Drosophila population inhibition biological control method targeting CG8630 gene

Abstract

The invention discloses an application of a CG8630 gene in regulating and controlling the development and reproduction functions of attached glands of fruit flies and a fruit fly population inhibition biological prevention and control method of the CG8630 gene. A transgenic vector for expressing specific double-stranded RNA (dsRNA) is constructed through UAS-CG8630-IR, and a GAL4 / UAS system is used for carrying out targeted interference on the CG8630 gene in the accessory gland of the drosophila melanogaster. After the expression of the male fruit fly attach gland CG8630 gene is knocked down, the development of the male fruit fly attach gland is influenced, the reproductive function is reduced, the spawning of a mating female fly is reduced, the hatching rate is reduced, and the embryonic development death rate is increased, so that the number of fruit fly populations is inhibited. The drosophila population inhibition biological control method based on the RNA interference targeting CG8630 gene is high in specificity, only acts on male attached glands, is reversible in interference, has extremely small influence on non-target organisms, has an obvious inhibition effect on the number of drosophila populations, and has great application potential.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and agricultural pest control technology, specifically relating to a biological control method for suppressing fruit fly populations by targeting the CG8630 gene. Background Technology

[0002] Fruit flies, a significant agricultural pest distributed globally, cause serious damage to horticultural crops, especially fruit products. Against the backdrop of climate change, frequent international trade, and accelerated globalization, fruit fly populations are expanding their distribution areas and activity ranges, exhibiting an increasingly invasive and damaging trend. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), the economic losses caused by crop yield losses and quality degradation due to fruit fly infestations amount to billions of dollars annually worldwide. Furthermore, as an important quarantine pest, the presence of fruit flies severely restricts the international trade of agricultural products, increasing quarantine costs and trade barriers for exporting countries.

[0003] Currently, fruit fly control strategies mainly include chemical control, physical control, biological control, and integrated management, but existing control systems face multiple technical bottlenecks. While chemical control (such as organophosphates and pyrethroids) is fast-acting, years of long-term, large-scale use of these pesticides has led to significant resistance in fruit flies to multiple pesticides, resulting in a marked decline in field efficacy. Simultaneously, the non-target effects of chemical agents trigger cascading ecological problems, and the environmental pollution, ecological damage, and food safety risks caused by pesticide residues are becoming increasingly prominent. Physical control methods (such as pheromone trapping), while environmentally friendly, are limited by equipment coverage and maintenance costs, requiring 45-60 traps per hectare to achieve an 80% control threshold. Traditional biological control (such as natural enemy release) has made progress, but faces numerous technical challenges, including the adaptability and reproductive efficiency of natural enemies, and the timing of release. Therefore, developing novel fruit fly control technologies based on new mechanisms of action that combine high control efficiency, environmental compatibility, population specificity, and economic feasibility has become an important research direction in the field of agricultural pest control.

[0004] Existing technologies have drawbacks on non-target species, poor species specificity, require frequent application of chemical pesticides, and necessitate the continuous release of large numbers of male insects for SIT (Solar Insect Infestation), resulting in high costs. Furthermore, chemical control easily leads to resistance development, causing a decline in effectiveness year by year. Most existing technologies also cause environmental pollution and ecological damage. Considering cost, existing safe and environmentally friendly technologies are relatively expensive, leading to low farmer acceptance. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide the application of the CG8630 gene in regulating the development of accessory glands in Drosophila, wherein the nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1, or the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0006] Furthermore, the regulation involved reducing the expression of the CG8630 gene in the Drosophila accessory glands, resulting in developmental defects in the Drosophila accessory glands.

[0007] Furthermore, the developmental defects include reduced accessory gland volume, damaged accessory gland cell membrane structure, reduced accessory gland cell area, and impaired accessory gland cell maturation process.

[0008] The second objective of this invention is to provide the application of the CG8630 gene in regulating the reproductive function of Drosophila, wherein the nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1, or the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] Furthermore, the regulation involved reducing the expression of the CG8630 gene in the accessory glands of Drosophila, which led to a decline in the reproductive function of the fruit flies.

[0010] Furthermore, the decline in reproductive function includes a reduction in the number of eggs laid by mating female flies, a decrease in the hatching rate after egg laying, and an increase in mortality during embryonic development.

[0011] Furthermore, the decline in reproductive function is caused by a decrease in the secretory function of accessory glands and a decrease in the migration rate of sperm within the female reproductive tract.

[0012] The third objective of this invention is to provide a biological control method for suppressing fruit fly populations by targeting the CG8630 gene. The nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1 or the encoded amino acid sequence is shown in SEQ ID NO.2. Targeting the CG8630 gene involves reducing the expression of the CG8630 gene in the accessory glands of male flies, followed by the artificial release of male flies to suppress the fruit fly population.

[0013] Furthermore, the method for reducing the expression of the CG8630 gene in the accessory glands of Drosophila includes the following steps: based on the GAL4 / UAS system, UAS-CG8630-RNAi Drosophila is crossed with Prd-Gal4 Drosophila to obtain UAS-CG8630-RNAi;Prd-Gal4 Drosophila.

[0014] The fourth objective of this invention is to provide a fruit fly strain obtained by crossing UAS-CG8630-RNAi fruit flies with Prd-Gal4 fruit flies.

