A kit for detecting cold tolerance in honeybees

Abstract

The application discloses a kit for detecting cold resistance of bees, and belongs to the technical field of biology. The kit is prepared by screening 30 important cold resistance metabolic markers of bees in metabolites of worker bees in overwintering period and non-overwintering period. The kit is used for comparing the content of the cold resistance markers in a to-be-detected sample with the content of corresponding markers in a control sample, and determining whether the source individual of the to-be-detected sample has cold resistance according to the comparison result. According to the verification experiment result, if the to-be-detected bee sample contains at least one of the 30 cold resistance metabolic markers, it is determined that the bee individual has cold resistance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a kit for detecting the cold resistance of bees. Background Art

[0002] The Oriental honeybee (Apis cerana) is widely distributed in Asia and nearby islands, with a range covering tropical, subtropical, and temperate climates. Before the introduction of the Western honeybee to China, it was the only bee species that could be artificially bred for production, and its breeding history in China spans over thousands of years. The Changbai Mountain Oriental honeybee (Apis cerana in Changbai Mountain, abbreviated as the Changbai Mountain Chinese honeybee) belongs to the genus Apis, the species Apis cerana, and the subspecies Apis cerana. The Changbai Mountain Oriental honeybee, also known as the Changbai Mountain Chinese honeybee, belongs to one of the ecotypes of the subspecies Apis cerana (there are nine ecotypes of the subspecies Apis cerana, divided by geographical location into Northern Chinese honeybee, Central China Chinese honeybee, Hainan Chinese honeybee, etc.). It is generally referred to as the Changbai Mountain Oriental honeybee in foreign literature and as the Changbai Mountain Chinese honeybee (abbreviated as the Changbai Mountain Chinese honeybee) in domestic literature. The Changbai Mountain Chinese honey bee (Apiscerana in Changbai Mountain, abbreviated as Changbai Mountain Chinese honey bee) is the only ecotype of the Oriental honey bee in the Changbai Mountain flora. Distributed at varying altitudes throughout the mountainous region, it contributes significantly to maintaining and enriching terrestrial ecosystem biodiversity, providing pollination and other valuable ecosystem services. The Changbai Mountain Chinese honey bee exhibits morphological and behavioral adaptations to the long, cold winters, such as its larger wingspan and body length, darker body coloration, high cold tolerance, strong foraging ability, and low swarming tendency. It can naturally overwinter in temperatures as low as -40°C, making it an exceptional representative of my country's native cold-resistant bee species and highly valuable for conservation and development.

[0003] Metabolomics, an emerging discipline that developed in the mid-1990s, is a key component of systems biology. It can reveal differences in metabolite profiles between species, between different tissues of the same species, and within the same tissue of the same species under varying stress conditions, thereby helping to explain the physiological and biochemical characteristics of insect responses to adversity. Currently, metabolomics has been widely applied to the study of the physiological and biochemical mechanisms of insects under adverse stress environments.

[0004] It is well known that the safe overwintering of honeybees is crucial for the survival of bee colonies and subsequent beekeeping production. Overwintering ability is a key indicator of a bee species' superiority and is closely linked to the economic benefits of the beekeeping industry. However, the overwintering ability of honeybees depends largely on their cold tolerance. Compared with Western honeybees, Oriental honeybees are more cold-tolerant and more diligent during the overwintering period. Therefore, identifying key metabolites involved in overwintering bee cold tolerance will help elucidate the physiological and biochemical basis of cold tolerance in bee species, provide scientific guidance for the development of safe overwintering strategies for bee colonies, and offer a theoretical basis for the selection and breeding of new cold-resistant honeybee strains. However, the molecular mechanisms underlying the cold tolerance of Oriental honeybees remain largely unexplored. As the only ecotype of Oriental honeybees in the Changbai Mountain ecosystem, the Changbai Mountain Oriental honeybee has long adapted to the harsh winters and short frost-free periods of the Changbai Mountain ecosystem. This adaptation to the harsh winters and short frost-free periods is essential for the long evolutionary process, and seasonal cold acclimation has enhanced the cold tolerance of the Changbai Mountain population. However, no metabolomic markers of cold tolerance in overwintering worker bees have been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a kit for detecting the cold resistance of honeybees to solve the problems existing in the above-mentioned prior art. The 30 most important honeybee cold resistance metabolic markers are obtained by screening the metabolites of worker bees in the wintering and non-wintering periods. By comparing the content of cold resistance markers in the test sample with the amount of corresponding markers in the control sample, if at least one cold resistance metabolite is contained, the individual honeybee is judged to be cold-resistant.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention also provides a kit for detecting the cold resistance of honeybees, comprising a reagent for detecting the content of at least one of the following metabolites in honeybees: deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, levodopa, 7-O-methyleriodictyol, 9-oxo-10,12-octadecadienoic acid (FA 18:3+1O), 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid, L-β-homothrexane, syringin, kynurenine, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid, and 4-hydroxyquinoline.

