CN114569592B - The application of meclofenamic acid in the control of bee cyst larvae - Google Patents

Abstract

The invention relates to the technical field of bee breeding, in particular to application of meclofenamic acid in preventing and treating Chinese bee sacbrood. The research of the invention finds that the meclofenamic acid has certain function of inhibiting the Chinese bee sacbrood virus, and further can effectively prevent and treat the Chinese bee sacbrood. In addition, meclofenamic acid has certain effect of improving RNA N of Chinese bee larvae ⁶ -methyladenosine (m) ⁶ A) The effect of methylation levels, which may be precisely through this pathway, is to inhibit the Chinese sacbrood virus. The invention further provides bee feed comprising meclofenamic acid, which can effectively control the infection of Chinese bee sacbrood and ensure the health of bee colonies.

Description

The application of meclofenamic acid in the control of bee cyst larvae

technical field

The invention relates to the technical field of bee breeding, in particular to the application of meclofenamic acid in the prevention and treatment of mid-bee cyst larvae.

Background technique

Chinese Sacbrood larvae disease is a disease caused by Chinese Sacbrood Virus (CSBV) infection, which not only leads to the death of larvae, but also shortens the lifespan of adult bees, eventually leading to the destruction of the entire bee colony. There is a serious threat to the breeding of bees, and there is currently no effective control drug. In order to reduce losses, the bee farms in the outbreak of bee sac larvae use drugs indiscriminately and abuse drugs, which may lead to excessive veterinary drug residues in the honey of the bee, which greatly affects the quality of honey and the health of consumers. Therefore, it is urgent to develop drugs for the prevention and treatment of Chinese honeybee cystic larvae, improve the health situation of bees, and escort the healthy development of the bee industry.

Meclofenamic acid (MA) is a non-steroidal anti-inflammatory drug with anti-inflammatory, analgesic and antipyretic properties, approved for arthritis (osteoarthritis and rheumatoid arthritis), relief Pain (mild to moderate pain), dysmenorrhea, and severe menstrual blood loss (menorrhagia). This drug is commonly used in human medicine and rarely used in the treatment of bee diseases.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the present invention provides the application of meclofenamic acid in the prevention and treatment of mid-bee cyst larvae.

In a first aspect, the present invention provides the application of meclofenamic acid in the prevention and treatment of Chinese honeycomb larvae.

The present invention further provides the use of meclofenamic acid in inhibiting the amplification of the medulla virus.

The present invention further provides the use of meclofenamic acid in increasing the methylation level of RNA N ⁶ -methyladenosine (m ⁶ A) in Chinese honeybee.

The present invention further provides the application of meclofenamic acid in the preparation of a medicine or feed for preventing and treating Bee sac larvae.

The present invention further provides the use of meclofenamic acid in the preparation of a medicine or feed for inhibiting the amplification of the mesothelioma virus.

The present invention further provides the use of meclofenamic acid in the preparation of a medicine or feed for improving the methylation level of RNA N6 ^- methyladenosine of Chinese honeybee.

Further, the consumption of described meclofenamic acid is 200～600 μM.

Further, the feed also includes royal jelly, fructose, glucose, yeast extract and water.

In a second aspect, the present invention provides a bee feed containing 200-600 μM meclofenamic acid.

Further, in parts by weight, also include:

2~10 parts royal jelly, 0.1~1 part fructose, 0.1~1 part glucose, 0.01~1 part yeast extract, 2~8 parts water.

The present invention has the following beneficial effects:

In the present invention, a compound meclofenamic acid that can effectively inhibit the amplification of the medaka larvae virus is found, which can significantly reduce the level of the medlar larvae infected by the medlar larvae, thereby preventing and treating the medlar larvae. The role of larvae. In the present invention, it is found that meclofenamic acid may play a role in inhibiting the mesophila larvae virus by increasing the methylation level of RNA N ⁶ -methyladenosine (m ⁶ A) in the larvae of S. spp.

The meclofenamic acid provided by the present invention can be used in medicines or as an additive for bee feed, which can effectively reduce the infection of the larvae of the bee medusa virus, which is of great significance in the field of apiculture.

