A kit for the prevention and control of bee coccidioidomycosis and its application

Abstract

This invention relates to the field of biotechnology, specifically to a kit for the prevention and control of *Gastromyxobinus* in bees and its application. The kit includes an interfering agent composed of at least one of si-lnc6140 and milR5658-x inhibitors. The si-lnc6140 is double-stranded, with the sequence of the sense strand as shown in SEQ ID NO:1 and the sequence of the antisense strand as shown in SEQ ID NO:2. The sequences of the milR5658-x inhibitors are shown in SEQ ID NO:3. This invention reveals for the first time and targets the lnc6140–milR5658-x–ATPase regulatory axis: This invention is the first to discover that non-coding RNAs (lnc6140 and milR5658-x) in *Gastromyxobinus* affect fungal proliferation and infection by regulating the expression of the energy gene ATPase, and uses this regulatory axis as an intervention target for the treatment of chalkbrood in bees. The combined application of two non-coding RNA interference agents can synergistically reduce the incidence of chalkbrood disease. Compared with traditional chemical drugs, this method works through RNA interference, avoiding the problem of drug residues. It is also simple to use (mix with sugar water and feed) and does not affect the survival rate of larvae or the immune response (expression of host immune genes).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a kit for the prevention and control of bee cysticercosis and its application. Background Technology

[0002] Chalk disease in bees is a fungal disease caused by Ascosphaera apis infecting bee larvae. It is one of the most serious diseases affecting Western honeybees and severely impacts the beekeeping industry. The spores of the chalk disease fungus can survive in the natural environment for more than 15 years, making it difficult to eradicate completely.

[0003] Currently, the main preventive measures for chalkbrood include: strengthening bee colony management and cultivating strong colonies to enhance the colonies' own disease resistance, as well as strengthening the disinfection of apiary sites, beehives, and bee feed to reduce the risk of infection. However, these measures can only reduce the probability of disease to a certain extent, and their overall effectiveness is very limited.

[0004] Currently, mebendazole can be used to treat chalkbrood, and while it has some therapeutic effect, it easily leaves residues in bee products, thus limiting its practical application. Its use in bee colonies is now strictly prohibited in my country. Besides chemical drugs, some people also use bee probiotics, traditional Chinese medicine preparations, and other biological agents and natural extracts to treat chalkbrood in bees, but these methods are cumbersome to administer and their therapeutic effects are generally limited. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for the prevention and treatment of bee coccidioidomycosis and its application, which can be mixed with sugar water and fed to bees.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a kit for the prevention and control of apigeninella, the kit comprising an interfering agent; the interfering agent is at least one selected from si-lnc6140 and milR5658-x inhibitors; The si-lnc6140 is a bistranded chain, wherein the sequence of the sense chain is shown in SEQ ID NO:1 and the sequence of the antisense chain is shown in SEQ ID NO:2; The sequence of milR5658-x inhibitors is shown in SEQ ID NO:3.

[0007] Furthermore, the ends of both strands of the si-lnc6140 are modified with two deoxythymuses.

[0008] Furthermore, the interfering agent is composed of a 1:1 mixture of si-lnc6140 and milR5658-x inhibitors.

[0009] The present invention also includes the use of any of the kits described herein in the preparation of bee feed.

[0010] Furthermore, the interferon in the kit is mixed with sugar water as a feeding solution for bees.

[0011] Compared with existing technologies, this invention reveals for the first time and targets the lnc6140–milR5658-x–ATPase regulatory axis: This invention is the first to discover that non-coding RNAs (lnc6140 and milR5658-x) in *Gastromyxobolus apis* affect fungal proliferation and infection by regulating the expression of the energy gene ATPase, and uses this regulatory axis as an intervention target for the prevention and control of *Gastromyxobolus apis*.

