural gut microbiota have 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 coumaphos can negatively affect the cellular homeostasis of probiotics

[14] . Pesticide exposure inhibits the growth of beneficial bacteria within the colony, but targeted probiotic supplementation can overcome this limitation by maintaining bacterial concentrations at effective levels. Summary of the invention

[0005] The purpose of the present invention is to provide an intestinal probiotic for improving the resistance of honey bees to nitenpyram and application thereof.

[0006] The intestinal probiotics for improving the resistance of bees to acetamiprid are Gilliamella sp. G0441, which were preserved in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 6, 2025, at the 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the collection number is: GDMCC NO: 65723.

[0007] The second object of the present invention is to provide the use of the above-mentioned Gilliamella sp. G0441 in the preparation of a drug for improving the pesticide resistance of honey bees.

[0008] Preferably, the pesticide is nitenpyram.

[0009] The third object of the present invention is to provide a drug for improving the pesticide resistance of honey bees, which contains Gilliamella sp. G0441 as an active ingredient.

[0010] The invention also provides a method for improving the pesticide resistance of honey bees, which comprises inoculating Gilliamella sp. G0441 into the intestinal tract of honey bees.

[0011] Preferably, Gilliamella sp. G0441 is inoculated into the bee intestine by feeding.

[0012] The bees can be various bees, such as Apis cerana and Apis mellifera.

[0013] Beneficial effects:

[0014] a) Safety. The chemical probiotic supplement developed this time, Gilliamella sp.G0441, is the core bacteria in the bee's intestines, so the use of this substance will not pollute bee products and the natural environment, nor will it harm bees.

[0015] b) High efficiency. Experiments have shown that Gilliamella sp.G0441 can significantly reduce the harm of nitenpyram to bees.

[0016] c) Availability. Gilliamella sp.G0441 is a core bacterium in the bee intestine, which is easy to isolate, cultured, low-cost and widely available.

[0017] Gilliamella sp.G0441 was deposited on January 6, 2025 in the Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC NO: 65723. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a phylogenetic tree based on 16S gene sequences.

[0019] Figure 2 It is a phylogenetic tree based on the pan-genome core genes.

[0020] Figure 3 It is a phylogenetic tree based on SNPs.

[0021] Figure 4 Effects of oral exposure to Gilliamella sp. G0441 and nitenpyram on food consumption in honey bees.

[0022] Figure 5 The effects of oral Gilliamella sp. G0441 and nitenpyram exposure on the body weight of honey bees.

[0023] Figure 6 This is the percentage ratio of the effects of different sucrose concentrations on PER of bees in the four treatment groups.

[0024] Figure 7 The effects of oral administration of Gilliamella sp. G0441 and nitenpyram exposure on the PER score of honey bees.

[0025] Figure 8 Survival curves of sterile bees and bees colonized with Gilliamella sp.G0441 exposed to high concentrations of nitenpyram.

[0026] Fig. 9 It is a flowchart of experimental design. DETAILED DESCRIPTION

[0027] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0028] Example 1: Isolation and identification of Gilliamella sp. G0441

[0029] Select healthy and strong colonies from the apiary, and use soft tweezers to randomly collect 10 worker bees in a test tube. Place in a refrigerator at 4°C for anesthesia for 10 to 15 minutes. Soak the surface of the worker bees in 75% alcohol for 1 minute, rinse with sterile water, and filter out excess water with sterile filter paper. In the clean bench, cut off the head with sterile dissecting scissors, and then use sterile tweezers to clamp the end of the tail of the worker bee, gently pull out the entire intestine (including honey sac, foregut, midgut, and hindgut) into a sterile small tube, collect 10 intestines in the same tube, add sterile 1mL 1×PBS to grind and mix, and shake with a vortex oscillator for 1 minute. The resulting suspension is diluted 10 times with 1×PBS solution. 3 , 10 4 , 10 5 Three concentrations, 100 μL of each dilution was applied to TSA medium (containing 5% defibrinated sheep blood), and each concentration was repeated 3 times. The coated plate was sealed with a sealing film and placed in an anaerobic incubator and cultured at 35°C and 60% RH for 3 days. Round, smooth, and translucent colonies were picked for purification, and pure strain G0441 was obtained after 3 rounds of purification using the plate streak method. Purified colonies were picked from the plate for PCR amplification. The 16SrRNA gene was amplified using bacterial universal primers (27F, 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R, 5'-GGTTACCTTGTTACGACTT-3') for identification. The PCR amplification reaction system was: 2×Taq HiFi PCR mixenzymes 25μL, upstream primer 1μL, downstream primer 1μL, DNA template 10μL, deionized water 13μL. The PCR program was: 95℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 60℃ annealing for 30s, and 72℃ extension for 30s. The number of amplification cycles was 35, and 72℃ was maintained for 10min. The PCR product was checked by 1% agarose gel electrophoresis. The target band was cut and recovered for bidirectional sequencing. Sequencing was completed by Sangon Biotechnology (Shanghai) Co., Ltd. The nucleic acid sequences obtained by sequencing were compared and identified in the NCBI database.