Description of drawings

Fig. 1 is the result picture that the meclofenamic acid provided in Example 1 of the present invention significantly reduces the CSBV infection after feeding the larvae of the bee; wherein, A is the degree of infection of the larvae of the bee of the bee fed 400 μM meclofenamic acid CSBV virus infection degree Contrast, B is the virus copy number results of CSBV larvae fed with 400 μM meclofenamic acid at 48 hours and 72 hours of CSBV virus infection; C is the result of CSBV infection for 24 hours of CSBV larvae fed with meclofenamic acid and 48 hours RNA m ⁶ A methylation level; 24 h in the figure represents 24 hours after CSBV virus infection; 48 h represents 48 hours after CSBV virus infection; 72 h represents 72 hours after CSBV virus infection.

Figure 2 is a schematic diagram showing the results of the effect of feeding meclofenamic acid on the survival rate of C. bee larvae infected with CSBV provided in Example 1 of the present invention.

3 is a schematic diagram of the comparison results of the RNAm ⁶ A methylation levels of the infected larvae of the bee sac virus and the larvae of the healthy Chinese honey bee provided in Example 1 of the present invention; wherein, the bar graph on the left is the absolute quantitative qPCR comparison of the CSBV-infected honey bee Result plot of CSBV virus copy number in larvae and healthy S. spp. larvae, and the right bar graph is the result of LC-MS/MS quantitative measurement comparing RNA m ⁶ A methylation levels in CSBV-infected S. spp. larvae and healthy S. spp. larvae .

Figure 4 is the LC-MS/MS analysis provided in Example 1 of the present invention for the m ⁶ A/total methylation (expressed as m ⁶ A/A ratio) of mRNA in the larval samples of Chinese honeybee treated with different concentrations of meclofenamic acid by LC-MS/MS ) of the results.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

The test methods involved in the following examples, unless otherwise specified, are conventional methods in the art.

The reagents and test materials involved in the following examples can be purchased commercially unless otherwise specified.

Example 1

1. The application of meclofenamic acid in reducing the prevalence of mesococcus larvae in honeybees

Meclofenamic acid is a non-steroidal anti-inflammatory drug with anti-inflammatory, analgesic and antipyretic properties approved for arthritis (osteoarthritis and rheumatoid arthritis), pain relief ( mild to moderate pain), dysmenorrhea, and severe menstrual blood loss (menorrhagia). Meclofenamic acid is a drug applied to humans and has never been used in honeybees. The present invention intends to use meclofenamic acid to carry out the treatment related to bee cyst-like larvae, and the specific process is as follows:

The bees used were Apis cerana , which were purchased from bee farms and raised by beekeepers.

The CSBV virus used was the virus liquid extracted, separated and purified from the pests, and the concentration was 10 ⁹ copies/μL.

1-day-old worker bee larvae were transferred from the hive to a 48-well culture plate and cultured in an incubator at a temperature of 35°C and a humidity of 95% HR. Each larva was fed 20 µL of worker bee basal feed (the formula for each 10 g feed was: 5 g royal jelly, 0.6 g fructose, 0.6 g glucose, 0.1 g yeast extract, 3.7 g water).

On the second day, the worker bee larvae were divided into 2 groups of 100 each. The experimental group added meclofenamic acid to the feed of the worker bee larvae to make the concentration of 40 µM, and the control group was still fed the basal diet.

On the third day, CSBV virus infection was carried out, and the CSBV virus solution was added to the feed of worker bees, and the amount of CSBV virus infection in each larva was 10 ⁹ copies. The feed formula is as follows:

CSBV infection feed formulation: CSBV virus solution was added to worker bee basal feed so that the final concentration of CSBV was 5×10 ¹⁰ copies/ml. Since each larva was fed 20 µL of CSBV-infected diet, the final infection of CSBV virus per larva was 10 ⁹ copies.

The 4, 5, and 6-day-old larvae were fed with the basic feed of worker bees without virus infection. Undead larvae were collected at 24 hours, 48 hours and 72 hours after CSBV infection, respectively. RNA was extracted and mRNA was isolated. Absolute quantitative qPCR analysis was performed.

The primers used for qPCR are as follows:

CSBV primers:

Forward primer: TATTCAGGGGGACGCTACAC;

Reverse primer: GCGTGAGTTGACAGAAAATC.

