Analysis of genetic diversity and structure of endangered Dengchuan cattle population using a single-nucleotide polymorphism chip

Abstract

This study aimed to evaluate the genetic diversity and structure within the Dengchuan cattle population and effectively protect and utilize their germplasm resources. Herein, the single-nucleotide polymorphisms (SNPs) of 100 Dengchuan cattle (46 bulls and 54 cows) were determined using the GGP Bovine 100K SNP Beadchip. The results showed that among the Dengchuan cattle, a total of 101,220 SNPs were detected, and there were 83,534 SNPs that passed quality control, of which 85.7% were polymorphic. The average genetic distance based on identity-by-state (IBS) within the conservation population of Dengchuan cattle was 0.26 ± 0.02. A total of 3,999 genome-length runs of homozygosity (ROHs) were detected in the Dengchuan cattle, with ROH lengths primarily concentrated in the range of 1–5 Mb, accounting for 87.02% of the total. The average inbreeding coefficient based on ROHs was 4.6%, within the conservation population of Dengchuan cattle, whereas it was 4.9% for bulls, and the Wright inbreeding coefficient (F_IS) value was 2.4%, demonstrating a low level of inbreeding within the Dengchuan cattle population. Based on neighbor-joining tree analysis, the Dengchuan cattle could be divided into 16 families. In summary, the conservation population of Dengchuan cattle displays relatively abundant diversity and a moderate genetic relationship. Inbreeding was observed among a few individuals, but the overall inbreeding level of the population remained low. It is important to maintain this low level of inbreeding when introducing purebred bloodlines to expand the core group. This approach will ensure the long-term conservation of Dengchuan cattle germplasm resources and prevent loss of genetic diversity.

Keywords:

• Dengchuan cattle
• genetic diversity
• population structure
• ROH
• SNP chip

Introduction

China, with its vast territory and rich history of animal husbandry development, has various cattle breeds. Among these, Dengchuan cattle are a local breed found in Yunnan Province and are mainly distributed in Eryuan County. They were first raised in the Dengchuan area, after which they were named. Dengchuan cattle serve the dual purpose of milk and meat production. The 305-day yield and lactation length of the animals in the herd were 872 kg, 243 days, respectively. Dengchuan milk has high-nutrient content, boasting an average fat content of 6.89% and a protein content exceeding 4%,¹ making it an excellent raw material for producing dairy products. Moreover, this breed exhibits strong adaptability, stress and disease resistance and drought and roughage tolerance.² To cultivate dairy cattle varieties with good lactation performance to meet the demands of milk production, Holstein cattle have been introduced in Eryuan County for cross-breeding and to improve Dengchuan cattle since 1954. This initiative has led to the development of an improved population known as Dengchuan black and white dairy cattle, substantially contributing to local economic growth. The increase in the resulting improved population of Dengchuan black and white dairy cattle has substantially promoted local economic development. However, although this cross-breeding effort has provided economic benefits, it has led to a reduction in the numbers of the original Dengchuan cattle breed, even to the point of endangerment.³ Owing to the increasing attention paid to protecting and utilizing native cattle germplasm resources in China, Dengchuan cattle were included in the National Catalogue of Livestock and Poultry Genetic Resources.⁴ As of 2019, only 212 Dengchuan cattle existed in Dali Prefecture.¹ Due to the lack of detailed breeding pedigree records, the inevitable inbreeding in the population will lead to an imbalanced population structure and create severe and complex challenges for Dengchuan cattle in terms of survival and breeding. Thus, protecting the unique germplasm resources of Dengchuan cattle is crucial.

The differences in DNA sequences among individuals in a specific species form the genetic diversity of that species, which is crucial to its survival, evolution and protection⁵ Genetic markers can be used to infer relationships among individuals within a species.⁶ Single-nucleotide polymorphisms (SNPs) are third-generation molecular markers with coverage and high genetic stability.⁷ Gene chips, also known as DNA chips or microarrays, have become pivotal tools in genomic research, facilitating genotyping and expression profile analysis.⁸ Gene chip technology is among the most prevalent methods for SNP detection, attracting considerable attention in many fields. In animal husbandry, gene chip technology is typically used for germplasm identification and population traceability, aiding the study of population genetic diversity, genetic relationships and family structures.^9–11 Such insights offer a theoretical reference for conservation and breeding efforts by research groups. Protecting the unique germplasm resources of Dengchuan cattle requires sufficient knowledge about the genetic diversity and population structure.¹² In this study, the genetic diversity and genetic structure within the Dengchuan cattle population were analysed at the molecular level using the GGP Bovine 100K Beadchip, laying a foundation for utilizing and preserving the resources of the original Dengchuan cattle population.