[0008] Preferably, the honey bee comprises Apis cerana cerana from Changbai Mountain.

[0009] The present invention provides a use of a reagent for detecting the content of a metabolite marker related to the cold resistance of honey bees in any of the following:

[0010] (1) Application in the preparation of a test kit for detecting the cold resistance of bees;

[0011] (2) Application in breeding cold-resistant bees;

[0012] (3) Application in screening new cold-resistant bee strains;

[0013] The metabolic markers include at least one of deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, levodopa, 7-O-methyleriodictyol, FA 18:3+1O, 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid, L-β-homothreonine, syringin, kynurenine, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid and 4-hydroxyquinoline.

[0014] Preferably, the honey bee comprises Apis cerana cerana from Changbai Mountain.

[0015] The present invention also provides a method for screening cold-resistant bees, comprising the following steps:

[0016] Metabolites are extracted from the bee tissue samples to be tested, the metabolites are annotated and analyzed using LC-MS, and the metabolite content is compared with that of the control. Whether the bees to be tested are cold-resistant is determined based on the changes in the metabolites; among them, worker bees in August of the same year are used as the control.

[0017] Preferably, the judgment method is: when the metabolite is at least one of deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, levodopa, 7-O-methyleriodictyol, FA 18:3+1O, 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid and L-β-homothreonine, and the corresponding metabolite content is reduced compared with the control, it indicates that the tested bee has cold resistance;

[0018] When the metabolite is at least one of syringin, kynurenine, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid and 4-hydroxyquinoline, and the content of the corresponding metabolite is upregulated compared with the control, it indicates that the tested bee has cold resistance.

[0019] The present invention also provides a method for screening metabolite markers related to the cold resistance of honey bees, comprising the following steps:

[0020] Collect honeybee tissue samples in the middle of wintering. Every 20-30 honeybee tissue samples were mixed into a composite sample. Each composite sample was set up with 3-6 biological replicates. At the same time, non-overwintering honeybee workers in August of the same year were used as the control group.

[0021] Metabolites were extracted from mixed samples and analyzed using liquid chromatography-mass spectrometry to screen for differential metabolites between mid-wintering bees and the control group.

[0022] The metabolites that were significantly different between the bees in the mid-wintering period and the control group were identified as metabolite markers related to the cold resistance of bees.

[0023] Preferably, the metabolite markers associated with the cold resistance of honey bees include deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, L-dopa, 7-O-methyleriodictyol, FA 18:3+1O, 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid, L-β-homothreonine, syringin, kynurenine, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid and 4-hydroxyquinoline.