[0030]

[0031] After comparison, the strain had a similarity of 99% with the core genus Gilliamella in the bee gut. The bacterial genome was further sequenced by Bioengineering (Shanghai) Co., Ltd. NCBI Blast was used for genome alignment, and the homologous strain information was obtained based on the matching score. The reference representative strains with complete annotations were sorted and collected for subsequent analysis. The 16S rRNA sequences of the strains were collected, multiple sequences were aligned, and a phylogenetic tree was constructed ( Figure 1 ). Roary was used to perform pan-genome analysis and homologous gene cluster analysis based on the gene information of homologous strains, and a phylogenetic tree was constructed based on homologous genes ( Figure 2 Snippy was used to detect mutations and a phylogenetic tree was constructed based on the mutation SNP information ( Figure 3 ).

[0032] The strain was named Gilliamella sp. G0441 and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 6, 2025. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the collection number is: GDMCC NO: 65723.

[0033] Example 2: Analysis of the protective effect of Gilliamella sp. G0441 on honey bees.

[0034] 1) Methods and steps:

[0035] (1) Preparation of Gilliamella sp. G0441 supplement solution:

[0036] Gilliamella sp.G0441 was isolated from the intestine of Apis cerana cerana using tryptic soy agar (TSA) containing 5% (v / v) defibrinated sheep blood. The culture conditions were completely anaerobic, 35°C, 60% RH. The preserved strain was expanded in liquid culture medium for 48 h. The bacterial concentration was obtained by counting under a microscope and diluted with 50% (w / v) sterile sucrose solution to prepare 10 6 CFU / mL intestinal bacteria supplement (prepared and replaced daily).

[0037] (2) Prepare the nitenpyram test solution:

[0038] Nitenpyram (purity 96%) was mixed with sterile water to prepare a 1000 mg / L stock solution, which was then diluted to the final concentration using 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] In order to strictly control variables such as age and growth environment, we selected 6 honeycombs from three healthy Chinese honey bee colonies, each consisting of half brood and half pollen and honey, shook off all the worker bees on the honeycombs, and transferred the honeycombs to an artificial climate incubator, adjusting the incubation conditions to darkness, 34°C, and 70% RH. After 0.24 hours, the newly emerged worker bees were collected and marked on their backs with a marker. We marked about 800 newly emerged worker bees, and these marked worker bees were put back into the same hive and raised in the hive for 7 days to allow them to complete the colonization of intestinal flora in a natural environment.

[0042] After 7 days, the marked bees (7 days old) were collected and randomly assigned to 20 cups, 30 bees in each cup, with feeders inserted on top. All were evenly divided into two groups. One group was fed with Gilliamella sp.G0441, recorded as Group G. The other group was fed with sterile sucrose solution, recorded as Group S. After 7 days, half of the bees in Group G (14 days old) were orally exposed to a sublethal concentration of 1 mg / L nitenpyram sucrose solution, recorded as GN. The other half were fed with 50% sucrose solution as a control, recorded as GS. The treatment of Group S (14 days old) was the same as above, recorded as SN and SS. There were 4 treatments in the whole experiment, 5 replicates for each treatment, and the pesticide exposure lasted for 7 days (21 days old). During the whole process, dead bees were recorded and cleaned up every 24 hours. Pollen has been shown to regulate the cytochrome P450 (CYP) detoxification enzymes of newly emerged bees. In order to avoid interference, bees were only fed with sucrose solution throughout the experiment.

[0043] (4) Food consumption and body weight test

[0044] Calculate the average daily sugar water consumption of each bee (A), A = G / L-(S / 2), where G represents the daily sugar water consumption; L represents the number of surviving bees on the previous day; and S represents the number of bees that die each day.

[0045] Body weights were recorded on day 21. Three cages were randomly selected for each treatment, and 10 workers from each treatment were randomly selected from each cage. Thus, 30 bees were tested in each group. The bees were stunned and weighed to determine their individual body weights.