Materials and methods

Test animals and sampling

A sample of Dengchuan cattle was randomly selected from a total of 257 in October 2022. Hair follicles were collected from the selected cattle, including 68 individual samples (33 bulls and 35 cows) from Guipi Village, Dengchuan Town, Yunnan Province and 32 individual samples (13 bulls and 19 cows) from Jiaoshi Village, Yousuo Town. The samples were collected following specific principles: (1) purebred Dengchuan cattle were selected according to their appearance characteristics; (2) samples were fully harvested to meet the required sample size and gender balance. (3) Each sample contains 10-15 hair follicles. These collected samples were placed in 10-mL centrifuge tubes containing 95% alcohol and stored at −80 °C.

Test method

Extraction and quality determination of bovine hair follicle genomic DNA

The extraction steps mainly involved the following: (1) An appropriate amount of hair follicle sample was added into a 2.0-mL cracking tube, along with 20-μL Proteinase K and 300-μL Buffer WL, followed by vortex mixing for 30s and incubation in a 56 °C constant temperature water bath for 40 min, with oscillation every 20 min. (2) The samples were placed in a centrifuge for instantaneous centrifugation after cracking, put all supernatant into 96-well deep-well plate, Lysate, Buffer KL, and Magbeads PN solution were added. (3) Spin tips Pack was inserted into a 96-well deep-well plate for the DNA extraction program and gDNA extraction. DNA integrity was determined using 0.8% agarose gel electrophoresis, while purity (OD_260 nm/OD_280 nm) and concentration were determined using ultraviolet spectrophotometry (NanoDrop 2000).

SNP typing and quality control

Samples from the 100 individuals were genotyped using the GGP Bovine 100K Beadchip (Illumina Inc., CA, USA). It contains 101,220 SNPs uniformly spanning the bovine genome with an average SNP spacing of about 34 kb. Plink (V1.90) software was used for sample and SNP locus quality control.¹³ Loci that did not meet the requirements were excluded according to the quality control standards: using autosomal loci, eliminating loci with an SNP call rate and individual detection rate DNA of < 90%, Hardy–Weinberg P-value of <10⁻⁶, and minimum allele frequency [MAF] of < 0.01).

Analysis of genetic diversity in the conserved population of Dengchuan cattle

The expected heterozygosity (He), observed heterozygosity (Ho), effective number of alleles, MAF and polymorphism information content (PIC) of the cattle were calculated using Plink (V1.90) software. The effective population size (Ne) of Dengchuan cattle was estimated by calculating r² values between pairs of SNPs using the SNeP (V1.1) software.¹⁴ The results are presented as mean ± SD.

Genetic relationship analysis of the conservation population of Dengchuan cattle

GCTA (V1.94) software was used to calculate the genetic coefficient of the Dengchuan cattle and construct a G-matrix. Plink (V1.90) software was used to calculate the genetic distance between Dengchuan cattle individuals and the runs of homozygosity (ROH) length of each sample and to construct an identity-by-state (IBS) matrix. R software was used to visualize the results of the kinship and genetic distance analyses. By counting the continuous homozygous segments of each Dengchuan cattle, the inbreeding coefficient value (F_ROH) based on ROH was obtained. Finally, R software was used to create an F_ROH violin diagram. The Wright inbreeding coefficient was estimated as $F_{\mathit{\text{IS}}}=1-\frac{H_O}{H_E}$^.15

Pedigree structure analysis of the conservation population of Dengchuan cattle

Cluster analysis based on genetic distance was conducted using the neighbour-joining (NJ) method, employing Mega X (V10.0) software to determine the genetic relationship between Dengchuan cattle individuals.

Results

SNP quality control and typing

The genome-wide SNPs of Dengchuan cattle were detected using the SNP chip, and the average call rate was 98.64%. After quality control, 83,534 SNPs were used to analyse genetic diversity, genetic relationships, family structure, and inbreeding levels in Dengchuan cattle (Table 1). Before and after quality control, the SNP data on the 29 autosomes showed that the highest number of SNPs (377 in total) removed were from chromosome 2. On chromosome 3, 369 SNPs were eliminated. The fewest SNPs (111) were removed from chromosome 27 (Figure 1).

