Yeast and application thereof in relieving damage of pesticides on bumblebees

Abstract

The application discloses a kind of yeast bacteria (Yeast) Starmerella sorbosivorans ) 114, and the preservation number of the strain is CGMCC NO.32805 in the China General Microbiological Culture Collection Center, and the strain is applied in relieving the damage of pesticide to bumblebee, and the experimental results show that the strain can significantly prolong the life of bumblebee, improve the food intake; In terms of movement ability and social behavior, the moving distance, maximum speed, contact time and frequency of bumblebee can be significantly improved; In terms of sucrose sensitivity and learning memory, the sensitivity of bumblebee to sucrose and learning memory ability can be improved, and the yeast bacteria 114 provided by the application is non-toxic and harmless, safe and healthy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a yeast strain isolated from the intestine of bumblebees and application thereof in relieving damage of pesticides to bumblebees. BACKGROUND

[0002] Bumblebees ( Bombus ) are indispensable pollinators in agriculture due to their large size, dense hair, and ability to carry large amounts of pollen. Compared with honeybees, bumblebees exhibit stronger pollination ability and are widely used in the pollination of greenhouse crops, berries, and some economic crops. However, in recent years, the number of bumblebee populations is experiencing a significant decline, which has attracted widespread attention. Among the many influencing factors, the extensive use of pesticides is considered one of the most important reasons for the decline in the number of bumblebees. Studies have shown that pesticides not only directly cause the mortality rate of bumblebees to rise, but also affect their nervous system, delay foraging behavior, reduce motor ability, and severely impair their learning and memory functions. These problems further weaken the ability of bumblebees to navigate, recognize flowers, and efficiently pollinate in complex environments. In response to this challenge, some measures have been taken, such as reducing pesticide use or using chemical detoxification agents. However, the effects of these measures are limited, and the cost is relatively high. In view of the above situation, how to effectively prolong the lifespan and improve the health level of bumblebees, and thus enhance their pollination efficiency, has become an important issue that needs to be addressed.

[0003] Currently, the health management of bumblebees by breeders focuses on antibiotic and chemical drug intervention, but the long-term use of such drugs can easily cause intestinal flora disorder and immune decline in bumblebees, causing certain negative impacts on the beekeeping industry. Therefore, it is urgent to find green and healthy drug substitutes during the breeding process. Probiotics, as a class of active microorganisms beneficial to the host, exhibit significant effects in improving the microecological balance of the host, enhancing immune function, and improving stress resistance. In recent years, the application of probiotics in the health management of bee colonies has attracted widespread attention. Studies have shown that specific probiotics can enhance the immune function, antioxidant capacity, and improve the digestive system of bee colonies, thereby improving the stress resistance and lifespan of bee colonies. However, the probiotic products for bumblebees on the market are relatively limited, and most of them are focused on common bacteria such as lactic acid bacteria and bifidobacteria. In contrast, the application of probiotic fungi in the health of bumblebees is relatively scarce.

[0004] Previous studies have shown that fungal probiotics have unique advantages in maintaining intestinal microecological balance and improving host physiological functions. In particular, certain fungi can secrete enzymes, antibacterial peptides and other bioactive substances, which not only help to enhance the digestion and absorption capacity of bumblebees, but also may enhance their physical strength and endurance. More importantly, certain probiotic fungi have the ability to alleviate pesticide damage. They can reduce the toxic effects of pesticides on bumblebees through mechanisms such as regulating intestinal flora and enhancing the immune system. SUMMARY

[0005] In view of the problems existing in the prior art, the present application provides a kind of yeast (Saccharomyces cerevisiae) Starmerella sorbosivorans It was deposited at the China General Microbiological Culture Collection Center on November 26, 2024, with the accession number CGMCC NO. 32805, and the deposit address is No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard 3rd.

[0006] Another object of the present application is to apply the above-mentioned yeast 114 in relieving the damage of pesticides to bumblebees. The yeast 114 can prolong the life span of bumblebees, improve their food intake, exercise capacity, social behavior ability and learning and memory ability.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] 1. The strain 114 is obtained by isolating and purifying from the intestinal tract of bumblebees;

[0009] 2. The strain 114 presents a round colony on YPD medium, the colony color is white, the edge is neat, and the surface is smooth. Under scanning electron microscope, the strain is observed as gray-white spherical shape and gathered together.

