Use of juvenile hormone in mitigating the risk of poisoning with thiamethoxam in apis mellifera ligustica

Abstract

The application relates to application of juvenile hormone in relieving thiamethoxam poisoning risk of Apis mellifera ligustica, and the juvenile hormone is used for accelerating the metabolism rate of thiamethoxam in the body of the honeybee; the juvenile hormone is JH III. The application method is as follows: JH III is first dissolved with methanol to prepare a JH III mother liquor with a concentration of 5 ng / muL, is blown dry with nitrogen, is re-dissolved with an equal volume of ultrapure water, is then added into sugar water feed to prepare a mixed solution with a JH III concentration of 1 ng / muL, and is fed to the honeybee. The application can significantly accelerate the metabolism of thiamethoxam by regulating the hormone level in the body of the honeybee, and can further enhance the detoxification capacity, so as to improve the survival rate. The JH III selected in the application is a hormone secreted by the honeybee itself, has the characteristics of strong targeting and low toxicity to the honeybee, and has a precise application dose, and has no significant influence on the normal growth and development, reproduction and foraging behavior of the honeybee, and the quality and safety of the bee product.

Description

Technical Field

[0001] This invention relates to the field of bee ecological protection and pesticide metabolism regulation technology, and in particular to the application of juvenile hormone in alleviating the risk of thiamethoxam poisoning in Italian honeybees. Background Technology

[0002] Honeybees, as one of the most important pollinating insects, play a vital role in ensuring the safety, value, and yield of crops. However, with the widespread use of pesticides in agricultural production, honeybees are highly susceptible to pesticide residues while collecting nectar and pollen. Thiamethoxam, a commonly used neonicotinoid insecticide, has significant toxic effects on honeybees.

[0003] Thiamethoxam is highly acutely toxic to bees. When ingested by bees, it easily accumulates in their bodies and is difficult to metabolize and eliminate quickly, thus severely disrupting the bees' nervous, endocrine, and immune systems. Studies have shown that thiamethoxam can severely interfere with bees' flight ability, learning and memory abilities, and metabolic capacity, leading to abnormal behavior, stunted growth and development, decreased reproductive capacity, and shortened lifespan, posing a serious threat to bee colony safety.

[0004] Juvenile hormones are important endocrine hormones secreted by the lateral pharyngeal body in insects, widely involved in regulating physiological processes such as growth, development, metamorphosis, reproduction, and metabolism. In honeybees, juvenile hormones form a complex regulatory module with vitellogenin (Vg), influencing behavioral changes and energy metabolism. Even minute changes in juvenile hormones can significantly regulate the expression of metabolism-related genes in honeybees. Studies have found that juvenile hormones can affect the metabolism of exogenous substances in insects by regulating metabolic enzyme activity and related signaling pathways; however, there are currently no reports on technologies that utilize juvenile hormones to accelerate the metabolism of thiamethoxam or mitigate its toxicity in honeybees.

[0005] Meanwhile, traditional methods such as reducing pesticide use, developing low-toxicity pesticides, and using buffers are constrained by the needs of agricultural production and cannot fundamentally solve the problem of thiamethoxam accumulation in bees. In recent years, some studies have attempted to enhance bees' detoxification abilities and P450 enzyme metabolism systems using plant extracts and probiotics, but these methods suffer from slow effects, narrow applicability, and high costs, making large-scale application difficult. Therefore, developing an efficient, convenient, and low-cost method to accelerate the metabolism of thiamethoxam in bees, reduce its residue levels, and alleviate toxic damage has become a pressing technical challenge in bee conservation. Summary of the Invention

[0006] Technical problem solved: In view of the shortcomings of the existing technology, the present invention provides the application of juvenile hormone in alleviating the risk of thiamethoxam poisoning in Italian honeybees. JH III is added to sugar water feed and fed to worker bees, thereby accelerating the metabolism of thiamethoxam in the worker bees, improving metabolic efficiency, increasing the detoxification ability of the bee colony, reducing the risk of thiamethoxam intake, and thus improving the survival rate of the bee colony.

[0007] Technical solution: Application of juvenile hormone in alleviating the risk of thiamethoxam poisoning in Italian honeybees, wherein the juvenile hormone is used to accelerate the metabolic rate of thiamethoxam in honeybees; the juvenile hormone is JH III.