[0024] These metabolites were analyzed using high-performance liquid chromatography-mass spectrometry-coupled metabolomics techniques. The composition and content of metabolites in the gut tissues of overwintering and non-wintering worker bees (Apis orientalis) from Changbai Mountain were compared. The metabolites and metabolic pathways were identified, and the 30 most significant differential metabolites were identified. These differential metabolites are associated with resistance to oxidative damage, microbial infection, cold tolerance, and the synthesis and metabolism of nutrients and energy. These metabolites contribute significantly to the differences between the guts of overwintering and non-wintering bees. By consuming or accumulating bioactive antioxidant metabolites such as isorhamnetin and syringin, the body participates in oxidative stress defense responses, mitigates oxidative damage caused by stressors such as low temperature, and improves overwintering resistance. Furthermore, the most significantly downregulated KEGG pathways were purine metabolism, the pentose phosphate pathway, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis, while the most significantly upregulated KEGG pathways were sulfur relay system, niacin and nicotinamide metabolism, and alanine, aspartate, and glutamate metabolism. This indicates that the body enhances its tolerance to low-temperature stress and maintains homeostasis of the internal environment by regulating related pathways such as intracellular and extracellular substance transport, cofactor synthesis, and energy metabolism, thereby providing nutritional and energy support for the bee colony's wintering behavior. Using these cold-resistant metabolites, a kit for detecting the cold tolerance of honeybees is provided. The kit can be used to measure the cold tolerance markers in the test sample. By comparing the cold tolerance marker content in the test sample with the amount of the corresponding marker in the control sample obtained using the same determination method, it is determined whether the individual bee from which the test sample is derived is cold-resistant based on the comparison results. If at least one cold-resistant metabolite is present, the individual bee is determined to be cold-resistant.

[0025] The present invention discloses the following technical effects:

[0026] The ultra-high performance liquid chromatography combined with quadrupole-orbitrap mass spectrometry technology used in the present invention can study biological problems in a more fundamental and specific manner, and conduct in-depth analysis of the molecular mechanisms of substance metabolism, providing a stable and reliable detection method for the metabolomics analysis of the intestinal tissue of the Oriental honey bees in Changbai Mountain during the wintering period.

[0027] This invention provides fundamental data for studying the molecular mechanisms by which the Changbai Mountain oriental honey bee population adapts to cold weather, and offers a theoretical basis for the molecular breeding of new cold-resistant honey bee strains. Furthermore, based on the obtained cold-resistant metabolites of overwintering bees, the invention provides a kit for testing the cold tolerance of honey bees. By measuring the content of these metabolites, the cold tolerance of individual honey bees can be determined, allowing the screening of bee colonies with high cold tolerance to be obtained. This provides effective assurance and a reliable basis for the safe overwintering of bee colonies and the molecular breeding of cold-resistant bee species. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is the Base Peak overlay spectrum of the QC sample in positive ion mode of Example 1;

[0030] Figure 2 This is the Base Peak overlay spectrum of the negative ion mode of the QC sample in Example 1;

[0031] Figure 3 This is the PCA score graph of the sample in positive ion mode of Example 1;

[0032] Figure 4 This is the PCA score graph of the negative ion mode of the sample in Example 1;

[0033] Figure 5 The volcano plots of differential metabolites in the positive and negative ion modes of Example 1; A: Volcano plot of differential metabolites in the positive ion mode; B: Volcano plot of differential metabolites in the negative ion mode;

[0034] Figure 6 The bar graph is for importance analysis of differential metabolites (top 30 VIP values);

[0035] Figure 7 Classification diagram of differential metabolites; A: KEGG classification result; B: HMDB classification result;

[0036] Figure 8 Results of KEGG pathway analysis of differential metabolites; A: KEGG pathway enrichment bar chart; B: DAscore shows the overall up- and down-regulation trend of metabolic pathways;

[0037] Figure 9 This is the Base Peak overlay spectrum of the positive ion mode of the QC sample in Example 2;

[0038] Figure 10 This is the Base Peak overlay spectrum of the negative ion mode of the QC sample in Example 2;

[0039] Figure 11 This is the PCA score graph of the sample in positive ion mode of Example 2;

[0040] Figure 12 This is the PCA score graph of the negative ion mode of the sample in Example 2;

[0041] Figure 13The volcano plots of differential metabolites in the positive and negative ion modes of Example 2; A: Volcano plot of differential metabolites in the positive ion mode; B: Volcano plot of differential metabolites in the negative ion mode;

[0042] Figure 14 This is the result of the cold resistance marker test of the cephalothorax tissue of the Changbai Mountain honey bee based on the honey bee cold resistance kit;

[0043] Figure 15 This is the result of cold resistance marker detection in the intestinal tissue of Italian honey bees;

[0044] Figure 16 These are the results of cold resistance marker detection in the cephalothorax tissue of the Italian honey bee. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] The main instruments and reagents involved in the following examples are:

[0051] Reagents: Acetonitrile, methanol, aqueous ammonia, ammonium acetate, and formic acid were all of chromatography grade (manufacturer: Millipore).