[0010] The DNA of the isolated and purified strain is extracted by fungal genome DNA rapid extraction kit, and the ITS rDNA sequence is obtained by amplification with universal primer of ITS region. The amplification and sequencing results are compared in NCBI database by BLAST tool. The results show that the strain is most closely related to Saccharomyces cerevisiae, with a similarity of 97.7%. Therefore, the strain is identified as Saccharomyces cerevisiae Starmerella sorbosivorans ; Starmerella sorbosivorans

[0011] ​3. The yeast strain 114 is mixed with a sucrose solution, a PBS buffer and pollen to prepare a yeast strain 114 suspension solution; the yeast strain 114 suspension solution is used to feed bumblebees, and the influence of the yeast strain 114 on the bumblebees after pesticide treatment is observed; the experimental results show that the yeast strain 114 can improve the survival rate of the bumblebees, prolong the life span of the bumblebees, improve the diet amount of the bumblebees, significantly improve the moving distance, the maximum speed, the contact time and the contact frequency of the bumblebees in terms of the movement ability and the social behavior, and improve the sucrose sensitivity and the learning and memory ability of the bumblebees in terms of the sucrose sensitivity and the learning and memory.

[0012] The yeast strain 114 can be prepared into a microbial agent or a feed for relieving the damage of pesticides to the bumblebees; the components (or effective components) of the microbial agent or the feed are the yeast strain 114 or the fermentation product thereof, and one or more auxiliary materials acceptable for the microbial agent or the feed can be added to prepare a dosage form suitable for the microbial agent or the feed, and the microbial agent or the feed is used in a conventional adding amount.

[0013] The yeast strain 114 has the functions of significantly improving the life span, the diet amount, the movement ability, the social behavior ability and the learning and memory ability, and the present application can solve the influence of pesticides on the population reproduction of the bumblebees and make up for the shortage of high-quality fungal probiotic strains in the market.

[0014] The yeast strain 114 has the functions of significantly improving the life span, the diet amount, the movement ability, the social behavior ability and the learning and memory ability, and the present application can solve the influence of pesticides on the population reproduction of the bumblebees and make up for the shortage of high-quality fungal probiotic strains in the market. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 FIG. 1 is a schematic diagram of the morphology of the yeast strain 114, wherein FIG. 1A is an imaging of the strain under a stereomicroscope, and FIG. 1B is an imaging of the strain under a scanning electron microscope;

[0016] Figure 2 FIG. 4 is the survival situation of the bumblebees in the control group, the pesticide treatment group and the yeast strain 114 intervention group within 7 days;

[0017] Figure 3 FIG. 5 is the sugar water (FIG. 5A) and pollen (FIG. 5B) consumption amount of the bumblebees in the control group, the pesticide treatment group and the yeast strain 114 intervention group per day;

[0018] Figure 4 FIG. 6 is the movement ability statistical result of the bumblebees in the control group, the pesticide treatment group and the yeast strain 114 intervention group after 7 days of treatment, wherein FIG. 6A is the moving distance, and FIG. 6B is the maximum speed;

[0019] Figure 5 FIG. 7 is the social behavior statistical result of the bumblebees in the control group, the pesticide treatment group and the yeast strain 114 intervention group after 7 days of treatment, wherein FIG. 7A is the contact frequency, and FIG. 7B is the contact time;

[0020] Figure 6 FIG. 8 is the taste reaction score statistical result of the bumblebees in the control group, the pesticide treatment group and the yeast strain 114 intervention group after 7 days of treatment.