[0008] The JH III described above was first dissolved in methanol to prepare a JH III stock solution with a concentration of 5 ng / μL. The JH III stock solution was dried with nitrogen and then reconstituted with an equal volume of ultrapure water. It was then added to sugar water feed to prepare a mixed solution with a JH III concentration of 1 ng / μL, which was fed to bees. The sugar concentration in the sugar water feed was 50%.

[0009] The feeding method described above is as follows: After removing the bees from the hive, fix them on a clean flat plate, and use a micropipette to feed the bees the prepared mixed solution.

[0010] Each bee was fed 4 μL of the mixed solution once as described above.

[0011] The bees were starved before feeding.

[0012] Beneficial Effects: The application of the juvenile hormone provided by this invention in alleviating the risk of thiamethoxam poisoning in Italian honeybees has the following beneficial effects:

[0013] 1. Significantly improves the metabolic efficiency of thiamethoxam in bees: This invention regulates hormone levels in bees through JH Ⅲ, which can significantly accelerate the metabolism of thiamethoxam, thereby enhancing detoxification ability and improving survival rate.

[0014] 2. High safety and no side effects: The natural JH Ⅲ selected in this invention is a hormone secreted by bees themselves. It has the characteristics of strong targeting, low toxicity to bees, and safe bee products. Moreover, the dosage is precise and has no significant impact on the normal growth, development, reproduction and foraging of bees.

[0015] 3. Simple operation and low cost: This invention uses JH Ⅲ preparation as the mother liquor, which is diluted with sugar water to form a mixed sample, and then fed to bees. It requires no complicated equipment, is easy to operate, and is suitable for large-scale application. The required dosage of JH Ⅲ and excipients is low, which can significantly reduce the input cost of bee protection and is easy to promote in beekeeping and agricultural production.

[0016] 4. Significant ecological value: The method of this invention can effectively alleviate the toxic threat of thiamethoxam to bees, protect bee populations, maintain the pollination function of agricultural ecosystems, promote crop yield and biodiversity conservation, and reduce ecological imbalance caused by bee mortality, thus having significant ecological and economic benefits. Attached Figure Description

[0017] Figure 1 The quantitative ion spectrum of thiamethoxam in Example 2 is shown.

[0018] Figure 2 This is a graph showing the dynamic changes in the residual amount of thiamethoxam in bees in Example 2. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0020] The sources of the raw materials used in the following examples are shown in Table 1 below.

[0021] Table 1

[0022]

[0023] The manufacturers and models of the instruments used in the following examples are shown in Table 2 below.

[0024] Table 2

[0025]

[0026] In this invention, juvenile hormone JH III can alleviate the risk of thiamethoxam poisoning in Italian honeybees. The method is as follows: JH III is first dissolved in methanol to prepare a JH III stock solution with a concentration of 5 ng / μL. The JH III stock solution is dried with nitrogen and then reconstituted with an equal volume of deionized water. Then, it is added to a 50% sugar water diet to prepare a mixed solution with a JH III concentration of 1 ng / μL. Before feeding, the bees are starved. Then, the bees are removed from the hive and attached to a clean plate with double-sided tape. The prepared mixed solution is fed to the bees using a micropipette. Each bee is fed 4 μL of the mixed solution once. After that, they are cultured normally with a 50% sugar water diet. After the bees finish eating, they are placed in an insect culture box that simulates the internal environment of the honeycomb for further culture.

[0027] Example 1

[0028] This example is an acute oral toxicity test to investigate whether juvenile hormone can reduce the sensitivity of bees to thiamethoxam.

[0029] In an experimental bee colony, Italian honeybee worker bees were selected as the experimental subjects, and the following steps were followed:

[0030] Preparation of sugar solution: Dissolve sugar in water to prepare a 50% sugar solution.

[0031] Preparation of mixed solution (juvenile hormone solution): JH III was first dissolved in methanol to prepare a JH III stock solution with a concentration of 5 ng / μL. The juvenile hormone JH III stock solution was dried with nitrogen and then reconstituted with an equal volume of deionized water. Then it was dissolved in sugar water solution to prepare a mixed solution with a JH III concentration of 1 ng / μL.