Figure 1. Number of SNPs on autosomes before and after quality control.

Table 1. Statistical results of SNP quality control.

Quality control standard	Number of SNPs
Total number of SNPs	101220
SNP with MAF <0.01	5195
SNP not in Hardy-Weinberg equilibrium P < 10^-6	271
SNP with call rate < 0.90	1228
SNPs on chromosome X	3785
SNPs on chromosome Y	330
SNPs on chromosome 0	6267
Insertion / deletion	172
SNPs used after quality control	83534

Genetic diversity analysis of the conservation population of Dengchuan cattle

The results of the genetic diversity analysis of the conservation population of Dengchuan cattle are shown in Table 2. Most of the MAF values were between 0 and 0.1, accounting for 26.39% of the total, while the lowest proportion ranged from 0.4 to 0.5, representing 14.78% (Figure 2(a)). The MAF of the conservation population of Dengchuan cattle was 0.22 ± 0.15. Figure 2(b) shows the proportion of PIC, ranging from 0 to 0.38, with an average of 0.24 ± 0.12. Furthermore, the analysis of heterozygosity in Dengchuan cattle showed that Ho (0.31) was slightly lower than He (0.32), but that both were similar (Figure 2(c)).

Figure 2. Genetic diversity of Dengchuan cattle. (a) Proportion of minimum allele frequency. (b) Proportion of polymorphic information content. (c) Graph of heterozygosity analysis.

Table 2. Results of population genetic diversity analysis.

Index	Value
Effective Population Content (Ne)	2.6
Proportion of Polymorphic Markers (PN)	0.857
Expected Heterozygosity (HE)	0.321
Heterozygosity Observed (HO)	0.313
Polymorphism Information Content (PIC)	0.244
Effective Numbers of Alleles	1.459
Minor Allele Frequency (MAF)	0.221

IBS distance matrix analysis of the conservation population of Dengchuan cattle

The IBS distance within the conservation population of Dengchuan cattle was calculated, revealing that the IBS distance between individuals ranged from 0.11 to 0.35, with an average of 0.26 ± 0.02. The genetic distance among the 46 breeding bulls ranged from 0.11 to 0.31, with an average of 0.26 ± 0.02, indicating relatively close genetic distances among some individuals was relatively close. Visualization results of the IBS distance matrix showed that only some Dengchuan cattle individuals had a short IBS genetic distance. The IBS genetic distance of most individuals was large, with great variability, reflecting substantial variability and a moderate degree of kinship (Figure 3).

Figure 3. Visualization results of the IBS distance matrix. This figure represents the genetic distance between two individuals. The closer the colour shifts toward green, the closer the genetic relationship between two individuals. The horizontal and vertical coordinates represent individual identity documents.

G-matrix analysis of the conservation population of Dengchuan cattle

To further evaluate the genetic relationships among Dengchuan cattle individuals within the conservation population, a G-matrix analysis was conducted, revealing that the kinship coefficients between individuals ranged from −0.11 to 0.71, with an average of −0.01 ± 0.09, indicating that most Dengchuan cattle individuals share a moderate to high degree of genetic relationship (Figure 4).

Figure 4. Visualization results of the G-matrix. This figure represents the kinship coefficient between two individuals. The closer the color is to purple, the closer the genetic relationship between two individuals.

Inbreeding coefficient analysis of the conservation population of Dengchuan cattle

ROH analysis showed that 3,999 ROHs were detected in this population, with an average of 39.99 ± 13.10 ROHs per individual. Individual ROH lengths ranged from 38.42 to 838.31 Mb, with an average of 151.47 ± 177.20 Mb. The number of ROHs per individual ranged from 16 to 78, with the majority falling within the range of 1–100 Mb, accounting for 64% of the total. The total ROH length of the three individuals (F45, F44 and F42) ranged from 800 to 900 Mb. The number of individuals with total ROH lengths between 600–700 Mb and 700–800 Mb was the least, each accounting for one individual (Figure 5(a)). The distribution of ROH length across the population is shown in Figure 5(b), with ROH lengths mainly concentrated in the 1–5 Mb range, representing 87.02% of the total. ROH lengths between 15 and 20 Mb were the least common, accounting for only 1.68% of the total. Analysis of ROH distribution across the 29 autosomes revealed 262, 251, and 44 ROHs on chromosomes 13, 8, and 27, respectively (Figure 5(c)). The F_ROH of the Dengchuan cattle genome was estimated based on ROH analysis. The results showed that the number of individuals with F_ROH values between 0.01 and 0.05 was the largest, and the average F_IS and F_ROH values were 2.4% and 4.6%, respectively. Among the 54 cows and 46 bulls, the F_ROH values of 3 cows and 1 bull exceeded 0.2, with the average inbreeding coefficient of bulls being 4.9% (Figure 5(d)).