[0012] The combined application of two non-coding RNA interference agents can synergistically reduce the incidence of chalkbrood disease. Compared with traditional chemical drugs, this method works through RNA interference, avoiding the problem of drug residues. It is also simple to use (mix with sugar water and feed) and does not affect the survival rate of larvae or the immune response (expression of host immune genes). Attached Figure Description

[0013] Figure 1 Identification of lnc6140 in the gut of worker bee larvae infected with *Gastrocystis aegyptiacus*; (A) Microscopic features of *Gastrocystis aegyptiacus* spores; (B) Schematic diagram of *Gastrocystis aegyptiacus* infection in larvae; (C) Expression levels of lnc6140 in *Gastrocystis aegyptiacus* spores and bee gut based on full-length transcriptome sequencing; (D) Expression profile of lnc6140 during *Gastrocystis aegyptiacus* infection in bees; AmCK / AcCK: control group (Italian bees / Chinese bees); AmT / AcT: *Gastrocystis aegyptiacus* infected (Italian bees / Chinese bees); Different lowercase letters above the bars indicate significant differences between groups (One-way ANOVA, P ≤ 0.05), and the same letter indicates no significant difference; the Spores group is a pure spore sample of *Gastrocystis aegyptiacus*.

[0014] Figure 2 To investigate the effect of interfering with lnc6140 of *Gastromyxobolus apis* on the expression of genes related to infection and proliferation of *Gastromyxobolus apis*; (A) expression level of lnc6140 at 1–3 dpi; (BG) expression levels of Chi3, Grif, AdmB, Pkia, Ste11, and Dmap1 at 1–3 dpi after interfering with lnc6140; si-scramble, interference control group; si-lnc6140, interference group; P ≤ 0.05; P ≤ 0.01; , P≤ 0.001; ns, non-significant.

[0015] Figure 3To investigate the effects of interfering with *Lnc6140* in bees on bee innate immunity, survival rate, and chalkbrood incidence; (A) Expression levels of the Toll signaling pathway receptor gene Dorsal at 1–3 dpi; (BC) Expression levels of the antimicrobial peptide genes Hymenoptaecin and Abaecin at 1–3 dpi; (D) Phenotypes of bee mortality and chalkbrood incidence; (E) Bee survival rate after interfering with lnc6140; (F) Chalkybrood incidence rate in bees after interfering with lnc6140; si-scramble, interference control group; si-lnc6140, interference group. P ≤ 0.05; P ≤ 0.01; , P≤ 0.001; ns, non-significant.

[0016] Figure 4 The effect of lnc6140 targeting milR5658-x on the infection of *Gnaphalium apiae*; (A) Four milRNAs targeted by lnc6140; (B) Schematic diagram of the targeting binding of lnc6140 and milR5658-x; binding free energy is -20 kcal / mol; (C) Dual-luciferase assay to verify the binding relationship between lnc6140 and milR5658-x; (D) Expression level of milR5658-x at 1-3 dpi after *Gnaphalium apiae* infection; (E) Expression level of milR5658-x at 1-3 dpi after interfering with lnc6140; si-scramble, interference control group; si-lnc6140, interference group; (F) Expression level of milR5658-x at 1-3 dpi after milR5658-x mimics treatment; (G) milR5658-x Incidence of chalkbrood after treatment with mimics; (H) Expression level of milR5658-x at 1-3 dpi after treatment with milR5658-x inhibitors; (I) Incidence of chalkbrood after treatment with milR5658-x inhibitors. P ≤ 0.05; P ≤ 0.01; , P≤ 0.001; ns, non-significant.