[0046] (5) Proboscis extension reflex (PER) test

[0047] The test was conducted 7 days after exposure to nitenpyram (21 days old). Three cups were randomly selected from each treatment group, and 15 bees were sampled from each cup, for a total of 180 worker bees used in the study. The sucrose sensitivity of honey bees was assessed by the proboscis reaction. The proboscis reaction of honey bees was observed by stimulation with 0.1%, 1%, 10% and 50% (w / vg / ml) sucrose solutions. A glass rod was placed close to the antennae of the bees, and increasing concentrations of sucrose solutions were applied through a dropper. If the bee's proboscis was fully extended, a score of 1 was obtained; if there was no reaction, a score of 0 was obtained. The sucrose sensitivity score of each group was calculated by the sum of the number of proboscis reactions of each bee in the group.

[0048] (6) Microbiota-free (MF) bee model test

[0049] Axenic bees were produced in the laboratory. Late pupae were removed from the honeycomb using sterile tweezers and placed in a sterile plastic box. Pupae emerged in an incubator at 35°C and 50% humidity. Newly emerged axenic bees (0 days old) were placed in sterile cups and raised for 24 hours, with 15 bees per cup, and supplemented with sterilized sucrose syrup (50%, w / v). Axenic bees (1 day old) were then divided into two groups, one group was fed with Gilliamella sp. G0441 supplement, and the other group continued to be fed with sterilized sucrose syrup. After 7 days, both groups were treated with the following 3 treatments (each treatment contained 3 cups): (1) CV: continued to be fed with sterilized sucrose syrup; (2) fed with 5 mg / L acetamiprid sucrose solution; (3) fed with 10 mg / L acetamiprid sucrose solution. Dead bees were cleaned and recorded every day.

[0050] (7) Raw data collection and organization

[0051] 2) Test results

[0052] like Figure 4 As shown: Within 14 days, the average food consumption per bee 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, nitenpyram exposure (SN) significantly reduced the food consumption of bees (P=0.0092) and had a negative impact on the appetite of bees. GN food consumption was higher than SN and lower than GS. Although this trend was not significant (P=0.1759, P=0.1907), it suggests that supplementation with Gilliamella sp.G0441 may alleviate the loss of appetite caused by nitenpyram exposure to some extent.

[0053] like Figure 5As shown: Comparison between SN and SS revealed that the weight of bees exposed to nitenpyram was significantly lower than that of healthy bees (P < 0.0001). Comparison between GS and SS, and between GN and SN revealed that supplementation with Gilliamella sp. G0441 significantly promoted the weight gain of the host (P < 0.0001).

[0054] like Figure 6 As shown: The PER response of SN to four sucrose concentrations was significantly lower than that of the control SS (P<0.0001). At 1% concentration, Gilliamella sp. G0441 supplement (GS) significantly increased sucrose sensitivity compared with the control SS. At 10% and 0.1% concentrations, an increasing trend was observed, 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 for the 1% concentration, GN was more sensitive to sucrose than SN. After stimulation with 50% sucrose solution, no significant difference was observed between GN and SS (P=0.8158), indicating that the PER response of GN to this concentration of sucrose was almost as strong as that of healthy bees.

[0055] like Figure 7 As shown: In order to fully evaluate the effects of nitenpyram and Gilliamella sp.G0441 on the recognition and learning ability of honey bees, the sucrose sensitivity of each group of honey bees was scored. The average sucrose sensitivity of SN, SS, GN and GS was 1.15, 2.73, 2.02 and 3.07, respectively. This shows that nitenpyram exposure caused impaired cognitive ability of honey bees, and Gilliamella sp.G0441 supplementation can effectively alleviate these negative effects.

[0056] like Figure 8 As shown: When the oral concentration of 10 mg / L of nitenpyram, the median survival of the MF group was 1 day, while that of the Gilliamella sp. G0441 group was 2.5 days. When the oral concentration of 5 mg / L of nitenpyram, the median survival of the MF group was 2 days, while that of the Gilliamella sp. G0441 group was 3 days. The results showed that Gilliamella sp. G0441 can enhance the host's resistance to nitenpyram and significantly prolong the host's median survival.

Claims

1. Gilliamella sp. G0441, deposit number: GDMCC NO: 65723.

2. Use of the Gilliamella sp. G0441 described in claim 1 in the preparation of a drug for improving the pesticide resistance of honey bees.

3. The use according to claim 2, characterized in that: The pesticide is nitenpyram.

4. The use according to claim 2, characterized in that: The honey bee is Apis cerana cerana.

5. A drug for improving the pesticide resistance of bees, characterized in that: Contains the Gillia sp. G0441 described in claim 1 as an active ingredient.

6. A method for improving the pesticide resistance of honey bees, characterized in that: The method comprises inoculating the Gillia sp. G0441 described in claim 1 into the intestine of honey bees.

7. The method according to claim 6, characterized in that Gilliamella sp.G0441 is inoculated into the bee intestines by feeding.

8. The method according to claim 6, characterized in that The honey bee is Apis cerana cerana.

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