Saccharomycetes and application thereof in relieving damage of pesticide to bumblebees

Abstract

The invention discloses saccharomycetes 114, the preservation number of the saccharomycetes 114 in the China General Microbiological Culture Collection Center is CGMCC NO.32805. When the saccharomycetes 114 is applied to relieving damage of pesticides to bumblebees, experimental results show that the saccharomycetes 114 can remarkably prolong the life of the bumblebees and improve food intake; in the aspects of athletic ability and social behaviors, the moving distance, the maximum speed, the contact time and the contact frequency of the bumblebees can be remarkably improved; the saccharomycetes 114 provided by the invention can improve the sensitivity and learning and memory ability of bumblebees to cane sugar in the aspects of cane sugar sensitivity and learning and memory, and the saccharomycetes 114 provided by the invention is non-toxic, harmless, healthy and safe.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a yeast isolated from the intestinal tract of a bumblebee and an application of the yeast in alleviating the damaging effect of pesticides on the bumblebee. Background Art

[0002] bumblebee( Bombus ) Because of its large size and dense hair, bumblebees can carry a lot of pollen, making them an indispensable pollinating insect in agriculture. Compared with honey bees, bumblebees show stronger pollination ability and are widely used in the pollination of greenhouse crops, berries and some cash crops. However, in recent years, the number of bumblebees has been experiencing a sharp decline, which has attracted widespread attention. Among the many influencing factors, the large-scale use of pesticides is considered to be one of the most important reasons for the decline in the number of bumblebees. Studies have shown that pesticides not only directly lead to increased mortality in bumblebees, but also affect their nervous system, delay foraging behavior, reduce motor ability, and seriously damage their learning and memory functions. These problems further weaken the ability of bumblebees to navigate in complex environments, identify flowers and pollinate efficiently. In order to meet this challenge, some measures have been taken, such as reducing the amount of pesticides or using chemical antidotes. However, these measures have limited effects and are relatively costly. In view of the above situation, how to effectively extend the lifespan and improve the health level of bumblebees, thereby enhancing their pollination efficiency, has become an important issue that needs to be solved urgently.

[0003] At present, breeders focus on antibiotics and chemical interventions to manage the health of bumblebees. However, the long-term use of such drugs can easily cause intestinal flora disorder and decreased immunity in bumblebees, which has a certain negative impact on the beekeeping industry. Therefore, it is urgent to find green and healthy drug substitutes in the breeding process. As a type of active microorganisms that are beneficial to the host, probiotics have shown significant effects in improving the host's microecological balance, enhancing immune function and improving stress resistance. In recent years, the application of probiotics in bee colony health management has received widespread attention. Studies have shown that specific probiotics can enhance the immune function and antioxidant capacity of bee colonies and improve their digestive system, thereby improving the stress resistance and lifespan of bee colonies. However, the current probiotic products for bumblebees on the market are relatively limited, and most of them are concentrated on common bacterial probiotics such as lactic acid bacteria and bifidobacteria. In contrast, research on the application of probiotic fungi in bumblebee health is relatively scarce.

[0004] Studies have shown that fungal probiotics have unique advantages in maintaining the balance of intestinal microecology and improving the physiological functions of the host. In particular, some fungi can secrete enzymes, antimicrobial peptides and other bioactive substances, which not only help enhance the digestion and absorption capacity of bumblebees, but also may improve their physical strength and endurance. More importantly, some probiotic fungi have the ability to alleviate the damage caused by pesticides. They can reduce the toxic effects of pesticides on bumblebees by regulating intestinal flora and enhancing the immune system. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a yeast ( Starmerella sorbosivorans )114, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on November 26, 2024, with the deposit number CGMCC NO. 32805, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Another purpose of the present invention is to apply the yeast 114 to alleviate the damage of pesticides to bumblebees. The yeast 114 can prolong the life of bumblebees and improve their eating capacity, exercise ability, social behavior ability and learning and memory ability.

[0007] The purpose of the present invention is achieved through the following technical solutions: 1. The present invention isolates and purifies the bacterial strain 114 from the intestine of Xiongfeng; 2. Strain 114 showed round colonies on YPD medium. The colonies were white in color, with neat edges, opaque and smooth surfaces. Under a scanning electron microscope, the strain was observed to be grayish white spherical and aggregated together.