[0017] Figure 5To investigate the role of lnc6140 in regulating the infection and proliferation of *Gastromyxobina bees* via the milR5658-x-ATPase axis; (A) Dual-luciferase assay to verify the binding relationship between milR5658-x and ATPase; (B) ATPase expression levels at 1–3 dpi after *Gastromyxobina bees* infection; (CD) ATPase expression levels at 1–3 dpi after overexpression and inhibition of milR5658-x; (E) ATPase expression levels at 1–3 dpi after lnc6140 knockdown; (F) ATPase expression levels at 1–3 dpi after inhibition of both lnc6140 and milR5658-x; (G) Expression of genes related to *Gastromyxobina bees* infection and proliferation at 6 dpi after inhibition of both lnc6140 and milR5658-x. (H) Inhibition of lnc6140 and milR5658-x, survival rate of bees infected with Coccidioidomyces apiaceus; (I) Inhibition of lnc6140 and milR5658-x, incidence of chalkbrood disease infected with Coccidioidomyces apiaceus; P ≤ 0.05; P ≤ 0.01; , P≤ 0.001; ns, non-significan. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0019] Example 1 Activation of *Gyrodactylus apis* spores and larval inoculation The *Gynostemma pentaphyllum* strain was inoculated onto sterile potato dextrose agar (PDA) medium and cultured at 33±0.5℃ for 10 days. Sporangia were then collected, 200 µL of sterile water was added, and the spores were purified by differential centrifugation after grinding. Spores were counted using a hemocytometer. Three-day-old worker bee larvae were fed 50 μL of a solution containing 5 × 10⁵ spores. 3 Artificial feed containing spores was changed every 24 hours until 6 days of age. The control group was fed a feed without spores.

[0020] Example 2 LNC6140 expression detection Intestinal tissue was harvested 1, 2, and 3 days after inoculation (fed with spore-containing artificial feed) in Example 1 (corresponding to larval ages 4, 5, and 6). Total RNA was extracted from the intestinal samples using an RNA extraction kit (Aikoray, China). The RNA was extracted using Hifair® III1. stStrand cDNA Synthesis Kit (gDNA digester plus) (Yisheng, China) was used for reverse transcription. The cDNA of lnc6140 was obtained by reverse transcription using Random Primers N6 from the kit, and another copy was processed using Oligo (dT). 18 cDNA of the internal control 5.8S rRNA (GenBank accession number: U68313.1) was obtained by reverse transcription with Random Primers N6. Real-time PCR was performed using TB Green Fast qPCR Mix with the following reaction program: 95℃, 30 s; 95℃, 10 s, 60℃, 30 s, 40 cycles. 2 -ΔΔCt The relative expression level is calculated using this method.

[0021] See results Figure 1 lnc6140 was expressed at low levels in spores of *Gastromyxobolus apis*, but its expression increased significantly after infecting larvae and continued to be upregulated with the duration of infection. It was not expressed in the intestines of larvae in the control group.

[0022] Example 3 Functional verification of si-lnc6140 (sequence shown in Table 1) Gene synthesis was performed using siRNA targeting the lnc6140 sequence (SEQ ID NO:4) (si-lnc6140) and a negative control siRNA (si-scramble). The siRNA was mixed with a 1:1 sugar aqueous solution at a volume ratio of 1:1000 to prepare a feeding solution containing RNA interference. After inoculating larvae with *Gnaphalium affine* spores, 50 µL of the feeding solution containing RNA interference was added to larval culture plates using a pipette. Larval survival rate, chalkbrood incidence, and changes in the expression of pathogen infection-related genes (chi3, griF, ADM-B, pkiA, STE11, DMAP1) were assessed.

[0023] See results Figure 2 and Figure 3 Feeding with si-lnc6140 significantly reduced lnc6140 expression and led to downregulation of pathogen infection and proliferation-related genes (chi3, griF, ADM-B, pkiA, STE11, DMAP1). The incidence of chalkbrood decreased by 9.72% and 12.50% at 6 dpi and 7 dpi, respectively, but did not affect the expression of host immune genes.

[0024] Table 1. Sequences of si-lnc6140 and milR5658-x inhibitors ; Example 4 Dual-luciferase assay of the interaction between lnc6140 and milR5658-x, and milR5658-x and ATPase. The milRNAs targeted by lnc6140 were predicted using mirTarBase, and the target mRNAs of milR5658-x were predicted using mireap, miRanda, and TargetScan software. Cytoscape software was used to plot the binding relationships between lnc6140 and its target miRNAs, and between milR5658-x and mRNAs. Wild-type and mutant luciferase reporter vectors with binding sites of lnc6140 and milR5658-x, and milR5658-x and ATPase, were constructed respectively. These vectors were co-transfected with milR5658-x mimics into HEK293T cells, and fluorescence activity was detected to verify the binding relationships.

