Method for alleviating the adverse effects of glufosinate on bumblebees using astaxanthin

Abstract

The application belongs to the technical field of biology, and specifically discloses a method for relieving the adverse effects of glufosinate-ammonium on bumblebees by using astaxanthin. It is found that feeding astaxanthin to bumblebees can effectively relieve the adverse reactions caused by glufosinate-ammonium residues; in terms of the content of active oxygen in the intestinal tract, astaxanthin can significantly eliminate the active oxygen caused by glufosinate-ammonium; in terms of feeding behavior, astaxanthin can significantly restore the reduced feeding amount of bumblebees caused by glufosinate-ammonium exposure; in terms of the integrity of the midgut, astaxanthin can significantly relieve the dissolution of the peritrophic membrane of the midgut of bumblebees caused by glufosinate-ammonium exposure; and in terms of the intestinal microbial layer, astaxanthin can restore the level of core bacteria of bumblebees caused by glufosinate-ammonium exposure. The application also provides bumblebee feed for relieving the toxicity of glufosinate-ammonium and the application of astaxanthin in the preparation of a preparation for relieving the adverse effects of glufosinate-ammonium on bumblebees.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for mitigating the adverse effects of glufosinate on bumblebees using astaxanthin. Background Technology

[0002] Bumblebees are important wild pollinators, providing pollination services for both wild and cultivated flowering plants. Globally, there are approximately 250 species of bumblebees, many of which are commercially bred and used for pollinating greenhouse solanaceous crops, demonstrating significant economic and ecological value. However, due to habitat destruction, pathogen infection, pesticide exposure, and other major environmental stressors, wild bumblebee populations have been declining in recent years. Pesticides play a crucial role in modern agricultural production systems, but their negative impact on pollinating insects cannot be ignored. Compared to aquatic organisms and mammals, pollinating insects exhibit greater sensitivity to pesticides due to their unique physiological structures (such as open circulatory systems and limited detoxification enzyme systems). With increasing emphasis on environmental safety, research on the toxic effects of pesticides on bumblebees is gaining attention. Currently, bumblebee toxicology research focuses primarily on insecticides, while studies on herbicide toxicity remain relatively scarce.

[0003] Glufosinate is the second most widely used herbicide for genetically modified crops globally. This herbicide disrupts plant nitrogen metabolism by inhibiting glutamine synthase activity, ultimately causing plant death. With the expanding use and increasing dosage of glufosinate, its potential ecological risks are receiving increasing attention. Based on the limited existing reports, although glufosinate does not directly cause the acute death of large numbers of pollinating insects, the chronic damage it induces, such as gut microbiota dysbiosis, downregulation of immune genes, and reprogramming of energy metabolism, could potentially weaken the overwintering survival rate of bee colonies and the production capacity of the next generation of queen bees through cumulative effects. This "latent toxicity" is more insidious and harder to trace than the rapid lethality of insecticides. Furthermore, glufosinate exhibits broad-spectrum toxicity to a variety of organisms, necessitating the screening of effective mitigators to reduce its toxicity and protect the health of non-target organisms.

[0004] Currently, there is still a lack of effective technologies or agents to mitigate the toxic effects of glufosinate on bumblebees. Existing research has attempted to use antioxidants and microbial preparations to alleviate the toxicity of insecticides to bees, but these methods are mostly targeted at other types of pesticides and suffer from low mitigation efficiency, high costs, and potential side effects on the physiological or behavioral aspects of bumblebees. To date, no effective safety protectants or mitigation methods have been found specifically for glufosinate.

[0005] Therefore, developing an efficient and safe method to mitigate the adverse effects of glufosinate on bumblebees is of great significance for protecting wild bumblebee populations, maintaining agricultural ecosystems, and promoting the sustainable application of glufosinate. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides a method for mitigating the adverse effects of glufosinate on bumblebees using astaxanthin. This invention discovers that astaxanthin can alleviate the adverse effects of glufosinate on bumblebees. By exogenously feeding astaxanthin, the expression of genes related to reactive oxygen species production is reduced, the content of antioxidant metabolites is increased, and the relative abundance of core bacteria is enhanced, thus achieving comprehensive mitigation of glufosinate damage.

[0007] In a first aspect, the present invention provides a method for mitigating the adverse effects of glufosinate on bumblebees, the method comprising feeding bumblebees a feed containing an effective dose of astaxanthin.