[0008] The isolated and purified strain was used to extract DNA using a fungal genomic DNA rapid extraction kit, and the ITS rDNA sequence was obtained by amplification using universal primers in the ITS region. The amplified sequencing results were compared in the NCBI database using the BLAST tool. The results showed that the strain was similar to yeast. Starmerella sorbosivorans The closest relationship is 97.7%, so the strain is identified as yeast ( Starmerella sorbosivorans ); 3. The strain 114 was mixed with sucrose solution, PBS buffer and pollen to prepare a yeast 114 suspension solution; the yeast 114 suspension solution was used to raise bumblebees, and the effect of yeast 114 on bumblebees after the action of pesticides was observed. The experimental results showed that yeast 114 could improve the survival rate of bumblebees, prolong the life of bumblebees, increase the diet of bumblebees, and significantly improve the movement distance, maximum movement speed, contact time and frequency of bumblebees in terms of motor ability and social behavior; in terms of sucrose sensitivity and learning and memory, it could improve the sensitivity of bumblebees to sucrose and their learning and memory ability.

[0009] The yeast 114 of the present invention can be made into a microbial agent or feed for alleviating the damage of pesticides to bumblebees. The ingredients (or active ingredients) of the microbial agent or feed are yeast 114 or its fermentation products. One or more adjuvants acceptable to the microbial agent or feed can also be added to make a dosage form suitable for the microbial agent or feed, and added and used according to the conventional addition amount.

[0010] The beneficial effects of the present invention are: The yeast 114 of the present invention has the function of significantly improving life span, food intake, exercise ability, social behavior ability and learning and memory ability. The present invention can solve the impact of pesticides on the reproduction of the Xiongfeng population and make up for the shortage of high-quality fungal probiotic strains on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the morphology of yeast 114, wherein Figure A is the image of the strain under a stereo microscope, and Figure B is the image of the strain under a scanning electron microscope; Figure 2 The survival of the bumblebee control group, pesticide treatment group, and yeast 114 intervention group within 7 days; Figure 3 Results of daily sugar water (Figure A) and pollen (Figure B) consumption of bumblebee control group, pesticide treatment group, and yeast 114 intervention group; Figure 4 The statistical results of the movement ability of the bumblebee control group, pesticide treatment group and yeast 114 intervention group after 7 days of treatment, among which Figure A shows the moving distance and Figure B shows the maximum speed; Figure 5 The statistical results of social behavior of the bumblebee control group, pesticide treatment group, and yeast 114 intervention group after 7 days of treatment, where Figure A shows the contact frequency and Figure B shows the contact time; Figure 6 The statistical results of taste response scores of the bumblebee control group, pesticide treatment group and yeast 114 intervention group after 7 days of treatment; Figure 7These are the statistical results of learning and memory of the bumblebee control group, pesticide treatment group, and yeast 114 intervention group after 7 days of treatment. Figure A shows the proportion of bumblebees in each group extending their beaks during 10 rounds of learning, and Figure B shows the short-term memory of bumblebees in each group after completing 10 rounds of learning. DETAILED DESCRIPTION

[0012] The present invention is further described below by means of the accompanying drawings and examples, but the protection scope of the present invention is not limited to the contents described above. The methods in the present embodiments are operated according to conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified, and the culture media are commercially available products and are used according to the instructions for use; The medium used for culturing yeast 114 was yeast peptone dextrose medium (YPD): 20 g peptone, 20 g glucose, 10 g yeast extract, 10 g agar powder, and 0.22 g tryptophan, dissolved in 1 L distilled water; Example 1: Isolation and Identification of Yeast 114 Bumblebees were dissected, the complete intestines were taken out, the intestinal wall was ground and broken with a grinding rod in 100 μL 25% (v / v) glycerol, and stored in a -80°C ultra-low temperature medical refrigerator; the intestinal samples were streaked in three areas on YPD solid culture medium, cultured in a 30°C incubator for 2-3 days, the colony morphology was observed, and a single colony was picked out and streaked in four areas on YPD culture medium again, and continued to be purified and cultured in a 30°C incubator, and several single colonies were selected on the culture medium and numbered respectively, and 20 single colonies were selected for ITS rDNA sequencing; The total fungal DNA was extracted using the Fungal Genomic DNA Rapid Extraction Kit (Cat. No. B518229-0050) of Sangon Bioengineering, and the universal primers in the ITS region (ITS1F: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3') were used 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, ddH 2 O 11.6μL) for amplification; PCR conditions were: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; and finally 72℃ extension for 5min. The ITS amplification sequencing results of this example were compared with the NCBI database using the BLAST tool. Starmerella sorbosivoransThe closest relative, with a similarity of 97.7%, is strain 114, the ITS rDNA sequence of yeast 114 is shown in SEQ ID NO: 1; The morphological characteristics of yeast 114 are shown in Figure 1 , round colonies appeared on YPD medium, the colonies were white in color, with neat edges, opaque and smooth surface. Under scanning electron microscope, the strain was observed to be grayish white spherical and aggregated together.