[0021] Figure 7 Statistical results of learning and memory of bumblebees after 7-day treatment of the bumblebee control group, the pesticide treatment group, and the Saccharomycete 114 intervention group, wherein Figure A is the proboscis extension ratio of bumblebees in each group during 10 rounds of learning, and Figure B is the short-term memory of bumblebees in each group after 10 rounds of learning. DETAILED DESCRIPTION

[0022] The application is further illustrated by the accompanying drawings and examples, but the scope of protection of the application is not limited to the content described, and the methods in the examples are all operated according to the conventional method unless otherwise specified, the reagents used are all conventional commercially available reagents or reagents prepared according to the conventional method unless otherwise specified, and the culture medium is a commercially available product used according to the instructions;

[0023] The culture medium used for culturing Saccharomycete 114 is yeast extract peptone dextrose (YPD): 20 g of peptone, 20 g of glucose, 10 g of yeast extract, 10 g of agar powder, and 0.22 g of tryptophan are dissolved in 1 L of distilled water;

[0024] Example 1: Isolation and identification of Saccharomycete 114

[0025] The bumblebees were dissected, the complete intestinal tracts were removed, the intestinal walls were ground and broken with a grinding rod in 100 μL of 25% (v / v) glycerol, and were stored in a -80℃ ultra-low-temperature medical refrigerator; the intestinal tract samples were streaked in three regions on a YPD solid culture medium, and were cultured in a 30℃ incubator for 2-3 days, the colony morphology was observed, single colonies were picked from the colonies and were restreaked in four regions on the YPD culture medium, and were further purified and cultured in a 30℃ incubator, a number of single colonies were selected on the culture medium, were numbered respectively, and 20 single colonies were selected for ITS rDNA sequencing;

[0026] The total fungal DNA was extracted using the fungal genome DNA rapid extraction kit (item number B518229-0050) of Shengong Bioengineering, and was amplified in a 30 μL PCR system (2 × Phanta Max Master Mix 15 μL, Primer F 1.2 μL, Primer R 1.2 μL, DNA template 1 μL, and ddH2O 11.6 μL) using universal primers for the ITS region (ITS1F: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'); the PCR conditions were as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 1 min, for 30 cycles; and finally 72℃ extension for 5 min;

[0027] In this embodiment, the ITS amplification and sequencing results were compared with the NCBI database using the BLAST tool. This invention selected samples similar to yeast. Starmerella sorbosivorans The closest related strain is strain 114, with a similarity of 97.7%. The ITS rDNA sequence of yeast strain 114 is shown in SEQ ID NO: 1.

[0028] The morphological characteristics of yeast 114 are shown in [reference needed]. Figure 1 On YPD medium, the strain presents as round colonies, white in color, with neat edges, opaque in appearance, and a smooth surface. Under a scanning electron microscope, the strain can be observed to be grayish-white spherical and aggregated together.

[0029] Therefore, based on the morphological and molecular identification results, strain 114 was determined to be a yeast. Starmerella sorbosivorans .

[0030] Example 2: Preparation of freeze-dried bacterial agent of strain 114

[0031] Yeast 114 was activated with YPD solid medium. The activated strain was transferred to YPD liquid medium at an inoculation rate of 4%, cultured at 30°C for 25 hours, centrifuged at 5000g for 15 minutes, and the cells were harvested for later use.

[0032] The harvested mycelium was mixed with the protectant at a volume ratio of 3:1 and set aside. The protectant was an aqueous solution containing 7.5% trehalose, 7.5% skim milk powder, 4.5% gum arabic, 4.5% polyethylene glycol, and 6% L-glutamate.

[0033] Before freeze-drying, the sample needs to be pre-frozen at -80℃ for 3 hours, and then a three-stage freeze-drying process is adopted: the first stage is freeze-drying at 512Pa pressure for 6 hours, the second stage is freeze-drying at 256Pa pressure for 5 hours, and finally freeze-drying is continued at 103Pa pressure until the sample forms a powder product.

[0034] The freeze-dried bacterial agents were stored at 4℃ and 25℃ respectively, and the viable cell count of each bacterial powder was measured every 2 months. The experiment was repeated in triplicate, and the results are shown in Table 1. The table shows that after 10 months of storage at 4℃ and 25℃, the viable cell count of the freeze-dried bacterial agents remained at a high level. Although the stability at 25℃ was somewhat lower than that at 4℃, it still maintained a survival rate of over 90%. Specifically, the effective bacterial count of yeast 114 powder was not less than 3 × 10⁻⁶. 9 cfu / g;

[0035] Table 1

[0036] .