Figure 5. ROH analysis results. (a) Sample distribution of individual ROH lengths (the x-coordinate represents the length intervals of ROH, while the y-coordinate represents the number of individuals). (b) ROH length distribution across the population (the x-coordinate represents the length intervals of ROH, while the y-coordinate represents the population proportion). (c) distribution of ROH quantity on each chromosome (the x-coordinate represents the chromosome number, while the y-coordinate represents the ROH number). (d) distribution map of the inbreeding coefficient value (F_ROH) based on ROH (the white dots represent the median value of F_ROH within the Dengchuan cattle population. The upper and lower quartiles are represented by the upper and lower edges of the black box.).

Family structure analysis of the conservation population of Dengchuan cattle

The samples were clustered based on the genetic distance matrix obtained from the genetic distance analysis. Using Mega X (V10.0) software, an NJ tree was constructed to evaluate the genetic relationships among Dengchuan bull individuals and their families. Following the criteria that the genetic kinship coefficient between bulls should be ≥ 0.1, the 46 bulls were divided into 16 families. Families 5–12 and 16 had smaller numbers, containing only 1 bull each (Figure 6(a)). Moreover, Figure 6(b) shows the cluster analysis results for all Dengchuan cattle samples. In this analysis, individuals with close genetic relationships between cows and bulls were grouped into one family. Additionally, owing to the close relationships among some bulls in Families 2 and 3, there were multiple cross-family samples. Specifically, the genetic coefficients of seven cows and all bulls in the group were < 0.1, indicating a distant genetic relationship. Consequently, these samples were classified into the ‘other’ group.

Figure 6. Cluster analysis results. (a) Cluster analysis results of male samples (each colour represents a family). (b) Cluster analysis results for all samples (bull samples are coloured in the evolutionary tree, with each colour representing a lineage).

Discussion

As a rare native cattle breed used for dairy and meat purposes, Dengchuan cattle have a certain historical and cultural value, playing an important economic role. The number of existing pure-bred Dengchuan cattle in Eryuan County is about 257, and the lack of related genetic pedigrees has remarkably increased the risk of inbreeding depression within the population. Although conservation measures have been adopted for Dengchuan cattle as of 2023, the family structure and genetic relationship remained understudied. To achieve this objective in the case of Dengchuan cattle, we analyzed the genetic diversity, genomic kinship, family structure and inbreeding levels of this breed based on whole-genome SNP data. The average call rate of Dengchuan cattle showed the high performance of genotyping (98.64%), indicating that the chip was suitable for analysing the genetic diversity and genetic structure of the SNP analysis of this population.

Genetic diversity analysis

Assessment of genetic diversity is an important task in species conservation. Compared with traditional pedigree data, SNP chip data can more accurately reflect the diversity parameter.¹⁶ Most studies use third-generation molecular markers techniques to understand the genetic diversity of the breeds.

Ne is an important parameter for evaluating population genetic diversity and can be estimated based on the level of linkage disequilibrium, even without detailed population pedigree information.¹⁷ Research has shown that the long-term viability of a population is closely related to its Ne. When the Ne value ranges from 50 to 100, the population has long-term viability.¹⁸ However, conversely, if the Ne value is below 50, the short-term survival of the population is seriously endangered.¹⁹ Ne value was low when a closed group with limited migration, mutation and selection during its evolutionary history.²⁰ According to previous studies, the accuracy of Ne value estimation based on linkage disequilibrium is significantly correlated with the sample size.²¹ Moreover, the small population size of an endangered population can result in a low Ne value.²² Thus, it can be inferred that Dengchuan cattle may be a closed group with limited migration and selection, and the low Ne value could be attributed to the breed’s small population size or the limited sample size used in this study. Additionally, Ne is an estimated value based on an ideal mathematical model, and biological evolution processes are more complex than such models. To obtain more accurate estimates, it is essential to have a more extensive and representative test population and continuously optimizes mathematical models.²³ Therefore, the Ne value estimated in this study for Dengchuan cattle is only a reference for breeding and conservation efforts. According to Wang,²⁴ conservation populations with a low Ne value and a higher rate of inbreeding may experience reduced genetic diversity. To ensure the long-term conservation of the germplasm resources of Dengchuan cattle and prevent the loss of genetic diversity, efforts should be directed toward implementing a reasonable breeding plan. In this regard, proportion of polymorphic markers is an essential parameter to consider. This study found that 85.7% of the SNPs were polymorphic. PIC value is often used to measure the informativeness of a genetic marker for linkage studies. Generally, a higher PIC value suggests increased genetic information within the population.²⁵ The present study found that the average PIC value for Dengchuan cattle was 0.24, indicating low polymorphism (PIC < 0.25).²⁶ The relatively limited genetic variation in the small Dengchuan cattle population may be due to two main factors: the small population size and a certain degree of inbreeding within the population.