[0015] This invention provides a biological control method for Drosophila population suppression targeting the CG8630 gene. This targeting is based on RNA interference and the GAL4 / UAS system. Specifically, a transgenic vector expressing specific double-stranded RNA (dsRNA) is constructed using UAS-CG8630-IR to target and interfere with the expression of the CG8630 gene in the accessory glands of male Drosophila. This affects the development of the male accessory glands, inhibits their reproductive function, and leads to reduced egg production and hatching rate after mating with female Drosophila, thereby suppressing the Drosophila population. This invention's biological control method for Drosophila population suppression based on RNA interference targeting the CG8630 gene is highly specific, acting only on the male accessory glands. The suppression intensity can be controlled by adjusting the release ratio. The interference is reversible, does not produce toxic substances, has minimal impact on natural enemies and non-target organisms, and exhibits a significant effect on suppressing the Drosophila population, demonstrating great application potential. Attached Figure Description

[0016] Figure 1 This invention relates to the effect of CG8630 knockdown on accessory gland development in male fruit flies.

[0017] Figure 2 This invention relates to the effect of CG8630 knockdown on the development of accessory glands in Drosophila.

[0018] Figure 3 The effect of CG8630 knockdown on the chief and minor cells of the accessory glands in this invention;

[0019] Figure 4 This invention relates to the effect of CG8630 knockdown on the morphology and tissue structure of accessory gland cells.

[0020] Figure 5 The CG8630 knockdown of this invention leads to abnormal accumulation of lipid droplets in the accessory gland tissue;

[0021] Figure 6 The CG8630 knockdown of this invention leads to a decrease in the reproductive capacity of male fruit flies;

[0022] Figure 7 The CG8630 knockdown of this invention leads to a reduction in the secretion of proteins from the accessory glands of male fruit flies;

[0023] Figure 8 This invention relates to the effect of CG8630 knockdown on the structure of secretory vesicles and microvilli.

[0024] Figure 9 The CG8630 knockdown of this invention affects sperm migration rate but does not affect sperm count or morphology;

[0025] Figure 10 The CG8630 knockdown of this invention led to upregulation of Caspase3 expression in the accessory gland tissue;

[0026] Figure 11 This invention demonstrates that diap1 overexpression can rescue the apoptosis phenotype induced by CG8630 deficiency;

[0027] Figure 12 This invention relates to the effect of CG8630 knockdown on the reproductive competitiveness of male fruit flies.

[0028] Figure 13 This invention aims to evaluate the population suppression effect of knocking down male fruit flies with different proportions of CG8630. Detailed Implementation

[0029] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available.

[0030] Fruit fly strains and sources:

[0031] Prd-Gal4: Donated by the Lü Zhiyi Laboratory of Ocean University of China;

[0032] UAS-CG8630-IR (UAS-CG8630RNAi): Tsinghua Drosophila Center, No. TH04019.N;

[0033] UAS-MCD8-GFP: Tsinghua Drosophila Center, number THJ0081;

[0034] UAS-Syt1-GFP: BDSC, Stock#6925;

[0035] UAS-Diap-OE: BDSC, Stock#6657;

[0036] UAS-Protamin-GFP: BDSC, Stock#58406;

[0037] Roi / Cyo-GFP; TM3 / TM6B-GFP: preserved in the laboratory of the inventor's research group;

[0038] W 1118 Wild type, preserved in the laboratory of the inventor's research group.

[0039] Fruit fly rearing conditions: In a 25℃ light incubator, the relative humidity is 60%, and the light cycle is 12 hours of light and 12 hours of darkness.

[0040] Main research mechanism: This invention employs the GAL4 / UAS system for tissue-specific expression of different genes. Gal4 is a yeast protein that regulates galactose-induced genes. Gal4 binds to a 17-base-pair site, called the upstream activation sequence (UAS), to activate Gal10 and Gal1 target genes. Gal4 can be placed under the control of any promoter to produce tissue-specific expression of genes of interest. Drosophila strains carrying Gal4 and UAS are transcriptionally inactive before binding, avoiding any potential lethal effects. Once the two strains are bound, the offspring will express the UAS fusion protein according to the promoter activation pattern of the Gal4 fusion. Inducing endogenous point mutations in genes can be time-consuming; therefore, RNA interference (RNAi)-mediated gene expression knockdown using the GAL4 / UAS system can target mutations of interest both temporally and spatially.

[0041] Example 1: Fruit fly hybridization experiment

[0042] Collection of virgin fruit flies: When cultured at 17℃, adult fruit flies will not mate within 16 hours of emergence; when cultured at 25℃, they will not mate within 8 hours. Fruit flies about to emerge are alternately cultured at 17℃ and 25℃. Newly emerged fruit flies are anesthetized with CO2. Unmated female flies are collected in fruit fly culture tubes and stored in a 17℃ incubator for later use.