[0052] Instruments: Q Exactive Plus mass spectrometer (Thermo Scientific), Nexera X2 LC-30AD ultrahigh pressure liquid chromatograph (Shimadzu), chromatographic column (ACQUITY UPLC BEH Amide 1.8μm, 2.1×100mmcolumn), Bioruptor ultrasonic system (Diagenode), Concentrator plus vacuum centrifugal concentrator and 5430R centrifuge (all Eppendorf).

[0053] Example 1

[0054] 1. Sample collection

[0055] The test bee colonies are purebred Changbai Mountain honeybees, which were collected from Malugou Town, Changbai Korean Autonomous County, National Honeybee (Changbai Mountain Honeybee) Nature Reserve (N41°44′28″, E128°21′32″) in 2023. Three healthy Changbai Mountain honeybee colonies that overwinter naturally outdoors were selected, and samples were taken in the middle of the winter (December). The entire intestine of the overwintering worker bees was removed and quickly put into liquid nitrogen, marked as "CD", and stored at -80°C for later use. A mixed sample of the complete intestinal tissue of every 30 worker bees was used as one sample, and 6 biological replicates were set for each sample. The worker bees in August of that year were used as the control group, and the sampling method was the same as the December samples.

[0056] 2. Metabolite extraction

[0057] For quality control purposes, QC samples were prepared. These were equal amounts of all samples mixed together to equilibrate the chromatography-mass spectrometry system and instrument status, as well as to assess system stability throughout the experiment. After grinding the samples with liquid nitrogen, 60 mg of each sample was weighed and mixed with 200 μL of pre-chilled water and 800 μL of pre-chilled methanol / acetonitrile (1:1, v / v). The mixture was sonicated in an ice bath for 1 hour, allowed to stand at -20°C for 2 hours, and centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant was then removed and evaporated to dryness in a high-speed vacuum concentrator. For mass spectrometry analysis, the mixture was reconstituted with 450 μL of a methanol-water solution (1:1, v / v), centrifuged at 20,000 g for 40 minutes at 4°C, and the supernatant was sampled for analysis.

[0058] 3. LC-MS analysis

[0059] 3.1 Chromatographic separation

[0060] Throughout the analysis, samples were placed in an autosampler at 4°C and separated using a HILIC column on a SHIMADZU-LC30 ultra-high performance liquid chromatography (UHPLC) system. The injection volume was 3 μL, the column temperature was -25°C, and the flow rate was 0.3 mL / min. The mobile phases (A) were water + 25 mM ammonium acetate and B was acetonitrile. The gradient elution program was as follows: 95% B from 0 to 1 min; linear B from 95% to 65% from 1 to 7 min; linear B from 65% to 35% from 7 to 9 min; B maintained at 35% from 9 to 10.5 min; linear B from 35% to 95% from 10.5 to 11 min; and B maintained at 95% from 11 to 15 min.

[0061] 3.2 Mass spectrometry acquisition

[0062] Each sample was detected using electrospray ionization (ESI) in both positive (+) and negative (-) modes. After UPLC separation, the samples were analyzed by mass spectrometry on a QE Plus mass spectrometer using a HESI source. Ionization conditions were as follows: spray voltage: 3.8 kV (+) and 3.2 kV (-); capillary temperature: 320°C (±); sheath gas: 30°C (±); auxiliary gas: 5°C (±); probe heater temperature: 350°C (±); and S-Lens RF level: 50°C.