Heterozygosity is a measure of genetic variability and is often used to assess the presence of genetic variation in threatened taxa.²⁷ Ho represents the proportion of individuals within the population that are heterozygous at a certain locus compared to the total number of individuals. Conversely, He reflects the probability of any individual in the population being heterozygous at any given locus. A high He value indicates that inbreeding may occur among individuals in the population, whereas a high Ho value indicates that bloodlines from outside the population may have been introduced.²⁸ In this study, the average Ho for Dengchuan cattle was 0.313, which is higher than that reported for Jersey cattle (0.30) but lower than those reported for Italian Holstein cattle (0.34) and Swiss brown cattle (0.32).²⁹ Additionally, the average He value for Dengchuan cattle was 0.32, which is higher than that reported for Maremmana semi-feral cattle (0.26)³⁰ but lower than that reported for Egyptian Baladi cattle (0.75).³¹ These values are also similar to those reported for other native cattle breeds in southern China, such as Dabieshan (0.33), Wenshan (0.33) and Zhaotong cattle (0.33).³² One possible reason for the higher average Ho and He values of Dengchuan cattle compared with Jersey cattle or other cosmopolitan breeds is that cosmopolitan breeds may be affected by high selection pressure, resulting in these genetic indicators being lower than native breeds. The average Ho for the conservation population of Dengchuan cattle was slightly lower than its average He, indicating that no bloodlines from outside the population had been introduced, which signifies a high degree of genetic purity. Dengchuan cattle had a high genetic diversity within this population; this is consistent with the research conclusion of Jin et al.³³

Analysis of genetic relationships and structure

Using NJ tree and cluster analysis, the cattle were categorized into 16 families. Ten families (Families 1, 5–12, and 16) had few bulls, creating an unbalanced structure. The unique germplasm resources in Dengchuan cattle serve as a theoretical foundation and basis for their protection. Xu et al.³⁴ used genome-wide variants to study the genetic structure of Yiling yellow, Bashan, Wuling, and Zaobei cattle breeds from Hubei Province, China. They found that Yiling yellow cattle not only distinguished themselves from breeds in neighboring regions but also formed an independent branch. Similarly, Quan et al.³⁵ studied the population genetic structure of 1,163 native Chinese cattle, providing a scientific basis for protecting and utilizing genetic resources within China’s indigenous cattle population. Furthermore, Kawaguchi et al.³⁶ used an Illumina 50K SNP array to study the genetic structure and relationships among four Japanese cattle breeds. They found that the genetic similarity between Japanese Black cattle and European breeds was only 3.5%. The aforementioned studies used cluster analysis to offer theoretical evidence in favour of the conservation of indigenous breeds.

The numbers of bulls and cows in some Dengchuan cattle families exhibit considerable disparities. This imbalance poses a high risk of bloodline loss. Moreover, operating within a closed breeding system without introducing purebred Dengchuan cattle could hinder the long-term development of the population. Therefore, establishing reasonable, scientifically grounded breeding schemes and introducing new purebred Dengchuan cattle to expand the core group are necessary. This will help maintain the genetic diversity of the conservation population of Dengchuan cattle.