[0043] 1.1 Construction of Drosophila strains

[0044] Using the Roi / Cyo-GFP;TM3 / TM6B-GFP strain of Drosophila, different genotypes expressed by different strains were constructed on chromosomes 2 and 3 of the same strain. Five to ten virgin female flies of the Roi / Cyo-GFP;TM3 / TM6B-GFP strain were collected, and then three to five male flies of genotypes A (II) and B (III) located on chromosomes 2 and 3, respectively, were collected. A and B were crossed with virgin female flies of the Roi / Cyo-GFP;TM3 / TM6B-GFP strain, respectively. The F1 generation of the hybrids was selected based on phenotype (Roi: abnormal eyes; Cyo: curled wings; TM3: short bristles; TM6B: many bristles and short pupae). The selected F1 generation male flies containing the target genotype were then crossed with virgin female flies of the Roi / cyo-GFP;TM3 / TM6B-GFP line. Based on phenotype, the F2 generation A / Roi (or Cyo);TM3 / TM6B-GFP and Roi / Cyo-GFP;B / TM3 (or TM6B-GFP) lines were selected. Finally, the two selected F2 generation lines were crossed, and based on phenotype, the F3 generation A / Roi (or Cyo);B / TM3 (or TM6B-GFP) lines were selected, completing the construction of the fruit fly strains. The constructed fruit fly strains are as follows:

[0045] (1) UAS-MCD8-GFP; Prd-Gal4

[0046] (2)UAS-Protamin-GFP; Prd-Gal4

[0047] (3) UAS-Syt1-GFP; Prd-Gal4

[0048] (4)UAS-CG8630RNAi; UAS-Diap-OE

[0049] 1.2 Hybridization of Prd-Gal4 strain and target strain of Drosophila

[0050] The Prd-Gal4 strain and the target strain (Table 1) were anesthetized with CO2. Thirty virgin female flies of the Prd-Gal4 strain and 15 male flies of the target strain were placed in the same fruit fly culture tube for hybridization. The hybridized fruit fly culture tubes were placed in an incubator at 25°C with a relative humidity of 60%, and the incubator was set to a 12-hour light and 12-hour dark mode.

[0051] Table 1. Drosophila strains crossed with Prd-Gal4

[0052]

[0053] 1.3UAS-CG8630RNAi strain or W 1118 Hybridization with the target strain of fruit flies

[0054] Use UAS-CG8630RNAi strain or W 1118 The target strain (Table 2) was anesthetized with CO2, and 30 UAS-CG8630RNAi strain or W strain mice were taken. 1118 Virgin flies and 15 male flies of the target strain were placed in the same fruit fly culture tube for hybridization. The hybridized fruit fly culture tubes were placed in an incubator at 25°C with a relative humidity of 60%, and set to a 12-hour light and 12-hour dark mode.

[0055] Table 2. Comparison with UAS-CG8630RNAi or W 1118 Hybrid fruit fly strains

[0056]

[0057] Example 2: Immunofluorescence of male accessory glands in Drosophila

[0058] The immunofluorescence assay of male accessory glands in Drosophila is performed through the following steps:

[0059] (1) Place the fruit fly larvae and pupae to be dissected in 1×PBS buffer, and use tweezers to divide them into two parts from the middle, and simply remove excess tissue such as fat and intestines;

[0060] (2) Place the male reproductive system of Drosophila (including testes, ejaculatory ducts and accessory glands) into 500 μL of 4% paraformaldehyde general-purpose tissue fixative and fix it on a shaker at room temperature for 40-50 min.

[0061] (3) Remove the fixative, add 1 mL of 0.3% PBT solution and wash 3 times, 5-10 min each time;

[0062] (4) After removing the PBT solution, add 200 μL of blocking solution, place on a shaker, and treat at room temperature for 90 min;

[0063] (5) After removing the blocking solution, add 200 μL of primary antibody dilution solution (diluted at 1:200, primary antibody information is shown in Table 3), place on a shaker, and incubate overnight at 4°C;

[0064] (6) Remove the primary antibody diluent, add 1 mL of 0.3% PBT solution and wash 3 times, 5-10 min each time;

[0065] (7) After removing the PBT solution, add 90 μL of secondary antibody dilution buffer (diluted at 1:500 using blocking buffer; secondary antibody information is shown in Table 3) and 10 μL of LDAPI (nuclear fluorescent dye, purchased from Beyotime Biotechnology Co., Ltd.), place on a shaker, and incubate at room temperature in the dark for 3 h.

[0066] (8) Remove the secondary antibody dilution solution, add 1 mL of 1×PBS buffer and wash 3 times, 5-10 min each time;

[0067] (9) After cleaning, the male reproductive system was placed in 1×PBS buffer for dissection. The accessory glands were removed with forceps and placed on a glass slide. The buffer around the accessory glands was absorbed with filter paper. A small amount of anti-fluorescence quenching agent was added, and a coverslip was placed on top. The location of each accessory gland was marked on the back with a marker.

[0068] (10) Observe and collect images under a confocal fluorescence microscope;

[0069] (11) ImageJ was used to count the fluorescence intensity of different signals in the accessory glands, and GraphPad was used to draw a bar chart. The difference of the data was analyzed by t test.

[0070] Table 3 Antibody Information

[0071]

[0072]

[0073] Example 3: Differentially Expressed Gene Analysis

[0074] Using QIAGEN Plus Micro kit was used to extract RNA from the accessory glands of Drosophila. Prd-Gal4>W on day 5 post-emergence was also extracted. 1118 RNA from male accessory glands and Prd-Gal4>UAS-CG8630RNAi was extracted and then subjected to transcriptome sequencing and analysis (sequencing and data quality control were performed by Beijing Novogene Co., Ltd.). Differential expression analysis was performed using DESeq2. The screening criteria for differentially expressed genes were: corrected padj < 0.05 and |log2(fold change)| > 1. Genes meeting these criteria were considered differentially expressed. According to the results, for the CG8630 gene, log2FoldChange = -3.7, pvalue = 3.57E-68, and padj = 2.09E-66. This indicates that the expression of the CG8630 gene was significantly downregulated in the knockdown group compared to the control group, meaning the CG8630 gene was successfully knocked down.