[0032] Preparation of thiamethoxam solutions: Dissolve thiamethoxam in methanol to prepare a 5 ng / μL thiamethoxam stock solution. Then, dry the thiamethoxam stock solution with nitrogen gas and reconstitute it with an equal volume of deionized water. Then, dissolve it in a sugar solution to prepare thiamethoxam solutions with concentrations of 1 ng / μL, 0.75 ng / μL, 0.4 ng / μL, 0.2 ng / μL, and 0.1 ng / μL.

[0033] Group settings:

[0034] The control group was fed a sugar solution. A total of 30 bees were included, with 10 bees per replicate, for a total of 3 replicates.

[0035] The first group (thiamethoxam group) was fed thiamethoxam solution and sugar water solution. A total of 150 bees were divided into 5 groups, with 10 bees per group and one replicate, for a total of 3 replicates. Each group was fed thiamethoxam solution at concentrations of 1 ng / uL, 0.75 ng / uL, 0.4 ng / uL, 0.2 ng / uL, and 0.1 ng / uL once, followed by a sugar water solution.

[0036] The second group (thiamethoxam + JH III group) was fed thiamethoxam solution, a mixed solution (juvenile hormone solution), and a sugar solution. A total of 150 bees were divided into 5 groups, with 10 bees per group and one replicate, for a total of 3 replicates. Each group was fed thiamethoxam solution at doses of 1 ng / uL, 0.75 ng / uL, 0.4 ng / uL, 0.2 ng / uL, and 0.1 ng / uL once, the mixed solution (juvenile hormone solution) once, and then the sugar solution.

[0037] Feeding and culture:

[0038] First, the bees in the hive were captured using a rectangular, breathable plastic box (20cm long x 10cm wide x 8cm high) and quickly placed in an incubator (temperature: 25±1℃, humidity: 60%, light intensity: 6000 Lux) for 3 hours to starve them. Next, the bees were anesthetized with a small amount of carbon dioxide, then removed with tweezers and attached to a clean flat plate with double-sided tape, ensuring each bee was spaced at least 3cm apart to prevent contact. Once the bees were slightly awakened, a micropipette was used to draw the appropriate solution and feed it to their proboscis using a teasing motion, enticing them to eat. Afterward, the fed bees were carefully placed in a cylindrical, breathable plastic box (10cm in diameter at the bottom, 8cm high), with 10 bees of each duplicate placed in the same box. Finally, the bees in the box were placed in an insect culture box simulating the internal environment of a beehive (temperature: 25±1℃, humidity: 60%, light: darkness = 16h: 8h, light intensity: 6000Lux) and cultured with sugar solution.

[0039] The control group was fed a sugar solution throughout the entire culture process; the first group (thiamethoxam group) was initially fed 1 μL of the corresponding concentration of thiamethoxam solution, followed by feeding with a sugar solution; the second group (thiamethoxam + JH III group) was initially fed 1 μL of the corresponding concentration of thiamethoxam solution and 4 μL of a mixed solution (juvenile hormone solution), followed by feeding with a sugar solution. The number of sugar solution feedings was determined to ensure that the bees did not die from starvation.

[0040] Recording frequency: Record and observe bee mortality and mental state every 24 hours, including both the number of deaths and the number of survivors. Simultaneously, promptly remove dead bees from the feeding containers as needed, and replace them with new boxes.

[0041] Calculation method: SPSS statistical software was used for data statistical analysis. The actual mortality rate was calculated, and the median lethal concentration (LC50) of thiamethoxam for acute oral toxicity in bees was determined using the probability unit method. 50 ).

[0042] Record data and results:

[0043] The data records and results for 24 hours are shown in Table 3 below.

[0044] Table 3

[0045]

[0046] Table 3 shows that the acute oral toxicity of thiamethoxam to bees is concentration-dependent; the mortality rate of bees gradually increases with increasing treatment concentration: the mortality rate of bees in the 1 ng / μL and 0.75 ng / μL concentration groups reached 70%, while the mortality rates in the 0.4 ng / μL, 0.2 ng / μL, and 0.1 ng / μL concentration groups were 53.33%, 43.33%, and 36.67%, respectively. The mortality rate in the control group was 0%, with no deaths. Statistical analysis showed that the 24-hour acute oral toxicity LC50 of thiamethoxam to bees... 50 The concentration was 0.283 ng / bee, with a 95% confidence interval of 0.141–0.462 ng / bee.