[0008] According to some embodiments of the present invention, the effective dose of astaxanthin in the feed is 1~100 μg / kg.

[0009] According to some preferred embodiments of the present invention, the effective dose of astaxanthin in the feed is 10~100 μg / kg.

[0010] In a second aspect, the present invention provides a bumblebee feed for mitigating glufosinate toxicity, the bumblebee feed containing astaxanthin as an active ingredient.

[0011] According to some embodiments of the present invention, the bumblebee feed contains astaxanthin at a concentration of 1~100 μg / kg.

[0012] According to some embodiments of the present invention, the bumblebee feed also contains 40 wt.% to 60 wt.% sucrose.

[0013] A third aspect of the invention provides the use of astaxanthin in the preparation of formulations for mitigating the adverse effects of glufosinate on bumblebees.

[0014] According to some embodiments of the present invention, the adverse effects of glufosinate on bumblebees include: reduced bumblebee food intake, dissolution of the bumblebee periphery, increased levels of reactive oxygen species in the bumblebee gut, or decreased abundance of beneficial bacteria in the bumblebee gut.

[0015] A fourth aspect of the invention provides the use of astaxanthin in the preparation of formulations for increasing the abundance of beneficial bacteria in the gut of bumblebees.

[0016] According to some embodiments of the present invention, the beneficial bacteria include Lactobacillus.

[0017] A fifth aspect of the invention provides the use of astaxanthin in the preparation of formulations for increasing the content of antioxidant-related metabolites in the gut of bumblebees.

[0018] According to some embodiments of the present invention, the antioxidant-related metabolites include at least one of riboflavin, vitamin B5, taurine, 1,2,4-pyrogallol, or L-cysteine.

[0019] The beneficial effects of this invention are:

[0020] This invention has discovered that exogenously feeding bumblebees astaxanthin can effectively improve their food intake and restore the reduced food consumption behavior induced by glufosinate exposure; it can also alleviate the dissolution of the peritrophic membrane in the midgut of bumblebees caused by glufosinate exposure. Regarding bumblebee intestinal function, feeding astaxanthin can reduce the expression of genes related to reactive oxygen species (ROS) generation, thereby reducing the ROS content in the bumblebee gut, increasing the content of antioxidant metabolites in the gut, and further increasing the relative abundance of core gut bacteria (such as lactobacilli). Based on the above effects of feeding astaxanthin, comprehensive mitigation of glufosinate phytotoxicity is achieved.

[0021] The method provided by this invention can mitigate the adverse effects of glufosinate on bumblebees in a low-cost, efficient, and safe manner, which is of great significance for protecting wild bumblebee populations, maintaining agricultural ecosystems, and promoting the sustainable application of glufosinate.

[0022] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0024] Figure 1 This is a graph showing the results of different concentrations of astaxanthin intervention groups in Example 1 of the present invention in alleviating the reduced feeding caused by glufosinate.

[0025] Figure 2 This is a graph showing the relative abundance of Lactobacillus melliventris in the experimental and control groups in Example 2 of the present invention.

[0026] Figure 3 The results of daily sugar water consumption of bumblebees in the control group, glufosinate treatment group, and astaxanthin intervention group in Example 3 of this invention;

[0027] Figure 4 The results of intestinal hematoxylin-eosin staining in the control group, glufosinate treatment group and astaxanthin intervention group in Example 3 of this invention;

[0028] Figure 5 The images show the fluorescence intensity of reactive oxygen species in the intestines of the control group, glufosinate-ammonia treatment group, and astaxanthin intervention group in Example 3 of this invention; where (a) is a fluorescence image and (b) is a statistical graph of average fluorescence intensity.

[0029] Figure 6 This is a graph showing the abundance of core gut bacteria in the control group, glufosinate-ammonia treatment group, and astaxanthin intervention group in Example 3 of the present invention.

[0030] Figure 7 This is a volcano diagram of the intestinal differential metabolites in the glufosinate-ammonia treatment group and the astaxanthin intervention group in Example 3 of the present invention;

[0031] Figure 8 This is a heatmap showing the correlation between intestinal core bacteria and differential metabolites in the astaxanthin intervention group in Example 3 of the present invention. Detailed Implementation

[0032] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0033] The astaxanthin used in this example has a CAS number of 472-61-7 and is commercially available. The application of astaxanthin in mitigating the adverse effects of glufosinate on bumblebees will be described in detail below, based on examples and experimental data.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] Example 1

[0036] This embodiment provides the preparation of bumblebee feed containing different concentrations of astaxanthin, and uses the feed to feed bumblebees in order to screen the optimal concentration for alleviating glufosinate toxicity.