[0075] Example 4: Loss of CG8630 function leads to developmental defects in accessory glands and lipid accumulation in male fruit flies.

[0076] 4.1 CG8630 specifically regulates the development of accessory glands

[0077] 24 hours after hybridization, Prd-Gal4>W 1118Drosophila from the control group and the Prd-Gal4>UAS-CG8630-IR knockdown group were transferred to ovipositors containing grape juice-yeast agar plates (with an active yeast suspension pre-spread evenly on the grape juice plate) to collect eggs laid by the female flies. The ovipositors were continuously cultured under standard culture conditions.

[0078] 24 hours after placing the oviposition tube, when most of the larvae have hatched on the grape juice-yeast plate, remove the grape juice-yeast plate and place a new plate into the oviposition tube. Return the plate to the incubator for continued culture to collect newly produced eggs.

[0079] Collect the newly hatched Prd-Gal4>W from the extracted grape juice-yeast plate. 1118 First-instar larvae of Prd-Gal4>UAS-CG8630-IR were cultured in small, transparent glass tubes containing 30 larvae each, at the same time as Prd-Gal4>UAS-CG8630-IR, ensuring that at least 20 larvae simultaneously entered the pupal stage. To accurately observe the function of CG8630 in accessory gland development, individuals were dissected and analyzed at 60 hours post-pupa and subsequent time points. GraphPad software was used for statistical analysis and visualization of the experimental data, comparing the differences in accessory gland development between the control group and the CG8630-IR group.

[0080] On the 5th day after emergence, the reproductive systems of male fruit flies in the CG8630 knockdown group and the control group were dissected and observed. Figure 1 As shown in Figure A, after CG8630 knockdown, there were no significant differences in the morphology of the testes, ejaculatory ducts, and testes of male fruit flies. However, the accessory glands exhibited specific developmental defects, with a significantly reduced volume, only about one-third the size of the control group. To further quantify the effect of CG8630 knockdown on accessory gland morphology, the length, width, and area of ​​the accessory glands were measured and statistically analyzed. The results showed that compared with the control group, the length of the accessory glands in the knockdown group of male fruit flies was significantly reduced (…). Figure 1 B, the control group was approximately 406.2±7.271 μm, and the knockdown group was approximately 270.2±5.271 μm, P<0.0001). Simultaneously, the width of the accessory gland also showed a similar significant decrease. Figure 1 C, the control group was approximately 103.9±3.254μm, and the knockdown group was approximately 48.17±1.863μm (P<0.0001). Considering the combined effects of length and width, the total area of ​​the accessory gland was significantly reduced. Figure 1 D, control group approximately 33766±736.8μm 2 The knockdown group is approximately 10628±413 μm. 2The proportion of the control group was only about 31% (P<0.0001). This indicates that the CG8630 gene has a tissue-specific regulatory effect on the development of accessory glands in male fruit flies. The loss of its function leads to severe morphological disorders of the accessory glands, which in turn affects the reproductive function of male fruit flies. However, the development of other components of the male reproductive system, such as the testes, ejaculatory ducts and testes, was not significantly affected.

[0081] To further clarify the temporal role of CG8630 in accessory gland development, fluorescent staining observations were performed on the accessory glands of Drosophila in the knockdown group and control group at different developmental stages. Key time points were selected, including 60 hours and 80 hours of pupal development, 1 day after the end of pupal development (1 day after emergence), and 3 and 5 days after emergence, to create a complete atlas of accessory gland development. Figure 2 (Red indicates Actin staining, blue indicates DAPI nuclear staining), clearly showing the dynamic process of accessory gland development. Results showed that at 60 hours into the pupal stage, there was a significant difference in accessory gland size between the two groups. The volume of the accessory glands in the knockdown group was significantly smaller than that in the control group, and this difference persisted and became increasingly pronounced during subsequent development. From a developmental dynamics perspective, the accessory glands in the control group continued to enlarge after emergence, exhibiting a typical curved tubular structure by days 3 and 5, with neatly arranged cells, and Actin staining revealed a clear cytoskeleton. In contrast, although the accessory glands in the knockdown group also grew over time, their development rate was significantly reduced, resulting in a more elongated final shape, lacking the typical curvature and volume of the control group's accessory glands. Furthermore, Actin staining patterns revealed certain abnormalities in the cell arrangement and tissue structure of the accessory glands in the knockdown group, suggesting that CG8630 not only affects the overall size of the accessory glands but may also participate in regulating cell spatial arrangement and tissue structure establishment.

[0082] 4.2 CG8630 regulates the maturation process of accessory gland cells by coordinating membrane integrity and maintaining nuclear polarity.