[0013] Therefore, combined with the morphological and molecular identification results, it was determined that strain 114 was a yeast Starmerella sorbosivorans .

[0014] Example 2: Preparation of freeze-dried bacterial preparation of strain 114 The yeast 114 was activated with YPD solid medium, and the activated strain was transferred to YPD liquid medium at a 4% inoculum, cultured at 30°C for 25 h, centrifuged at 5000 g for 15 min, and the cells were harvested for later use; The harvested cells were mixed evenly with a protective agent in a volume ratio of 3:1 for later use. The protective agent was an aqueous solution containing 7.5% trehalose, 7.5% skim milk powder, 4.5% gum arabic, 4.5% polyethylene glycol, and 6% sodium L-glutamate.

[0015] Before freeze-drying, the sample needs to be pre-frozen at -80°C for 3 hours, followed by a three-stage freeze-drying process: the first stage is freeze-drying at a pressure of 512Pa for 6 hours, the second stage is freeze-drying at a pressure of 256Pa for 5 hours, and finally freeze-drying is continued at a pressure of 103Pa until the sample forms a powdered product.

[0016] The freeze-dried bacterial preparations were stored at 4°C and 25°C, respectively. The number of live bacteria of each bacterial powder was measured every 2 months. The experiments were repeated 3 times in parallel. The results are shown in Table 1. As can be seen from the table, the number of live bacteria of the freeze-dried bacterial preparations remained at a high level after being stored at 4°C and 25°C for 10 months. Although the stability at 25°C was somewhat lower than that at 4°C, it still maintained a survival level of more than 90%. The effective bacterial count of yeast 114 powder was not less than 3×10 9 cfu / g; Table 1 .

[0017] Example 3: Effects of Yeast 114 on Survival and Behavior of Bumblebees 1. Preparation of Yeast 114 Suspension Weigh 1 g of the freeze-dried bacterial agent of Example 2 (with an effective viable count of not less than 10 9cfu / g), placed in a 1L beaker, added with 450mL of a mixed solution of 50% sucrose solution and 450mL of 1×PBS buffer, stirred to fully suspend. Then, 100g of pollen was added and stirred evenly to obtain a yeast 114 suspension.

[0018] 2. Bumblebee breeding and experiments Twenty beehives were randomly selected and placed in a gas treatment box. They were anesthetized with carbon dioxide gas for 90 seconds, then the beehives were opened, and tweezers were used to pick out the pupae that had not yet left the cells. The black pupae selected from the 20 beehives were concentrated in one beehive for cultivation. Since bumblebees usually emerge from black pupae within 1-2 days, in order to ensure the survival rate of newborn bees, sugar water and pollen need to be provided in the beehive. The culture temperature was 29°C and the humidity was 65%. The 2-day-old bumblebees to be hatched were divided into containers, with 20 in each container. The experimental groups were divided into a control group, a pesticide treatment group, and a yeast 114 intervention group, with 3 parallel groups in each group.

[0019] After the experiment began, the three experimental groups were fed with tetracycline at a concentration of 450 μg / mL to reduce the interference of the bumblebees' original intestinal microorganisms. From the 6th day, the control group was fed with conventional sugar water and pollen, the pesticide treatment group was fed with sugar water and pollen containing 5 μg / mL acetamiprid, and the yeast 114 intervention group was fed with sugar water and yeast 114 suspension containing 5 μg / mL acetamiprid, while pollen continued to be fed as usual. The sugar water and pollen were replaced every three days, and the yeast 114 suspension also needed to be re-prepared and added to the hive every three days.

[0020] 3. Statistics on bumblebee deaths The daily number of bumblebee deaths in the control group, pesticide treatment group and yeast 114 intervention group was counted.

[0021] Results Figure 2 As can be seen from the figure, feeding started on 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 adding yeast 114 to the diet can reduce the mortality rate of bumblebees and prolong their lifespan.

[0022] 4. Detection of bumblebee diet, exercise and social indicators (1) The daily sugar water and pollen consumption of bumblebees in the control group, pesticide treatment group, and yeast 114 intervention group was counted and analyzed; Results Figure 3From the figure, it can be seen that 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 reduction in diet 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).