[0063] Mass spectrometry acquisition settings were as follows: MS acquisition time: 12 min. Precursor ion scan range: 80–1200 m / z, MS primary resolution: 70,000 at m / z 200, AGC target: 3e6, and maximum IT for primary analysis: 100 ms. Secondary mass spectrometry analysis was performed using the following protocol: triggering acquisition of the ten most intense precursor ion MS secondary spectra after each full scan. MS secondary resolution: 17,500 at m / z 200, AGC target: 1e5, maximum IT for secondary analysis: 50 ms, MS2 activation type: HCD, isolation window: 2 m / z, and normalized collision energy (Setpped): 10, 20, and 30.

[0064] 4. Data processing and analysis

[0065] Raw data were aligned, retention time corrected, and peak areas extracted using MSDIAL software. Positive and negative ion data were normalized for total peak area, integrated, and pattern recognition performed using R software. System stability was evaluated and analyzed using two strategies: mass spectrometry base peak alignment of QC samples and PCA statistical analysis of the overall sample. Metabolite ion peaks were extracted using MSDIAL software and subjected to principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) after unit variance scaling (UV). Univariate statistical analysis was performed using FC > 1.5 or FC < 0.667 and P value < 0.05 as screening criteria to visually demonstrate the significance of metabolite changes between the two groups. The variable weights (VIP) derived from the OPLS-DA model were used to measure the influence and explanatory power of each metabolite expression pattern on the classification and discrimination of each sample group. The present invention selects metabolites with both multivariate statistical analysis VIP>1 and univariate statistical analysis P value<0.05 as metabolites with significant differences, and performs expression change analysis and functional pathway analysis on them.

[0066] 5. Experimental quality evaluation

[0067] The system stability of this experiment was evaluated and analyzed by using two strategies: mass spectrum Base Peak graph comparison of QC samples and PCA statistical analysis of overall samples.

[0068] The total ion current (Base Peak) of the mass spectrometer of the QC sample in the positive and negative ion detection modes were superimposed and compared (see Figure 1 and Figure 2 ); The results showed that the response intensity and retention time of each chromatographic peak basically overlapped, indicating that the variation caused by instrument error during the entire experiment was small and the data quality was reliable.

[0069] The peaks extracted from all experimental samples and QC samples were subjected to PCA analysis, and the PCA model obtained by 7-fold cross-validation (7 cycles of interactive validation) (see Figure 3 and Figure 4 The results showed that the QC samples were clustered closely together, indicating good reproducibility in this experiment. Based on these results, the instrumental analysis system used in this experiment was stable, and the experimental data was robust and reliable. The differences in metabolic profiles observed in this experiment reflect the biological differences between the samples.

[0070] 6. Differential metabolite screening

[0071] Based on orthogonal partial least squares discriminant analysis (OPLS-DA), R 2 Y≥0.99, and Q 2 >0.95, indicating that the model is stable and can be used for subsequent analysis (see Table 1). A total of 344 differential metabolites were screened, of which 125 were differentially expressed between the overwintering bee and non-overwintering bee groups in the positive ion mode, 53 of which were upregulated and 72 were downregulated. 47 of them could be mapped to the KEGG and HMDB databases (see Figure 5 In the negative ion mode, 219 differential metabolites were obtained, 127 of which were upregulated and 92 were downregulated, and 94 of them could be mapped to the KEGG and HMDB databases (see Figure 5 Middle B).

[0072] Table 1 Evaluation parameters of OPLS-DA model

[0073]

[0074] Note: A: represents the principal component; R 2 : represents the model explanation rate; Q 2 : Indicates the model's predictive ability; R 2 and Q 2 The closer it is to 1, the more stable and reliable the model is. 2 If it is greater than 0.5, the model has good predictive ability. 2 Less than 0.5 indicates that the model has poor predictive ability; R 2 intercept and Q 2 intercept: represents R 2 and Q 2 The intercept of the regression line with the Y-axis.