[0037] Example 3: Effect of yeast 114 on the survival and behavior performance of bumblebees

[0038] 1. Preparation of yeast 114 suspension

[0039] 1 g of freeze-dried bacteria agent of Example 2 (not less than 10 9 cfu / g) was weighed into a 1 L beaker, 450 mL of a mixed solution of sucrose solution with a mass concentration of 50% and 1 × PBS buffer solution was added, and stirring was performed to fully suspend it. Then 100 g of pollen was added and stirring was continued until uniform, to obtain a yeast 114 suspension.

[0040] 2. Bumblebee breeding and experiment

[0041] Twenty beehives were randomly selected and placed in a gas treatment box, anesthetized with carbon dioxide gas for 90 s, then the beehives were opened, and the pupae that had not yet emerged from the house were selected using tweezers, and the black pupae selected from the 20 beehives were placed in one beehive for cultivation. Since bumblebees usually emerge from black pupae within 1-2 days, in order to ensure the survival rate of newly hatched bees, sugar water and pollen need to be provided in the beehive, the cultivation temperature is 29°C, and the humidity is 65%. The 2-day-old bumblebees that hatched were divided into containers, with 20 bees in each container. The experiment was divided into a control group, a pesticide treatment group, and a yeast 114 intervention group, with 3 parallel groups in each group.

[0042] After the start of the experiment, the three experimental groups were all fed tetracycline with a concentration of 450 μg / mL to reduce the interference of the original intestinal microorganisms of the bumblebees. From the 6th day, the control group was changed to feed conventional sugar water and pollen, the pesticide treatment group was changed to feed sugar water and pollen containing 5 μg / mL of acetamiprid, and the yeast 114 intervention group was fed sugar water and yeast 114 suspension containing 5 μg / mL of acetamiprid, while the pollen was continued to be fed conventionally. The sugar water and pollen were replaced every three days, and the yeast 114 suspension also needed to be reconfigured every three days and added to the beehive.

[0043] 3. Statistics of bumblebee death

[0044] The number of bumblebees that died each day in the control group, the pesticide treatment group, and the yeast 114 intervention group was counted.

[0045] The results are shown in Figure 2 As can be seen from the figure, after feeding from the 6th day, compared with the control group, the survival rate of bumblebees in the pesticide treatment group was significantly reduced, while the survival rate of bumblebees in the yeast 114 intervention group was significantly improved, indicating that the addition of yeast 114 to the diet can reduce the mortality rate of bumblebees and prolong their lifespan.

[0046] 4. Detection of bumblebee diet, exercise, and social indicators

[0047] (1) Statistics and analysis of the daily sugar water and pollen consumption of bumblebees in the control group, pesticide treatment group and yeast 114 intervention group;

[0048] Results are shown in Figure 3 As can be seen from the figure, the average daily diet of bumblebees in the pesticide treatment group was significantly lower than that in the control group, while the average daily diet of bumblebees in the yeast 114 intervention group was significantly higher than that in the pesticide treatment group, indicating that yeast 114 can increase the diet reduction caused by acetamiprid and improve the appetite of bumblebees. According to the statistical analysis of the total consumption of sugar water and pollen, the consumption of sugar water and pollen in the yeast 114 intervention group was significantly higher than that in the pesticide treatment group (p<0.05).

[0049] (2) After 7 days of yeast 114 intervention, 3 bumblebees in each group were placed in a culture dish, and after adapting to the environment, a 50-minute video was taken. The movement distance and maximum speed of bumblebees were analyzed and counted using EthoVision software;

[0050] Results are shown in Figure 4 From Figure 4 It can be seen that there are significant differences in the movement ability of bumblebees after 7 days of feeding (p<0.05), and the movement distance and maximum speed of bumblebees in the yeast 114 intervention group are significantly higher than those in the pesticide treatment group.