[0037] Astaxanthin powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 100 mg / kg. The stock solution was then diluted with 50 wt.% sucrose solution to prepare astaxanthin working solutions with concentrations of 100 µg / kg, 10 µg / kg, and 1 µg / kg, which are the astaxanthin-containing bumblebee feed.

[0038] Glufosinate powder was dissolved in a 50 wt.% sucrose solution to prepare a glufosinate working solution with a concentration of 10 mg / kg.

[0039] The experiment was divided into the following groups: Control group: fed with feed containing 50 wt.% sucrose solution and DMSO; Glufosinate treatment group: fed with feed containing 10 mg / kg glufosinate in 50 wt.% sucrose solution; 100 µg / kg astaxanthin group: fed with feed containing 100 µg / kg astaxanthin (without glufosinate) in 50 wt.% sucrose solution; 1 µg / kg astaxanthin intervention group: fed with feed containing 10 mg / kg glufosinate and 1 µg / kg astaxanthin in 50 wt.% sucrose solution; 10 µg / kg astaxanthin intervention group: fed with feed containing 10 mg / kg glufosinate and 10 µg / kg astaxanthin in 50 wt.% sucrose solution; 100 µg / kg astaxanthin intervention group: fed with feed containing 10 mg / kg glufosinate and 100 µg / kg astaxanthin in 50 wt.% sucrose solution.

[0040] The experimental group of bumblebees was fed with the above-mentioned sucrose solution, with the solution changed every two days for 10 days. The amount of food consumed by the bumblebees was recorded at fixed times each day.

[0041] The experimental and control groups of bumblebees were similar in size and were randomly selected. Each group had three parallel experiments with 48 bumblebees in each parallel experiment. They were raised and cultured in an artificial climate chamber (temperature: 25±2℃, relative humidity: 60±20%).

[0042] The results of bumblebee food intake in each group are as follows: Figure 1 As shown, treatment with 100 µg / kg astaxanthin had no significant effect on bumblebee feeding compared to the control group, indicating that astaxanthin is non-toxic to bumblebees. 10 mg / kg glufosinate significantly reduced bumblebee feeding. Interventions with 10 µg / kg and 100 µg / kg astaxanthin alleviated the reduction in feeding caused by glufosinate, restoring it to a level not significantly different from the control group. However, the low concentration of 1 µg / kg astaxanthin was ineffective. This indicates that low concentrations of astaxanthin are not very effective in alleviating glufosinate toxicity, while relatively high concentrations of astaxanthin have a good intervention effect.

[0043] Example 2

[0044] This embodiment provides the preparation of bumblebee feed containing astaxanthin and uses the feed to feed bumblebees.

[0045] Astaxanthin powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 10 mg / kg; the stock solution was then diluted with 50 wt.% sucrose solution to prepare an astaxanthin working solution with a concentration of 10 µg / kg, which is the astaxanthin-containing bumblebee feed.

[0046] The experimental group of bumblebees was fed with the above-mentioned sucrose solution containing astaxanthin, and the feeding solution was changed every two days. The control group of bumblebees was fed with the same volume of 50 wt.% sucrose solution and DMSO without the addition of astaxanthin. After 10 days of treatment, the bumblebees were anesthetized on ice and their intact intestines were dissected and separated.

[0047] The experimental and control groups of bumblebees were similar in size and were randomly selected. Each group had three parallel experiments with 48 bumblebees in each parallel experiment. They were raised and cultured in an artificial climate chamber (temperature: 25±2℃, relative humidity: 60±20%).

[0048] Total DNA was extracted from gut microbiota, and the DNA concentration and purity were detected using a micro spectrophotometer. Samples that met the quality standards were selected for subsequent experiments.

[0049] The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 341F (5'-CCTACGGGNGGCWGCAG-3') (SEQ ID No.1) and 806R (5'-GGACTACHVGGGTATCTAAT-3') (SEQ ID No.2).