[0075] 7. Differential metabolite expression analysis

[0076] The present invention annotated and analyzed the metabolites with high importance (VIP value top 30) in positive and negative ion modes and found that deoxyadenosine (C 10 H 13 N5O3), isorhamnetin (C 16 H 12 O7), isobutyric acid (C4H8O2), delphinidin (C 15 H 11 O7), Mipaline (C 23 H 30 ClN3O), melatonin (C 13 H 16 N2O2), γ-linolenic acid (C 18 H 30 O2), linoleic acid (C 18 H32 O2), levodopa (C9H 11 NO4), 7-O-methyleriodictyol (C 16 H 14 O6)、FA 18:3+1O(C 18 H 30 O3), 2'-deoxyinosine-5'-phosphate (C 10 H 13 N4O7P), tricoumarin spermidine (C 34 H 37 N3O6), anthranilic acid (C7H7NO2), L-β-homothreonine (C5H 11 NO3) decreased significantly, and the content of syringin (C 17 H 24 O9), kynurenine (C 10 H 12 N2O3), chrysanthemum aldehyde (C 16 H 10 O6), citraconic acid (C5H6O4), pyridoxine (C8H 11 NO3), thiamine ([C 12 H 17 N4OS] + ), S-adenosyl-L-homocysteine ​​(C 14 H 20 N6O5S), quinolinic acid (C7H5NO4), pentadecanoic acid (C 15 H 30 O2), N-fructosylpyroglutamate (C 11 H 17 NO8), Aloe Vera (C 19 H 22 O 10 ), 2,8-dihydroxyquinoline (C9H7NO2), D-fructose 1-phosphate (C6H 13 O9P), 3,4-di-O-caffeoylquinic acid (C 25 H 24 O 12 ), 4-hydroxyquinoline (C9H7NO) accumulates in large quantities in the intestine (see Figure 6 The above metabolites contribute significantly to the differences between the intestines of overwintering bees and non-overwintering bees. By consuming or accumulating bioactive metabolites with antioxidant functions, such as isorhamnetin and syringin, the body participates in the oxidative stress defense response, reduces oxidative damage caused by stress factors such as low temperature, and improves the body's overwintering resistance.

[0077] 8. Functional analysis of differential metabolites

[0078] 8.1 Classification of Differential Metabolites

[0079] Based on the structure and function of metabolites, the differential metabolites in the gut of overwintering bees and non-overwintering bees were classified and counted. The results of the substance classification in the KEGG database under the positive and negative example modes showed that the differential metabolites were mainly annotated into categories such as polyketides, peptides, nucleic acids, phenylpropanoids, carbohydrates and fatty acids (see Figure 7 The results of HMDB database showed that the main categories of differential metabolites were organic acids and their derivatives, organic heterocyclic compounds, phenylpropanoids and polyketides, lipids and lipid molecules, benzene ring compounds, etc. (see Figure 7 Middle B).

[0080] 8.2 KEGG pathway analysis of differential metabolites

[0081] The results of functional enrichment analysis of the KEGG database for the differential metabolites in positive and negative ion modes showed that the differential metabolites were mainly annotated to neuroactive ligand-receptor interaction, ABC transporter, valine, leucine and isoleucine biosynthesis, tyrosine metabolism, tryptophan metabolism, starch and sucrose metabolism, pyrimidine metabolism, purine metabolism, histidine metabolism, galactose metabolism, cofactor biosynthesis, amino acid biosynthesis, β-alanine metabolism, arginine and proline metabolism (see Figure 8 Middle A).

[0082] In order to systematically study metabolic changes, the overall trend analysis of metabolic pathways was performed using differential metabolite abundance. Differential abundance scores (DA score) can capture the trend of overall metabolite increase or decrease in the pathway relative to the control group. The results showed that purine metabolism, pentose phosphate pathway, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis were the most significantly downregulated KEGG pathways, while sulfur relay system, nicotinic acid and nicotinamide metabolism, alanine, aspartate, and glutamate metabolism were the most significantly upregulated KEGG pathways (see Figure 8 This suggests that the body enhances its tolerance to low-temperature stress and maintains internal environmental homeostasis by regulating pathways related to intracellular and extracellular substance transport, cofactor synthesis, and energy metabolism, thereby providing nutritional and energy support for the bee colony's wintering behavior.