[0083] To further investigate the effect of CG8630 knockdown on the structure of accessory gland cells, UAS-MCD8-GFP was used to label the cell membrane (via UAS-CG8630 RNAi line / W). 1118 (Hybridized with UAS-MCD8-GFP; Prd-Gal4 strains), and the cell nucleus was labeled with DAPI to observe changes in cell membrane and nuclear polarity. The effects of CG8630 knockdown on accessory gland chief cells and minor cells were as follows: Figure 3 As shown, knocking down CG8630 resulted in cell membrane fragmentation and irregular arrangement. Figure 3 A); Area of ​​accessory gland chief cells in the CG8630 knockdown group ( Figure 3 B) and secondary cell area ( Figure 3 C) All decreased significantly, from 115±10.54μm 2and 368±20.47μm 2 It decreased to 68.47±3.15μm 2 and 97.34±4.02μm 2 (n=18, p<0.0001). Effects of CG8630 knockdown on accessory gland cell morphology and tissue structure, as follows: Figure 4 As shown, the nuclei of the knockdown group of accessory gland cells exhibited characteristics of loss of polarity, such as fusion and displacement. Figure 4 A). Combined with three-dimensional confocal imaging, a cross-sectional image of the accessory gland was obtained. Figure 4 B) It can be seen that the CG8630 knockdown group cells could not complete the normal polarity remodeling and morphological "flattening" process, suggesting that this gene is involved in the spatial remodeling of cell structure and is a necessary regulatory factor for the maturation of accessory gland tissue. Figure 4 C further visually demonstrates the differences in this cell flattening process.

[0084] These results indicate that CG8630 plays a crucial role in the maturation process of accessory gland cells by coordinating the regulation of cell membrane integrity and nuclear polarity establishment. Knockdown of CG8630 leads to multiple defects in accessory gland cells: cell membrane structure disruption, reduced cell area, impaired binuclear polarization, and disruption of cell flattening.

[0085] 4.3CG8630 maintains lipid droplet homeostasis

[0086] To investigate the role of CG8630 in accessory gland lipid homeostasis, Nile Red staining of lipid droplets was combined with MCD8-GFP secondary cell labeling for imaging. Results are as follows: Figure 5 As shown, the number of lipid droplets in the accessory gland tissue of the CG8630 knockdown group was significantly increased. Figure 5 A) At every 100μm 2 The number of lipid droplets in the region was significantly higher than that in the control group (p<0.01). Figure 5 B), but the area of ​​individual lipid droplets did not change significantly. Figure 5 (C) This dense accumulation of lipid droplets suggests severe disruption of lipid metabolism in the accessory gland tissue, possibly due to impaired fatty acid processing caused by the inactivation of the desaturase CG8630. Given that excessive accumulation of lipid droplets is often associated with cellular stress and apoptosis signals, these results further support the close relationship between CG8630 and the metabolic health and functional stability of the accessory gland.

[0087] Experimental Case 5: Loss of CG8630 function leads to decreased fertility

[0088] 5.1 CG8630 knockdown leads to decreased reproductive capacity in male fruit flies.

[0089] To assess the reproductive capacity of male fruit flies, this study employed a standardized pairing experiment. In the experiment, male fruit flies from the knockdown group and the control group were paired with virgin W... 1118 Female fruit flies mated one-on-one, and males were removed immediately after mating. Fertilized females were then transferred to culture flasks containing fresh culture medium for fecundity and hatchability measurements. Specifically, fecundity and hatchability were measured over 10 days, with the culture flasks changed every 24 hours, and the number of eggs laid and offspring survival recorded. To compare fecundity and hatchability between the knockdown group and the control group, the Mann-Whitney U test was used for statistical analysis. All experiments were repeated three times, with at least 10 biological replicates per group.

[0090] The results of the egg-laying rate of mated female flies are as follows Figure 6 As shown in Figure A, compared to the control group, female flies mated with CG8630 knockdown males laid significantly fewer eggs, a trend that persisted throughout the experiment. The percentage of successfully hatched embryos is shown in Figure A. Figure 6 As shown in Figure B, the hatching rate of these eggs was also significantly lower than that of the control group, indicating that CG8630 knockdown not only affects the oviposition ability of male fruit fly mates but also negatively impacts the embryonic development of their offspring. Therefore, normal expression of CG8630 is crucial for the reproductive capacity of male fruit flies, and its knockdown significantly weakens their fertility.

[0091] 5.2 CG8630 knockdown leads to impaired accessory gland secretory activity.

[0092] To assess whether CG8630 knockdown affects accessory gland secretory activity, the accessory glands of Drosophila in the control and CG8630 knockdown groups were immersed in anhydrous ethanol for 10 minutes, and the protein flocculent matter was measured and the total secretory protein content was quantified. The results are as follows: Figure 7 As shown, significant differences in secretions were observed between the control group and the knockdown group. Figure 7 A) Quantitative analysis showed that the accessory gland secretions in the CG8630 knockdown group were significantly reduced, and the total protein content was only about 30% of that in the control group. Figure 7 B). TEM microscopy revealed a significant loss of filamentous secretory structures within the accessory gland cavity. Figure 7 C). Using the UAS-Syt1-GFP;Prd-Gal4 reporter system (via UAS-CG8630RNAi strain / W 1118Visualization of secretory vesicles was performed using hybridization with the UAS-Syt1-GFP and Prd-Gal4 strains. Synaptotagmin-1 (Syt1) is a calcium-sensing protein associated with secretory vesicles and is commonly used as a marker for exocytosis. Results are as follows: Figure 8 As shown, fluorescence microscopy and TEM microscopy observations revealed a sharp decrease in the number of Syt1-GFP-labeled secretory vesicles. Figure 8 A), while the apical microvilli structure is sparse and short ( Figure 8 (B) These changes all indicate that the absence of CG8630 affects the formation of secretory vesicles and the structure of the exocytosis apparatus. This defect directly leads to the ineffective transfer of sperm protein (Acp) into the female body, fundamentally impairing male reproductive potential.