[0047] After treatment with juvenile hormone, the 24-hour acute oral toxicity of thiamethoxam in honeybees was significantly reduced, but still in a concentration-dependent manner: the mortality rate was 70% in the 1 ng / μL group, 63.33% in the 0.75 ng / μL group, 46.67%, 13.33%, and 6.67% in the 0.4 ng / μL, 0.2 ng / μL, and 0.1 ng / μL groups, respectively, while the mortality rate in the control group was 0%. Statistical analysis showed that the LC50 of thiamethoxam for 24-hour acute oral toxicity in honeybees after juvenile hormone treatment was significantly reduced. 50 The concentration was 0.526 ng / bee, with a 95% confidence interval of 0.414–0.699 ng / bee.

[0048] The data records and results for 48 hours are shown in Table 4 below.

[0049] Table 4

[0050]

[0051] Table 4 shows that the acute oral toxicity of thiamethoxam to bees remained significantly concentration-dependent over 48 hours. Bee mortality gradually increased with increasing concentration: the mortality rate was 76.67% at 1 ng / μL, 70% at 0.75 ng / μL, 56.67% at 0.4 ng / μL, 43.33% at 0.2 ng / μL, and 36.67% at 0.1 ng / μL, respectively. The control group had a 0% mortality rate, with no deaths. Statistical analysis showed that the LC50 of thiamethoxam for acute oral toxicity to bees over 48 hours was [not specified in the original text]. 50 The concentration was 0.245 ng / bee, with a 95% confidence interval of 0.119–0.383 ng / bee.

[0052] Secondly, the sustained-release effect of juvenile hormone treatment on thiamethoxam toxicity remained stable. The mortality rate of bees from thiamethoxam gradually increased with increasing concentration: 70% in the 1 ng / μL group, 63.33% in the 0.75 ng / μL group, decreasing to 30% in the 0.4 ng / μL group, and only 13.33% and 10% in the 0.2 ng / μL and 0.1 ng / μL groups, respectively. No deaths were observed in the control group. Statistical analysis showed that the LC50 of thiamethoxam in bees after 48-hour acute oral toxicity following juvenile hormone treatment was... 50 The concentration was 0.537 ng / bee, with a 95% confidence interval of 0.151–0.774 ng / bee.

[0053] Conclusion: LC50 in the 24h thiamethoxam group 50 The LC in the 24h thiamethoxam + JH III group was 0.283 ng / bee (95% confidence interval: 0.141-0.462 ng / bee). 50 The LC50 concentration in the thiamethoxam group was 0.526 ng / bee (95% confidence interval: 0.414–0.699 ng / bee). 50 The LC50 concentration in the 48h thiamethoxam + JH III group was 0.245 ng / bee (95% confidence interval: 0.119–0.383 ng / bee). 50 The concentration was 0.537 ng / bee (95% confidence interval: 0.151–0.774 ng / bee).

[0054] That is: LC in the 24h thiamethoxam + JH III group 50 =0.526 ug / bee, thiamethoxam group LC 50 =0.283ug / bee

[0055] LC 50 Increase by multiple ≈1.857, indicating that the protection factor is 1.857.

[0056] LC in the 48h thiamethoxam + JH III group 50 =0.537 ug / bee, thiamethoxam group LC 50 =0.245ug / bee,

[0057] LC 50 Increase by multiple ≈2.192, indicating that the protection factor is 2.192.

[0058] Conclusion: This Example 1 demonstrates that juvenile hormone can reduce the sensitivity of bees to thiamethoxam, thereby reducing the toxicity of thiamethoxam to bees, thus reducing the mortality rate of bee colonies and the risk of poisoning. Moreover, juvenile hormone can exert its maximum protective effect within a certain period of time.

[0059] Example 2

[0060] This example is to investigate the effect of juvenile hormone on the metabolic rate of thiamethoxam in bees.

[0061] The preparation of the sugar solution, the mixed solution (juvenile hormone solution), and the thiamethoxam solution is the same as in Example 1.