[0083] Example 2

[0084] The present invention also extracts and analyzes metabolites from other tissues of the bee colony, specifically:

[0085] 1. Sample collection

[0086] Three healthy, naturally overwintering outdoor colonies of Apis cerana were selected. Sampling was performed in mid-winter (December). Cephalothorax muscle tissue from each overwintering worker bee was removed and quickly plunged into liquid nitrogen, labeled "ZZ," and stored at -80°C until further use. A pooled sample of cephalothorax tissue from 30 worker bees was used as one sample, with six biological replicates for each sample. Worker bees from August of the same year served as a control group, using the same sampling method as for the December samples.

[0087] 2. Metabolite extraction

[0088] Same as "2. Metabolite extraction" in Example 1.

[0089] 3. LC-MS analysis

[0090] Same as "3. LC-MS analysis" in Example 1.

[0091] 4. Data processing and analysis

[0092] Same as “4. Data processing and analysis” in Example 1.

[0093] 5. Experimental quality evaluation

[0094] Same as "5. Experimental quality evaluation" in Example 1.

[0095] The total ion current (Base Peak) of the mass spectrometer of the QC sample in the positive and negative ion detection modes were superimposed and compared respectively. Figure 9 and Figure 10 The results showed that the response intensity and retention time of each chromatographic peak basically overlapped, indicating that the variation caused by instrument error during the entire experiment was small and the data quality was reliable.

[0096] The peaks extracted from all experimental samples and QC samples were subjected to PCA analysis after UV treatment. The PCA model obtained by 7-fold cross-validation (7 cycles of interactive validation) is shown in Figure 11 and Figure 12 .like Figures 9-12 As shown in the figure, the QC samples are clustered together closely, indicating that the repeatability of the experiment in this project is good.

[0097] 6. Differential metabolite screening

[0098] Based on orthogonal partial least squares discriminant analysis (OPLS-DA), R 2 Y≥0.99, and Q 2 >0.92, indicating that the model is stable and can be used for subsequent analysis (see Table 2). A total of 325 differential metabolites were screened, of which 188 differential metabolites were obtained between the overwintering bee and non-overwintering bee groups in the positive ion mode (see Figure 13A), 137 differential metabolites were obtained in negative ion mode (see Figure 13 Middle B).

[0099] Table 2 Evaluation parameters of OPLS-DA model

[0100]

[0101] Note: A: represents the principal component; R 2 : represents the model explanation rate; Q 2 : Indicates the model's predictive ability; R 2 and Q 2 The closer it is to 1, the more stable and reliable the model is. 2 If it is greater than 0.5, the model has good predictive ability. 2 Less than 0.5 indicates that the model has poor predictive ability; R 2 intercept and Q 2 intercept: represents R 2 and Q 2 The intercept of the regression line with the Y-axis.

[0102] Application Example 1

[0103] Overwintering bee colonies of Apis cerana cerana (Changbai Mountain) in natural outdoor overwintering conditions were selected, and the non-cold-tolerant Italian honey bee species (Apis mellifera L.) was used as a control. This kit was used to detect cold-resistance markers in intestinal tissue samples of the two bee species.

[0104] As shown in Table 3, 30 cold-resistant metabolites were detected in the intestines of the overwintering honeybee colonies of the Changbai Mountain Chinese honeybee. However, no cold-resistant metabolites were detected in the intestines of the Italian honeybee, such as Figure 15 shown.

[0105] Table 3 Cold-resistant metabolites detected in the intestines of overwintering honeybee colonies of Apis cerana cerana in Changbai Mountain

[0106]

[0107]

[0108] Note: Dec-C1 to Dec-C6 represent 6 biological technical replicates.

[0109] Application Example 2

[0110] Overwintering colonies of Chinese honey bees (Apis cerana cerana) from Changbai Mountain, which were naturally overwintering outdoors, were selected. The non-cold-tolerant Italian honey bees (Apis mellifera L.) were used as controls. This kit was used to detect cold-resistance markers in cephalothorax tissue samples of the two bee species.