[0093] 5.3 CG8630 knockdown affects sperm migration rate but not sperm count or morphology.

[0094] Sperm were specifically labeled using UAS-Protamin-GFP (via UAS-CG8630RNAi strain / W) 1118 The sperm count and morphological characteristics were compared between the control group and the CG8630-IR group (hybridized with UAS-Protamin-GFP and Prd-Gal4 lines). Fluorescence microscopy results are shown below. Figure 9 As shown in Figure A, both groups of sperm exhibited typical filamentous structures, and there were no significant differences in quantity and morphology, indicating that CG8630 knockdown does not affect sperm production and development.

[0095] The distribution of sperm in the female reproductive system was examined 2 hours post-mating (2h ASM), and the results are as follows: Figure 9 As shown in Figure B, sperm from both the control group and the CG8630-IR group successfully migrated into the female reproductive tract, indicating that CG8630 knockdown does not hinder sperm migration. However, a significant difference was observed when sperm migrated to the seminal vesicles: a large number of sperm had accumulated in the seminal vesicles of the control group females. Figure 9 (See Figure B above, indicated by the red arrow), while the sperm count in the seminal vesicles of females in the CG8630-IR group was significantly reduced. Figure 9 (See diagram B below, indicated by the red arrow).

[0096] This indicates that although CG8630 knockdown does not affect sperm production or morphological development, nor does it hinder sperm migration into the female reproductive tract, it significantly reduces the sperm migration rate within the female reproductive tract, preventing sperm from reaching the seminal vesicles within the normal timeframe. This reduced migration rate may be one of the key factors contributing to the decreased fertility in CG8630-knocked male fruit flies. This further corroborates the regulatory role of accessory gland secretions on sperm function, suggesting that CG8630 may affect fertilization efficiency and reproductive success by influencing the composition of accessory gland secretory proteins, thereby regulating sperm migration behavior within the female reproductive tract.

[0097] Experimental Example 6: CG8630 deficiency affects accessory gland function by activating Caspase-3-dependent apoptosis.

[0098] 6.1 CG8630 knockdown induces early apoptosis activation in accessory gland development

[0099] Caspase3 expression was detected by immunofluorescence staining, and the results are as follows: Figure 10 A showed that Caspase 3 signaling was significantly enhanced in accessory gland cells of the CG8630 knockdown group, which was 3.65 times that of the control group (n=10, p<0.0001). Figure 10 B) indicates that the cells have entered a state of programmed cell death. This suggests the important role of CG8630 in maintaining the normal development of accessory gland tissue, and its loss of function may be caused by activation of the apoptosis pathway.

[0100] The developmental abnormality was subsequently investigated by co-expression of the anti-apoptotic factor Diap1, which revealed complete disappearance of Caspase3 expression and restoration of normal accessory gland morphology and area. These results confirm that CG8630 plays a crucial role in maintaining accessory gland survival signals, and its deficiency induces apoptosis by regulating Caspase3 activity, representing an important molecular mechanism of accessory gland dysfunction.

[0101] 6.2 Diap1 overexpression can rescue the apoptosis phenotype induced by CG8630 deficiency.

[0102] Whether overexpression of the anti-apoptotic factor diap1 (Death-associated inhibitor of apoptosis 1) can rescue accessory gland defects caused by CG8630 knockdown was investigated. In a CG8630 knockdown genetic background, a Drosophila strain co-expressing diap1 (UAS-CG8630RNAi; UAS-Diap-OE) was constructed, and its accessory gland morphology and apoptosis were systematically analyzed. Morphological observation ( Figure 11 A) It is evident that diap1 overexpression can effectively restore the accessory gland shrinkage phenotype induced by CG8630 knockdown, bringing its volume close to that of the control group. Furthermore, Figure 11Quantitative statistical analysis of the accessory gland area in sample B further validated this conclusion. Data showed no significant difference in accessory gland area between the diap1 overexpression group and the control group (P>0.05), indicating that diap1 overexpression can effectively rescue accessory gland developmental abnormalities caused by CG8630 knockdown. Further analysis was performed using anti-activated Caspase-3 immunostaining to detect apoptosis levels. Figure 11 C), the results showed that under the background of diap1 overexpression, apoptosis signals almost completely disappeared, reaching the level of the control group, indicating that diap1 overexpression can effectively inhibit CG8630 knockdown-induced apoptosis.