[0050] After gel extraction and purification, the amplified products were ligated with sequencing adapters and sequencing libraries were constructed. High-throughput sequencing was then performed using the Illumina PE250 platform. End-to-end reads were assembled based on sequence overlap relationships. After sample splitting, operational taxonomic unit (OTU) clustering and taxonomic analysis were performed.

[0051] Analysis results as follows Figure 2 As shown, compared with the control group, the relative abundance of Lactobacillus melliventris was significantly increased in the experimental group treated with astaxanthin-containing feed.

[0052] Example 3

[0053] This embodiment further explores the effects of astaxanthin on bumblebee feeding behavior, intestinal pathology, and abundance of core intestinal bacteria.

[0054] 1. Bumblebee rearing experiment:

[0055] Three beehives were randomly selected. Under a light-protected environment with red light illumination, bumblebees of similar size were chosen and placed in homemade plastic rearing cages, four bumblebees per cage. The experimental group consisted of a control group, a glufosinate treatment group, and an astaxanthin intervention group, with three replicates in each group. The experimental bumblebees were fed a 50% sucrose solution and reared in an artificial climate chamber. After 24 hours of rearing, dead individuals were removed and healthy bumblebees were added, and the experiment was then formally conducted.

[0056] After the experiment began, the control group was fed a conventional 50 wt.% sucrose solution, the glufosinate treatment group was fed a 50 wt.% sucrose solution containing 10 mg / kg glufosinate, and the astaxanthin intervention group was fed a 50 wt.% sucrose solution containing 10 mg / kg glufosinate and 10 μg / kg astaxanthin. The feeding solution was changed every two days for a total of 10 days.

[0057] 2. Statistics on daily food intake of bumblebees:

[0058] After 10 days of treatment, the daily sugar water consumption of bumblebees in the control group, glufosinate treatment group and astaxanthin intervention group was statistically analyzed.

[0059] See results Figure 3 As shown in the figure, the average daily saccharide consumption of bumblebees in the glufosinate-treated group was significantly lower than that in the control group (p < 0.05), while the average daily saccharide consumption of bumblebees in the astaxanthin intervention group was higher than that in the glufosinate-treated group. There was no significant difference in the average daily saccharide consumption between the astaxanthin intervention group and the control group (p > 0.05). This indicates that astaxanthin can effectively alleviate the reduction in average daily saccharide consumption caused by glufosinate and improve the appetite of bumblebees.

[0060] 3. Analysis of pathological sections of the midgut of bumblebees:

[0061] Ten days after treatment, bumblebees in each group were anesthetized on ice, and the midgut tissue was carefully dissected and separated using forceps. The extracted midgut tissue was fixed in 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, and then embedded in paraffin. The embedded samples were prepared into 5 μm thick tissue sections using a microtome. Subsequently, hematoxylin-eosin (H&E) staining was performed according to standard experimental methods. The samples were observed and images were acquired using an optical microscope.

[0062] See results Figure 4 As can be seen from the figure, compared with the control group, the glufosinate-ammonium treatment group showed degradation damage to the periphery membrane; while no periphery membrane dissolution was observed in the astaxanthin intervention group.

[0063] 4. Experiment on detection of reactive oxygen species in the intestines of bumblebees:

[0064] Ten days after treatment, bumblebees in each group were anesthetized on ice, and their intact intestines were dissected and separated. The intestines were then placed in PBS buffer containing 10 mM 3-amino-1,2,4-triazole, and Malpighian tubes were removed. Next, the intestines were added to buffer containing 1×CM-H2DCFDA probe and incubated in the dark for 30 min. After incubation, the intestines were washed twice with deionized water. Bright-field and fluorescence images were acquired using a fluorescence stereomicroscope, with the excitation wavelength set to 488 nm and the emission wavelength to 525 nm. ROS-stained fluorescence images were analyzed using ImageJ software. The images were converted to 8-bit grayscale, and after background subtraction, the average grayscale value of the entire intestinal region was measured. The results are expressed in arbitrary units and are used to reflect the relative ROS content of the organism.