[0111] The results are shown in Table 4 and Figure 14 As shown in Figure 2, seven cold tolerance markers were detected in the cephalothorax tissue samples of overwintering honeybees from Changbai Mountain: fusoflavone, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid, thiamine, S-adenosyl-L-homocysteine, mepalene and tricoumarin spermidine; while no cold tolerance markers were detected in the non-cold-tolerant honeybee species, Apis mellifera (see Figure 16 ), which proved the specificity and reliability of this kit for detecting bee cold resistance.

[0112] Table 4 Seven cold resistance markers detected in the cephalothorax tissue samples of overwintering Apis cerana cerana bees in Changbai Mountain

[0113]

[0114]

[0115] As can be seen from the above examples, the present invention uses intestinal tissue from overwintering worker bees to screen for cold-resistance biomarkers. This is because during the long winter, bees cannot obtain food from the outside world and can only consume stored overwintering bee food. They also face multiple survival pressures such as pathogen invasion, hunger, and malnutrition. The intestine is a key immune organ in bees and a major tissue organ for food digestion, detoxification, and metabolism, playing a crucial role in resisting exogenous stimuli. Furthermore, numerous studies have shown that the intestinal flora of bees plays a crucial role in the digestion and metabolism of host food, activation of host immunity, resistance to pathogens, and regulation of host physiology. The intestinal microbiome of bees is closely related to the health of their host. Therefore, under the adverse conditions of the overwintering period, the intestinal health of bees has a significant impact on the survival of the bee colony. Therefore, the inventors believe that sampling intestinal tissue from worker bees is more typical and representative than other tissues and organs, and that the intestinal metabolite profiles of overwintering bees can better reflect the environmental adaptability of a bee species to the local winter climate. Furthermore, there are currently no reports analyzing the mechanisms of cold-resistance adaptation in overwintering bees from the perspective of intestinal nutrient metabolism. In addition, in the verification experiment, samples from different tissue sites were collected for verification, indicating that this method of screening cold-resistant metabolites is not limited to intestinal tissue, but can also screen cold-resistant metabolites in other tissue sites, and the results are consistent.

[0116] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the content of a metabolite marker associated with bee cold tolerance in the preparation of a kit for detecting bee cold tolerance metabolites; the metabolites are bee intestinal metabolites; in, The metabolite markers include deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, L-dopa, 7-O-methyleriodictyol, 9-oxo-10,12-octadecadienoic acid, 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid, L-β-homothreonine, syringin, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid, and 4-hydroxyquinoline; The bee is the Changbai Mountain Chinese honey bee.

2. A method for screening metabolite markers related to bee cold resistance, characterized in that: The following steps are involved: Collect honeybee tissue samples in the middle of wintering. Every 20-30 honeybee tissue samples were mixed into a composite sample. Each composite sample was set up with 3-6 biological replicates. At the same time, non-overwintering honeybee workers in August of the same year were used as the control group. Metabolites were extracted from mixed samples and analyzed using liquid chromatography-mass spectrometry to screen for differential metabolites between mid-wintering bees and the control group. The metabolites with significant differences between the bees in the mid-wintering period and the control group were identified as metabolite markers related to the cold tolerance of bees. The bee is Apis cerana cerana from Changbai Mountain; the metabolites are bee intestinal metabolites; The metabolite markers related to honey bee cold tolerance include deoxyadenosine, isorhamnetin, isobutyric acid, delphinidin, mepaline, melatonin, γ-linolenic acid, linoleic acid, levodopa, 7-O-methyleriodictyol, 9-oxo-10,12-octadecadienoic acid, 2'-deoxyinosine-5'-phosphate, tricoumarin spermidine, anthranilic acid, L-β-homothrexane, syringin, calciferol, citraconic acid, pyridoxine, thiamine, S-adenosyl-L-homocysteine, quinolinic acid, pentadecanoic acid, N-fructosylpyroglutamic acid, aloin, 2,8-dihydroxyquinoline, D-fructose 1-phosphate, 3,4-di-O-caffeoylquinic acid and 4-hydroxyquinoline.

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