[0103] Experimental Example 7: Developing a biological control strategy for male reproductive defects regulated by CG8630

[0104] 7.1 Evaluation of the competitive advantage of CG8630 knockdown in male sperm

[0105] To assess the impact of CG8630 expression in male accessory glands on sperm competitiveness in a population, this study designed a multiple mating experiment with female Drosophila to compare the reproductive success rates of CG8630 knockdown groups and wild-type males under different mating sequences. Three groups were set up: in the first group, females mated with two wild-type males sequentially (WT / WT); in the second group, females mated first with a wild-type male and then with a CG8630-IR male (WT / CG8630-IR); and in the third group, females mated first with a CG8630-IR male and then with a wild-type male (CG8630-IR / WT). To compare the differences in egg production and hatching rates among different mating combinations, one-way ANOVA combined with Tukey's post-hoc test was used for statistical analysis among multiple groups. When the data did not conform to the normality assumption, the nonparametric Kruskal-Wallis test combined with Dunn's post-hoc test was used. All experiments were repeated three times, with each group containing at least 10 biological replicates. Data are expressed as mean ± standard error, and P < 0.05 was considered statistically significant.

[0106] The effect of mating order on female egg production results are as follows: Figure 12 As shown in Figure A, females exhibited the highest egg production when wild-type males mated first (WT / WT); egg production decreased when wild-type males mated first followed by CG8630-IR males (WT / CG8630-IR); and the lowest egg production was observed when CG8630-IR males mated first followed by wild-type males (CG8630-IR / WT). The effect of mating order on hatching rate is as follows. Figure 12 As shown in B, the WT / WT group performed best, followed by the WT / CG8630-IR group, and the CG8630-IR / WT group performed worst. Mating order significantly affected both female egg production and hatching rate.

[0107] These results indicate that CG8630 expression in male accessory glands significantly impacts mating competitiveness in Drosophila. Knockdown of CG8630 via RNA interference significantly reduced reproductive success in male Drosophila under multiple mating conditions, regardless of whether they mated first or last. Notably, when CG8630-IR males acted as the first mating partner, their reproductive contribution was significantly impaired even after subsequent mating with wild-type males, suggesting that CG8630 may play a crucial role in sperm competition and / or sperm migration.

[0108] 7.2 Assessment of Male Mating Competitiveness and Population Suppression Effect

[0109] To evaluate the effects of CG860-IR on the mating competitiveness and population suppression of male fruit flies, and to explore its feasibility as a novel biological control strategy, this experiment employed a simulated ecosystem approach and incorporated the principles of SIT (Self-Induced Influence) technology to systematically assess the role of CG8630-IR male flies in population dynamics.

[0110] (1) Establishment of experimental population

[0111] The experiment used wild-type fruit flies (W 1118 Transgenic fruit flies, including CG8630-IR, were used. Five-day-old virgin female and male flies were collected before the experiment to ensure that the experimental individuals were sexually mature but had not yet mated.

[0112] (2) Simulated ecosystem construction

[0113] Transparent plexiglass cages (5 cm in diameter and 10 cm in height) were used as population containers in the experiment. A standard culture medium was placed at the bottom of the container for fruit flies to lay eggs and for larval development. A breathable sponge plug was placed at the top of the container to ensure ventilation and prevent fruit flies from escaping. Experimental conditions were maintained at 25±1℃, relative humidity 60±5%, and a light / dark cycle of 12h light / 12h dark.

[0114] (3) Experimental group design

[0115] To evaluate the effects of different release ratios of CG8630-IR male flies on population reproduction, four experimental groups were set up, with a total of 60 fruit flies in each group and three biological replicates in each experimental group:

[0116] Knockdown group 1 (high proportion group): 25 male CG8630-IR flies, 5 wild-type male flies, and 30 wild-type female flies (5:1:6).

[0117] Knockdown group 2 (equal proportion group): 15 male CG8630-IR flies, 15 wild-type male flies, and 30 wild-type female flies (3:3:6).

[0118] Control group 1 (pure wild type group): 0 male CG8630-IR flies, 30 wild type male flies, and 30 wild type female flies (0:6:6).

[0119] Control group 2 (pure CG8630-IR group): 30 male CG8630-IR flies, 0 wild-type male flies, and 30 wild-type female flies (6:0:6).

[0120] (4) Evaluation of mating competitiveness and population suppression effect

[0121] For the F0 generation (parents), the rearing cages were placed in an environment of 25℃ and 60% humidity, allowing free mating for 48 hours. One hundred eggs were randomly selected and transferred to fresh culture medium, incubated at 25℃ for 5 days, and the number of hatched larvae was counted to calculate the hatching rate and obtain the Fried competitiveness index. After 8 more days of free mating, the F0 generation fruit flies were removed, and the total number of F1 generation fruit flies was counted after 10 days to evaluate the population suppression effect.

[0122]

[0123] The mating competitiveness of male flies of CG8630-IR was assessed by calculating the Fried Competitiveness Index (C), as follows:

[0124]

[0125] Wherein, Ha is the hatching rate of pure wild type (control group 1), Ee is the hatching rate of mixed population (knockdown groups 1 and 2), Hs is the hatching rate of pure CG8630-IR (control group 2), N is the number of wild type male flies, and S is the number of CG8630-IR male flies.

[0126] (5) Data Analysis Methods

[0127] Experimental data included key indicators such as egg production and hatching rate. Statistical analysis methods employed ANOVA combined with Tukey HSD post-hoc tests to assess differences between experimental groups.