Inbreeding level analysis

The F_IS coefficient is often used to measure the degree to which a group deviates from Hardy-Weinberg equilibrium. Dengchuan cattle exhibited a positive F_IS value of 2.4%, suggesting a potential small Wahlund effect within this population. ROH refers to consecutive segments of homozygous genotypes inherited by offspring when parents pass on the same haplotype.³⁷ The distribution patterns of ROH quantity and length can shed light on differences in breed formation and recent breed management practices.³⁸ Therefore, ROH can be used to assess population inbreeding, and the F_ROH is likely to be a more precise approach for detecting inbreeding compared to others.³⁹ Studies have shown that inbreeding increases ROH segment lengths.^40,41 In this study, the ROH lengths of 87% of the population were concentrated in the 1–5 Mb range. This is consistent with the results reported by Xu et al.⁴² regarding cattle breeds in southern China and by Liu et al.⁴³ regarding Italian Mediterranean buffalo breeds, whose ROH lengths were mostly <5 Mb. The average inbreeding coefficient (calculated to be 4.6% for F_ROH) was slightly lower than that of German red Holstein cattle (5.3%),⁴⁴ but slightly higher than that of Polish Red-and-White cattle (4.2%).⁴⁵ It was also similar to that of Chinese Simmental beef cattle (4.7%),^[46] indicating that the inbreeding coefficient is within the acceptable level. It is worth noting that there are only two conservation centres for Dengchuan cattle, located in Dengchuan Town and Yousuo Town, which are more than 30 kilometres apart. One possible reason for Dengchuan cattle being able to maintain a low level of inbreeding is that the distance between the breeding centres of the Dengchuan cattle limits gene exchange between individuals. Future breeding plans for Dengchuan cattle should focus on individual breeding schemes to prevent a rapid increase in the inbreeding coefficient and maintain population stability.

Sustainable conservation strategies for Dengchuan cattle population

The F_ROH of the conservation population of Dengchuan cattle was determined to be 4.6%. To maintain the low inbreeding rate and ensure the ability of this group to continue breeding, the mating of bulls and cows with distant genetic relationships between families should be prioritized. Conversely, breeding bulls and cows within the same family is not recommended. Notably, the seven cows categorized under the ‘other’ group in the family construction results were distantly related to all individual bulls in the group (coefficient of kinship of < 0.1). Thus, they can be paired with any bull for breeding. Furthermore, increasing the number of breeding farms can help preserve and propagate different genotypes, reducing genetic bottlenecks and the risk of inbreeding, thus maintaining the overall health and adaptability of the population. This study also found that some families had a limited number of bulls; to prevent bloodline loss, increased attention should be directed toward breeding offspring in these families during subsequent stages. Increasing the population of Dengchuan cattle through cross-family breeding is identified as an effective measure to reduce inbreeding. The selection and retention of suitable breeding cattle should be a priority to enhance the long-term viability of Dengchuan cattle. To facilitate the genetic management and assessment of the population, using modern science and technology such as big data technology to monitor the health of breeding cattle in real time and establishing accurate breeding cattle records and pedigree databases are important steps to achieve sustainable management and growth of Dengchuan cattle.

Conclusions

In this study, we assessed genetic diversity and structure through a SNP chip. We observed that the genetic diversity within the Dengchuan cattle conservation population was relatively high and most individuals demonstrated a moderate genetic relationship, some families had a shortage of bulls, increasing the risk of bloodline loss, they require more attention for conservation. Future conservation management should aim at maintaining genetic diversity and expanding the population by introducing new purebred lineages.

Author contribution

Conceived, drafted, and approved this research article: T.Z.Z. Conceptualization: P.P.W., G.Y.O. and T.Z.Z. Data curation: P.P.W., G.Y.O. and T.Z.Z. Formal analysis: P.P.W., G.Y.O. and T.Z.Z. Funding acquisition: T.Z.Z. Investigation: P.P.W., G.Y.O., H.Y.L., G.C.L. and T.Z.Z. Methodology: P.P.W., G.Y.O. and T.Z.Z. Project administration: T.Z.Z. Resources: T.Z.Z. and G.C.L. Visualization: P.P.W. and G.Y.O. Roles/Writing—original draft: P.P.W. and G.Y.O. Writing—review & editing: P.P.W., G.Y.O., H.Y.L. and T.Z.Z. All coauthors reviewed the final version and approved the manuscript before submission.

Ethics approval and consent to participate

All animal experiments were conducted according to the Regulations and Guidelines for Experimental Animals established by of Dali University.

Consent for publication

Not applicable.

Acknowledgements

Thanks to all authors with the help of various aspects.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Additional information

Funding

This research was supported by the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities (202301BA070001-033); Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D (AG2024003); Key projects of the Science and Technology Plan Doctoral Research Foundation Project of Dali University (KYBS2021072) and Basic Research of Dali Prefecture (2024).