[0051] (3) After 7 days of yeast 114 intervention, 3 bumblebees in each group were placed in a culture dish, and after adapting to the environment, a 50-minute video was taken. The body contact frequency and time of bumblebees were analyzed and counted using EthoVision software;

[0052] Results are shown in Figure 5 From Figure 5 It can be seen that there are significant differences in the social behavior of bumblebees after 7 days of feeding (p<0.05), and the body contact frequency and time of bumblebees in the yeast 114 intervention group are significantly higher than those in the pesticide treatment group.

[0053] 5. Sucrose sensitivity detection experiment of bumblebees

[0054] After 7 days of yeast 114 treatment, the test bumblebees in different groups were starved for 3 hours, frozen for 5 minutes on ice, or tied up after their activity slowed down. A cotton swab was dipped in deionized water and extended to the front of the bumblebee's head to test whether the bumblebee's proboscis flexion was due to thirst or habituation. After 4 minutes of deionized water testing, different concentrations of sugar water (0.1, 0.3, 1, 3, 10, 30, and 50% sterile sugar water mass / volume concentration) were used to test the proboscis flexion response of each bumblebee in turn, from low to high concentration. The sugar response threshold (SRT) of each bumblebee was recorded, which corresponds to the lowest sugar water concentration at which the bumblebee's proboscis flexion response was observed. The gustatory response score (GRS) ranged from 1 to 7, with 1 point for a response to the highest concentration of sugar water (50%) and 7 points for a response to all concentrations of sugar water.

[0055] The results are shown in Figure 6 From Figure 6 As can be seen, the gustatory response score of the bumblebees in the pesticide treatment group was significantly lower than that of the control group, and the gustatory response score of the bumblebees in the yeast 114 intervention group was significantly higher than that of the pesticide treatment group, indicating that yeast 114 can alleviate the damage to the bumblebee's sucrose sensitivity caused by acetamiprid.

[0056] 6. Learning and memory ability detection experiment of bumblebees

[0057] After the sucrose sensitivity experiment, a single bumblebee was placed in front of an exhaust fan, and a lemon odor syringe was aimed at the front of the bumblebee's antennae. The bumblebee was given a uniform speed of 5 seconds of lemon odor, and 3 seconds of sugar at the 6th second of lemon odor. Bumblebees that extended their proboscis when given lemon odor were marked as having learned. Each bumblebee needed to undergo 10 rounds of learning, with a 15-minute interval between each round. After 10 rounds of learning, the bumblebee was placed in an incubator for 45 minutes, and then a short-term memory rate test was performed. During the test, the bumblebee was given a lemon odor syringe, and its proboscis flexion response was tested. Bumblebees that extended their proboscis when given lemon odor were marked as having remembered. After the experiment, the learning and memory of each group were statistically analyzed.

[0058] The results are shown in Figure 7 As can be seen from the figure, the learning and memory of the bumblebees in the pesticide treatment group were significantly lower than those of the control group, and the learning and memory of the bumblebees in the yeast 114 intervention group were better than those of the pesticide treatment group, indicating that yeast 114 can alleviate the decline in learning and memory ability of bumblebees caused by acetamiprid.

[0059] In conclusion, the yeast 114 provided by the application can significantly prolong the life of bumblebees, increase the diet amount, significantly improve the moving distance, maximum speed, contact time and frequency of bumblebees in terms of movement ability and social behavior, and improve the sensitivity to sucrose and learning and memory ability of bumblebees in terms of sucrose sensitivity and learning and memory. Although the application is illustrated by the above examples, those skilled in the art should understand that the improvements and modifications made to the application without departing from the spirit and essence of the application shall belong to the protection scope of the application.

Claims

1. A type of yeast ( Starmerella sorbosivorans )114, its accession number at the China General Microbiological Culture Collection Center is CGMCC NO.32805.

2. The use of Saccharomyces 114 in the preparation of a feed or a fungicide for alleviating the damage of pesticides to bumblebees.

3. Use according to claim 2, characterized in that: Saccharomyces 114 can improve the survival rate of bumblebees, improve the diet, exercise ability, social behavior ability and learning and memory ability of bumblebees.

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