[0065] See results Figure 5 Using reactive oxygen species (ROS)-specific fluorescent probes, the distribution and accumulation levels of ROS in the gut of bumblebees in the control group, glufosinate-treated group, and astaxanthin intervention group were detected and analyzed (e.g., ...). Figure 5 As shown in Figure a). The results showed that the reactive oxygen species (ROS) level was significantly increased in the glufosinate-ammonia treatment group (p < 0.05); while compared with the glufosinate-ammonia treatment group, the ROS content in the astaxanthin intervention group was significantly decreased (p < 0.05) (as shown in Figure a). Figure 5 (As shown in Figure b).

[0066] 5. Relative abundance analysis of core gut bacteria in bumblebees:

[0067] Ten days after treatment, bumblebees in each group were anesthetized on ice, and their intact intestines were dissected and separated. Total DNA from the intestinal microorganisms was extracted. DNA concentration and purity were measured using a micro-spectrophotometer, and samples meeting quality standards were selected for subsequent experiments. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 341F and 806R. After gel extraction and purification, sequencing adapters were ligated, and sequencing libraries were constructed. High-throughput sequencing was performed using the Illumina PE250 platform. End-unit reads were spliced ​​based on sequence overlap, and after sample splitting, operational taxonomic unit (OTU) clustering and species taxonomic analysis were performed.

[0068] See results Figure 6 Compared with the control group, the relative abundance of the core bacteria genus Lactobacillus in the glufosinate-ammonia-treated group decreased by approximately 74.4%. Compared with the glufosinate-ammonia-treated group, the relative abundance of the core bacteria genus Lactobacillus in the gut increased by approximately 71.5% in the astaxanthin-intervention group.

[0069] 6. Experiment on the analysis of intestinal metabolites of bumblebees:

[0070] Ten days after treatment, bumblebees in each group were anesthetized on ice, and their intact intestines were dissected and separated. After removing the intestinal contents, an appropriate amount of methanol-water solution was added to the intestinal tissue, and the mixture was homogenized using a grinder. The resulting mixture was centrifuged, and the supernatant was used for subsequent analysis. The supernatant was analyzed using an Agilent 1290 Infinity LC system in series with an ABTriple TOF 6600 mass spectrometer, with acetonitrile as the mobile phase. Data were acquired in both positive and negative ion modes, and the datasets from both ionization modes were integrated in subsequent analyses.

[0071] See results Figure 7 Compared with the glufosinate-ammonia treatment group, the astaxanthin intervention group showed significantly higher levels of differential metabolites such as riboflavin, vitamin B5, taurine, 1,2,4-pyrogallol, and L-cysteine ​​in the intestines.

[0072] Correlation analysis between differentially metabolites and gut core bacteria showed the following results: Figure 8 Riboflavin content showed a significant positive correlation with Lactobacillus abundance, suggesting that riboflavin may be produced by Lactobacillus and then used for host life activities. 1,2,4-Phenylopyrrolidone content showed a significant negative correlation with Gilliamella abundance, which is believed to be because Gilliamella participates in the degradation of 1,2,4-Phenylopyrrolidone, preventing its concentration from becoming too high.

[0073] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for mitigating the adverse effects of glufosinate on bumblebees, characterized in that, The method involves feeding bumblebees a diet containing an effective dose of astaxanthin.

2. The method according to claim 1, characterized in that, The effective dose of astaxanthin in the feed is 1~100 μg / kg.

3. A bumblebee feed for mitigating glufosinate toxicity, characterized in that, It contains astaxanthin as an active ingredient.

4. The bumblebee feed according to claim 3, characterized in that, The bumblebee feed also contains 40 wt.% to 60 wt.% sucrose.

5. Application of astaxanthin in the preparation of formulations to alleviate the adverse effects of glufosinate on bumblebees.

6. The application according to claim 5, characterized in that, The adverse effects of glufosinate on bumblebees include: reduced food intake, dissolution of the bumblebee periphery, increased reactive oxygen species levels in the bumblebee gut, or decreased abundance of beneficial bacteria in the bumblebee gut.

7. Application of astaxanthin in the preparation of formulations for increasing the abundance of beneficial bacteria in the gut of bumblebees.

8. The application according to claim 7, characterized in that, The beneficial bacteria include Lactobacillus.

9. Application of astaxanthin in the preparation of formulations for increasing the content of antioxidant-related metabolites in the gut of bumblebees.

10. The application according to claim 9, characterized in that, The antioxidant-related metabolites include at least one of riboflavin, vitamin B5, taurine, 1,2,4-pyrogallol, or L-cysteine.

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