[0062] Group setup: 150 Italian honeybee worker bees were divided into two groups of 75 bees each. The following treatments were administered: Group 1 (0.4 ng thiamethoxam group): Bees were fed a 0.4 ng / uL thiamethoxam solution once, followed by a sugar water solution. Group 2 (0.4 ng thiamethoxam + JH III group): Bees were fed a 0.4 ng / uL thiamethoxam solution once, a mixed solution (juvenile hormone solution) once, followed by a sugar water solution.

[0063] Feeding and rearing: First, the bees in the hive were captured using a rectangular, breathable plastic box (20cm long x 10cm wide x 8cm high) and quickly placed in an incubator (temperature: 25±1℃, humidity: 60%, light intensity: 6000Lux) for 3 hours to starve them. Next, the bees were anesthetized with a small amount of carbon dioxide, then removed with tweezers and attached to a clean flat plate with double-sided tape, ensuring each bee was spaced at least 3cm apart to prevent contact. After the bees slightly regained consciousness, a micropipette was used to draw the appropriate solution and feed it to their proboscis using a teasing motion, enticing them to eat. Afterward, the fed bees were carefully placed in cylindrical, breathable plastic boxes (10cm in diameter at the bottom, 8cm high), with 15 bees placed in each box. Finally, the bees in the box were placed in an insect culture box simulating the internal environment of a beehive (temperature: 25±1℃, humidity: 60%, light: darkness = 16h: 8h, light intensity: 6000Lux) and cultured with sugar solution.

[0064] The first group (0.4 ng thiamethoxam group) was initially fed 4 μL of a 0.4 ng / μL thiamethoxam solution, followed by a sugar solution for incubation. The second group (0.4 ng thiamethoxam + JH III group) was initially fed 4 μL of a 0.4 ng / μL thiamethoxam solution and 4 μL of a mixed solution (juvenile hormone solution), followed by a sugar solution for incubation. The frequency of sugar solution feeding was adjusted to ensure the bees did not die from starvation.

[0065] Sampling frequency: Samples were taken at 3 h, 6 h, 9 h, 12 h, and 24 h after feeding. During sampling, a box containing 15 bees that had already been fed was randomly selected from each treatment group and quickly dropped into liquid nitrogen for 1 minute. Afterward, the box was removed, and the bees inside were randomly assigned to three replicate groups using tweezers. Five bees from each replicate group (consisting of one sample) were placed in the same cryovial, labeled with a marker and label paper, and then frozen at -80℃.

[0066] Sample processing and extraction:

[0067] (1) Sample pretreatment

[0068] Weighing: Weigh a single sample (5 bees per sample) using a 0.01 g electronic balance, and place each sample into a grinding tube.

[0069] Grinding: Add 4 mL of acetonitrile (analytical grade) and 5 steel balls to each grinding tube and grind for three minutes.

[0070] (2) Chemical extraction:

[0071] ① Add 0.4 g of sodium chloride to each tube and shake for three minutes using a shaker.

[0072] ② Centrifuge for 5 minutes and take 1.5 mL of the supernatant.

[0073] ③ Add 75 mg of octadecylsilane-bonded silica gel to each tube of supernatant and shake for three minutes.

[0074] ④ Take 1.2 mL of supernatant liquid nitrogen and blow it dry.

[0075] ⑤ Add 0.2 mL of acetonitrile (chromatographic grade), shake for three minutes, filter through a 0.22 μm double-layer organic filter membrane, and wait for injection.

[0076] LC-MS injection and detection analysis: Instruments and methods are shown in Table 5 below. Gradient elution program is shown in Table 6 below. Compound mass spectrometry parameters and retention times are shown in Table 7 below.

[0077] Table 5

[0078]

[0079] Table 6

[0080]

[0081] Table 7

[0082]

[0083] Software Analysis: Thiamethoxam was quantitatively detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS) in multiple reaction monitoring (MRM) mode. The characteristic ion pair of thiamethoxam (mother ion / daughter ion: 292.000 / 211.100) was selected for monitoring. The system's built-in Analyst® Software was used to analyze and export the quantitative ion spectrum of thiamethoxam, as shown below. Figure 1 As shown. By Figure 1 As can be seen, thiamethoxam exhibits a characteristic sharp chromatographic peak at 2.63 min, with a symmetrical peak shape and stable baseline, without significant interference from other peaks, and a maximum response intensity of 2.0 × 10⁻⁶. 4 The cps result indicates that the detection method has good specificity, resolution, and sensitivity, enabling accurate qualitative and quantitative analysis of thiamethoxam.