[0128] 7.2.1 Analysis of mating competitiveness of CG8630 knockdown male fruit flies and its application value in SIT control

[0129] To evaluate the application potential of CG8630-IR male Drosophila in sterile male insemination technique (SIT), we systematically measured its mating competitiveness at different release ratios. The results are shown in Table 3. With increasing CG8630-IR male ratio, the observed hatching rate after mating showed a significant decreasing trend, decreasing from 74.85±2.65% in the control group (0:6:6) to 34.56±4.09% in the pure CG8630-IR group (6:0:6).

[0130] The competitiveness coefficient (C) calculated using the Fried formula shows that the low-ratio release group (3:3:6) has a competitiveness of 0.32, exceeding the minimum application standard and maintaining a sufficient competitive advantage; while the high-ratio release group (5:1:6) reaches 0.55, which meets the good standard for practical application of SIT technology. This indicates that the strategy of targeting the CG8630 gene based on RNAi technology can effectively reduce fruit fly reproductive capacity while maintaining appropriate field competitiveness.

[0131] Table 3. Effects of CG8630 knockdown on mating competitiveness in male fruit flies.

[0132]

[0133]

[0134] Note: C≥0.3: Has practical application value; C≥0.5: Has a good competitive level and is suitable for large-scale field application.

[0135] 7.2.2 Evaluation of the population suppression effect of CG8630 knockdown on male fruit flies

[0136] To further verify the practical application effect of CG8630-IR male fruit flies in population control, this study designed experiments involving the mixed release of CG8630-IR males and wild-type males at different ratios, and statistically analyzed the number of offspring as a direct indicator of population suppression effect. The results are as follows: Figure 13 As shown, with the increase in the proportion of male fruit flies in CG8630-IR, the number of offspring showed a significant downward trend.

[0137] In the control group (0:6:6), the average number of offspring was approximately 280, while in the low-ratio release group (3:3:6), the number of offspring decreased slightly to approximately 255, but this difference did not reach a statistically significant level (ns). However, when the male ratio of CG8630-IR was further increased to the high-ratio release group (5:1:6), the number of offspring significantly decreased to approximately 185 (p<0.05), a reduction of approximately 34% compared to the control group.

[0138] This result is highly consistent with the analysis of competitiveness coefficient and hatching rate in the previous section, further confirming that CG8630-IR male fruit flies have a certain mating competition ability and can effectively reduce the population reproduction rate by inhibiting CG8630 gene expression. Particularly noteworthy is the significant population suppression effect exhibited by the high-proportion release group (5:1:6), indicating that when CG8630-IR males account for a high proportion (>80%) of the total males, they can effectively interfere with the normal reproductive process of wild-type females, thereby achieving the goal of controlling population growth.

[0139] In summary, this invention uses *Drosophila melanogaster* as a model organism and employs RNAi technology to specifically knock down the expression of the CG8630 gene. The results demonstrate that loss of function of this gene leads to defective accessory gland development, lipid accumulation, and decreased reproductive capacity in male *Drosophila*. Furthermore, it was verified that CG8630 knockdown in male *Drosophila* has a suppressive effect on population size. This provides experimental evidence for the practical application of SIT technology (based on RNAi targeting the CG8630 gene) in *Drosophila* population control.

[0140] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The application of the CG8630 gene in regulating the development of accessory glands in Drosophila, characterized by, The nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1 or the encoded amino acid sequence is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, Lowering the expression of the CG8630 gene in the Drosophila accessory glands resulted in developmental defects in the Drosophila accessory glands.

3. The application as described in claim 2, characterized in that, The developmental defects include reduced volume of the accessory glands, damage to the cell membrane structure of the accessory glands, reduced area of ​​the accessory gland cells, and obstruction of the maturation process of the accessory gland cells.

4. The application of the CG8630 gene in regulating reproductive function in Drosophila, characterized by, The nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1 or the encoded amino acid sequence is shown in SEQ ID NO.

2.

5. The application as described in claim 4, characterized in that, Decreased expression of the CG8630 gene in the accessory glands of Drosophila led to a decline in the reproductive function of the fruit flies.

6. The application as described in claim 5, characterized in that, The decline in reproductive function includes a decrease in the number of eggs laid by mating female flies, a lower hatching rate after egg laying, and an increased mortality rate during embryonic development.

7. The application as described in claim 5, characterized in that, The decline in reproductive function is caused by a decrease in the secretory function of accessory glands and a decrease in the migration rate of sperm within the female reproductive tract.

8. A biological control method for suppressing fruit fly populations by targeting the CG8630 gene, characterized in that, The nucleotide sequence of the CG8630 gene is shown in SEQ ID NO.1 or the encoded amino acid sequence is shown in SEQ ID NO.

2. The targeting of the CG8630 gene is to reduce the expression of the CG8630 gene in the accessory glands of male flies, and then artificially release male flies to suppress the fruit fly population.

9. The method as described in claim 8, characterized in that, The method for reducing the expression of the CG8630 gene in the accessory glands of Drosophila includes the following steps: based on the GAL4 / UAS system, UAS-CG8630-RNAi Drosophila is crossed with Prd-Gal4 Drosophila to obtain UAS-CG8630-RNAi;Prd-Gal4 Drosophila.

10. A fruit fly strain, characterized in that, It was obtained by crossing UAS-CG8630-RNAi Drosophila with Prd-Gal4 Drosophila.

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