The experimental results are shown in Figure 1. Compared with the larvae that were not fed with meclofenamic acid, the degree of CSBV infection in the larvae fed with meclofenamic acid at 2 days of age was significantly reduced (A in Figure 1); At 48 and 72 h post-infection, the CSBV copy numbers differed by 41-fold and 1.4-fold in unfed and meclofenamic acid-fed larvae, respectively (Fig. 1, B). LC-MS/MS detection of each sample showed that the larvae of the bee fed meclofenamic acid showed significantly elevated m ⁶ A methylation levels at both 24 and 48 hours after infection, although at 72 hours. The m ⁶ A level decreased, but still had anti-CSBV infection effect (C in Figure 1). Statistical analysis of the survival of larvae infected with CSBV showed that compared with larvae that were not infected with CSBV, the survival rate of CSBV infection decreased. The survival rate was improved after the treatment (Figure 2). Based on the above results, it was shown that meclofenamic acid can play a significant role in the control of CSBV virus, and at the same time, it can significantly increase the level of RNA m ⁶ A methylation in worker bee larvae. Therefore, meclofenamic acid can be used as a feed additive or a drug additive for the prevention and treatment of Chinese honeycomb virus.

2. CSBV-infected larvae have significantly lower m ⁶ A levels than healthy larvae

In the present invention, the infected larvae of the bee are picked from the beehives infected with the beehive. Healthy larvae were selected from the hives of other hives that were not infected with CSBV. The mRNAs were isolated separately, and the m ⁶ A methylation levels on the mRNA were quantitatively measured by liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS), and the CSBV virus copy number was detected by absolute quantitative qPCR with CSBV-specific primers. The results are shown in Figure 3: the virus copy number of the susceptible larvae is 10 ⁴ times that of the healthy larvae, while the m ⁶ A methylation level of the healthy larvae is 3 times that of the susceptible larvae. According to the above research results, the present invention speculates that the infection degree of CSBV virus can be reduced by reducing the m ⁶ A methylation level of the larvae of the bee, and meclofenamic acid may inhibit CSBV virus through this way.

3. Comparison of m ⁶ A methylation levels under different concentrations of meclofenamic acid. The specific experiments are as follows:

In the present invention, 1-day-old worker bee larvae are moved from the hive to a 48-well culture plate, and cultured in an incubator with a temperature of 35° C. and a humidity of 95% HR. Each larva was fed 20 µL of worker bee basal diet (formulation per 10 g of feed: 5 g royal jelly, 0.6 g fructose, 0.6 g glucose, 0.1 g yeast extract, 3.7 g water).

Different concentrations of meclofenamic acid were added to the feed for 2-day-old worker bee larvae, and the specific feed formula for each treatment was as follows:

Control: basic feed for worker bees (the formula for each 10 g feed is: 5 g royal jelly, 0.6 g fructose, 0.6 g glucose, 0.1 g yeast extract, 3.7 g water).

100 µM Treated Feed Formulation: Meclofenamic acid was added to worker bee basal feed to give a final concentration of meclofenamic acid of 100 µM.

200 µM Treated Feed Formulation: Meclofenamic acid was added to worker bee basal feed to give a final concentration of meclofenaic acid of 200 µM.

400 µM Treated Feed Formulation: Meclofenamic acid was added to worker bee basal feed to give a final concentration of meclofenamic acid of 400 µM.

After 48 hours of treatment, the larvae were collected and mRNA was isolated, and the m ⁶ A methylation level was quantified by LC-MS/MS, and the results are shown in Figure 4. Meclofenamic acid treatment at 400 µM significantly increased m ⁶ A modification compared to control samples. On the one hand, this result verifies the effect of meclofenamic acid on m ⁶ A methylation, and on the other hand, 400 μM is determined as the optimal dosage of meclofenamic acid for the control of mesococcus larvae.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (3)

1. Application of meclofenamic acid in preparing medicine for preventing and treating Chinese bee sacbrood.

2. Use of meclofenamic acid in the manufacture of a medicament for inhibiting amplification of a Chinese bee sacbrood virus.

3. Use according to claim 1 or 2, wherein meclofenamic acid is used in an amount of 200 to 600 μ M.

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