[0025] See results Figure 4 BC and Figure 5 B. Dual-luciferase assays confirmed that lnc6140 binds to milR5658-x, and milR5658-x binds to ATPase.

[0026] Example 5 Functional validation of milR5658-x inhibitors (sequences shown in Table 1) Genetically synthesized milR5658-x inhibitors targeting the milR5658-x sequence (SEQ ID NO:5), and mixed the milR5658-x inhibitors with a 1:1 sugar aqueous solution at a volume ratio of 1:1000 to prepare a feeding solution containing RNA interference. After inoculating larvae with *Gnaphalium affine* spores, 50 µL of the feeding solution containing RNA interference was added to larval culture plates using a pipette. Changes in milR5658-x expression, chalkbrood incidence, and larval survival rate were detected.

[0027] See results Figure 4 Feeding with milR5658-x inhibitors significantly reduced milR5658-x expression. The incidence of chalkbrood decreased by 10.13% and 19.38% at 6 dpi and 7 dpi, respectively, but did not affect the expression of host immune genes.

[0028] Example 6 Functional validation of the mixed interferon LNC6140 and milR5658-x Si-Lnc6140 and milR5658-x inhibitors were mixed at a 1:1 ratio to obtain a mixed interfering agent. This mixed interfering agent was then mixed with a 1:1 sugar solution at a volume ratio of 1:2000 to prepare a feeding solution containing the RNA mixed interfering agent. After inoculating larvae with *Gnaphalium affine* spores, 50 µL of the feeding solution containing the RNA mixed interfering agent was added to larval culture plates using a pipette. Larval survival rate, chalkbrood incidence, and changes in the expression of pathogen infection-related genes (chi3, griF, ADM-B, pkiA, STE11, DMAP1) were detected.

[0029] See results Figure 5 Feeding with a mixture of interfering agents led to downregulation of the expression of pathogen infection and proliferation-related genes (chi3, griF, ADM-B, pkiA, STE11, DMAP1). The incidence of chalkbrood decreased by 13.74% at 7 dpi, but did not affect the expression of host immune genes.

[0030] Example 7 Statistics on the incidence and survival rate of chalkbrood disease After inoculating larvae with *Gnaphalium affine* spores, they were fed daily with 50 µL of a single RNA interference agent containing either si-lnc6140 or milR5658-xinhibitors, or a mixed RNA interference agent containing both. Larval mortality and chalkbrood disease incidence were observed and recorded daily. Incidence rate = (number of chalkbrooded larvae / total number of larvae) × 100%, survival rate = (number of surviving larvae / total number of larvae) × 100%. Each group had three biological replicates, with 24 larvae per replicate.

[0031] See results Figure 3 , Figure 4 , Figure 5 Interference with lnc6140 or milR5658-x inhibitors led to a decrease in ATPase expression and a reduction in the incidence of chalkbrood disease. Furthermore, interference with lnc6140 and milR5658-x further inhibited the expression of ATPase and infection-related genes, resulting in a significant reduction in the incidence of chalkbrood disease at 5 dpi and 7 dpi.

[0032] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A kit for the control of Nosema apis, characterized in that it comprises: The kit comprises an interfering agent; the interfering agent is at least one of si-lnc6140 and milR5658-x inhibitors; The si-lnc6140 is double-stranded, wherein the sequence of the sense strand is shown as SEQ ID NO: 1, and the sequence of the antisense strand is shown as SEQ ID NO: 2; The sequence of the milR5658-x inhibitors is shown as SEQ ID NO:

3.

2. The kit for controlling Ascosphaera apis according to claim 1, characterized in that: The double-stranded ends of the si-lnc6140 are both modified with two deoxythymidines.

3. The kit for controlling Ascosphaera apis according to claim 1, characterized in that: The interfering agent is composed of si-lnc6140 and milR5658-x inhibitors mixed in a 1:1 ratio.

4. Use of the kit according to any one of claims 1-3 in the preparation of a feed for bees.

5. Use according to claim 4, characterized in that: The interfering agent in the kit is mixed with sugar water as a feeding liquid for bees.

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