[0084] Residue analysis: Residue levels at different time points (3 h, 6 h, 9 h, 12 h, 24 h) were derived using the above stabilization method. One-way ANOVA was performed using SPSS (V20.0) software, and graphs were generated using Origin 2018 software. The resulting dynamic variation graph of thiamethoxam residue levels in bees is shown below. Figure 2 As shown. By Figure 2 As can be seen, in the first group (0.4 ng thiamethoxam group): the residual amount of thiamethoxam in bees decreased significantly over time, about 3.8 ng / bee at 3h, about 2.2 ng / bee at 6h, about 0.8 ng / bee at 9h, and remained at a low level of about 0.4 ng / bee after 12h; in the second group (0.4 ng thiamethoxam + JH III group): the residual amount of thiamethoxam in bees remained at a low level, about 0.4 ng / bee at 3h, slowly decreased to about 0.1 ng / bee from 6h to 12h, and slightly rebounded to about 0.8 ng / bee at 24h, and the residual amount at each time point was significantly lower than that in the first group (0.4 ng thiamethoxam group).

[0085] The above results indicate that JH III treatment can significantly accelerate the metabolic clearance of thiamethoxam in bees and reduce its residual level. This is consistent with the conclusion in the acute oral toxicity test of Example 1 that juvenile hormone alleviates thiamethoxam toxicity, revealing the metabolic mechanism by which juvenile hormone regulates thiamethoxam toxicity. This provides experimental evidence at the metabolic level for the research and development of bee protection and safe pesticide use technologies.

[0086] Conclusion: Monitoring of the characteristic ion pair of thiamethoxam (parent ion / daughter ion: 292.000 / 211.100) showed that thiamethoxam exhibited a characteristic sharp chromatographic peak at 2.63 min, with symmetrical peak shape and stable baseline, no obvious interference from other peaks, and a maximum response intensity of 2.0 × 10⁻⁶. 4The CPS (Cost Per Second) indicates that the detection method has good specificity, resolution, and sensitivity, enabling accurate qualitative and quantitative analysis of thiamethoxam. Subsequently, statistical analysis was performed on the groups at each treatment time point. Specifically, within 24 hours, the second group (0.4 ng thiamethoxam + JH III group) showed a faster metabolic rate compared to the first group (0.4 ng thiamethoxam group), especially at 3 hours, where the second group (0.4 ng thiamethoxam + JH III group) exhibited a higher metabolic rate and the lowest relative residue of thiamethoxam. Overall, administering 4 ng / bird of JH III to Italian honeybees accelerated the metabolism of thiamethoxam, mitigating its toxicity to honeybees to some extent and providing protection.

[0087] The embodiments of the present invention have been described in detail above. For those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The application of juvenile hormone in alleviating the risk of thiamethoxam poisoning in Italian honeybees, characterized by: This juvenile hormone is used to accelerate the metabolic rate of thiamethoxam in bees; the juvenile hormone is JH III.

2. The application of the juvenile hormone according to claim 1 in alleviating the risk of thiamethoxam poisoning in Italian honeybees, characterized in that... The method of this application is as follows: JH III is first dissolved in methanol to prepare a JH III stock solution with a concentration of 5 ng / μL. The JH III stock solution is dried with nitrogen and then reconstituted with an equal volume of ultrapure water. Then it is added to sugar water feed to prepare a mixed solution with a JH III concentration of 1 ng / μL, which is then fed to bees. The sugar concentration in the sugar water feed is 50%.

3. The application of the juvenile hormone according to claim 2 in alleviating the risk of thiamethoxam poisoning in Italian honeybees, characterized in that... The feeding method is as follows: After removing the bees from the hive, fix them on a clean flat plate, and use a micropipette to feed the bees the prepared mixed solution.

4. The application of the juvenile hormone according to claim 3 in alleviating the risk of thiamethoxam poisoning in Italian honeybees, characterized in that: Feed each bee 4 μL of the mixed solution once.

5. The application of the juvenile hormone according to claim 4 in alleviating the risk of thiamethoxam poisoning in Italian honeybees, characterized in that: The bees were starved before being fed.

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