[0023] (2) Seven days after the yeast 114 intervention, three bumblebees in each group were placed in a culture dish. After they adapted to the environment, a 50-minute video was recorded and the moving distance and maximum speed of the bumblebees were analyzed and counted using EthoVision software. Results Figure 4 ,from Figure 4 It can be seen that there are significant differences in the movement ability of bumblebees after seven days of feeding (p<0.05). The moving distance and maximum speed of the bumblebees in the yeast 114 intervention group are significantly higher than those in the pesticide treatment group.

[0024] (3) Seven days after the yeast 114 intervention, three bumblebees in each group were placed in a culture dish. After they adapted to the environment, a 50-min video was recorded and the frequency and duration of physical contact of the bumblebees were analyzed and counted using EthoVision software. Results Figure 5 ,from Figure 5 It can be seen that there are significant differences in the social behavior of bumblebees after seven days of feeding (p<0.05). The frequency and duration of physical contact of bumblebees in the yeast 114 intervention group are significantly higher than those in the pesticide treatment group.

[0025] 5. Sucrose sensitivity test of bumblebees After 7 days of yeast 114 treatment, the test bumblebees of different groups were starved for 3 hours, placed on ice for 5 minutes until they were stunned or slow in activity, and then tied up. First, a cotton swab was dipped in deionized water and extended to the front of the bumblebee's head to test whether its proboscis extension was due to thirst or a habit. 4 minutes after the deionized water test, different concentrations of sugar water (mass volume concentration of sterile sugar water 0.1, 0.3, 1, 3, 10, 30, 50%) were used to test the proboscis extension reaction of each bee in order from low to high concentrations, and the sugar response threshold and taste response score of each bee were recorded; among them, the sugar response threshold (Sugar response threshold, SRT) corresponds to the lowest sugar water concentration at which the bee has a proboscis extension reaction, and the gustatory response score (Gustatory response score, GRS) ranges from 1 to 7, with 1 point for only responding to the highest concentration of sugar water (50%) and 7 points for responding to all concentrations of sugar water; Results Figure 6 ,from Figure 6 It can be seen that the taste response score of bumblebees in the pesticide treatment group was significantly lower than that in the control group, while the taste response score of bumblebees in the yeast 114 intervention group was significantly higher than that in the pesticide treatment group, indicating that yeast 114 can alleviate the damage of acetamiprid to the sucrose sensitivity of bumblebees.

[0026] 6. Experiment on the learning and memory ability of bumblebees After the sucrose sensitivity experiment is completed, a single bumblebee is placed in front of an exhaust fan, and a lemon-scented syringe is aimed at the front of the bumblebee's antennae. Air is given at a uniform speed for 5 seconds. At the 6th second of the lemon odor, sugar is given for 3 seconds. The bees that can extend their beaks when given the lemon odor are marked as having learned. The above is the process of learning once. Each bumblebee needs to go through 10 rounds of learning, with an interval of 15 minutes between each round. After 10 rounds of learning are completed, the bumblebees are placed in an incubator for 45 minutes, and then a short-term memory rate test is conducted. During the test, air is given with a lemon-scented syringe to test whether the bumblebee has a beak extension reaction. The individuals that extend their beaks when given the lemon odor are marked as having remembered. After the experiment, the learning and memory of each group were counted.

[0027] Results Figure 7 From the figure, it can be seen that the learning and memory of bumblebees in the pesticide treatment group showed a significant decline compared with the control group, while the learning and memory of bumblebees in the yeast 114 intervention group were better than those in the pesticide treatment group, indicating that yeast 114 can alleviate the decline in learning and memory ability of bumblebees caused by acetamiprid.

[0028] In summary, the yeast 114 provided by the present invention can significantly prolong the life span of bumblebees and increase their food intake; in terms of motor ability and social behavior, it can significantly improve the moving distance, maximum speed, contact time and frequency of bumblebees; in terms of sucrose sensitivity and learning and memory, it can improve the sensitivity of bumblebees to sucrose and their learning and memory abilities. Although the present invention has been described above through the embodiments, those skilled in the art should understand that, without departing from the spirit and essence of the present invention, improvements and modifications made to the present invention should all fall within the scope of protection of the present invention.

Claims

1. A yeast ( Starmerella sorbosivorans )114, and its deposit number at the General Microbiology Center of China Culture Collection Administration is CGMCC NO.32805.

2. Use of the yeast 114 described in claim 1 in alleviating damage caused by pesticides to bumblebees.

3. The use according to claim 2, characterized in that: Yeast 114 can extend the lifespan of bumblebees and improve their appetite, exercise capacity, social behavior, and learning and memory abilities.

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