2. MATERIALS AND METHODS

2.1 Animal selection

Wild Catharsius molossus dung beetles were captured in their natural habitat in Xichuan County, Nanyang City, Henan Province, China, from April 2021 to June 2021. The captured beetles were transported to the laboratory facilities at the College of Life Sciences, Yangtze University, for further study. The laboratory was maintained at a constant temperature of 21°C with a soil moisture content of 26%. Adequate ventilation was ensured in the laboratory, and the beetles were provided with a daily diet of fresh cow dung.

2.2 Experimental procedures

2.2.1 Sample collection and grouping

The gut samples from dung beetles under starvation and refeeding conditions were collected on August 15th and August 22nd, respectively. Six samples were collected for each condition. Before collection, the dung beetles were artificially reared for one month to stabilize their physiological conditions. For the starvation treatment, the beetles were observed for defecation daily until their defecation frequency significantly decreased. Once the beetles exhibited minimal defecation, their gut samples were collected for the starvation condition group. Subsequently, the beetles were re-fed for seven days and their gut samples were collected for the refeeding condition group. The collected gut samples were placed in sterile tubes, sequentially numbered, and stored at -80°C for further analysis (Table 1).

2.2.2 DNA extraction and Illumina NovaSeq 6000 sequencing

DNA extraction of the entire gut microbial community genome was performed using the NovaSeq 6,000 SP Reagent Kit (Illumina, USA). For bacterial 16S rRNA gene V3-V4 hypervariable region amplification, genomic DNA served as the template, and the fusion primers 341F/805R were utilized(Tsou et al., 2020). The PCR reaction mixture consisted of 1 µL 10× Toptaq Buffer, dNTPs (2.5 mM), 0.2 µL Primer F/R (10 µM), 0.2 µL Toptaq DNA Polymerase, 1~3 µL Template DNA, with the final volume adjusted to 10 µL with supplementary ddH2O. The PCR cycling program comprised an initial denaturation step at 94 °C for 2 min, followed by 26 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Following amplification, the products were purified as per the Agencourt AMPure XP Nucleic Acid Purification Kit instructions. Subsequently, Illumina NovaSeq 6000 sequencing was performed by Shanghai Tianhao Biotechnology Co., Ltd. (Shanghai, China).

2.2.3 Analysis of 16S rRNA gene sequences and bioinformatics

We initially subjected the raw sequencing data to preprocessing steps to ensure the accuracy of subsequent analyses. The raw sequencing data may contain artificial additives such as adapter sequences and primers. To eliminate potential noise from these sources, the cutadapt plugin of QIIME2 software (v0.5.0) was employed to remove possible adapter sequences and primers. Subsequently, the raw data subjected to filtering were quality assessed to guarantee high data quality(Lima et al., 2021). For obtaining high-quality sequencing data and enhancing the accuracy of subsequent bioinformatics analyses, the DADA2 plugin of QIIME2 software was utilized for quality filtering, denoising, merging, and chimera removal, thereby generating Amplicon Sequence Variants (ASVs) to assess sample diversity(Liu et al., 2023). To evaluate the adequacy of sequencing depth for each sample, dilution curves were constructed using QIIME2 and the ggplot2 package (v3.3.0) in R, assessing sequencing saturation and determining the need for further sequencing. Furthermore, Mothur software (v1.41.1) was employed for taxonomic annotation, species composition was assessed, and Alpha and Beta diversity analyses were conducted using R software(Langbo et al., 2017). Leveraging amplicon sequencing data, PICRUSt2 analysis was employed for predictive and analytical functional profiling of microbial communities(C. Yang et al., 2023). Employing Wilcoxon rank-sum tests and Metastats analysis, a significance threshold of p-value <0.05 was used for differential significance screening, with the Bonferroni correction applied to the p-values to minimize the potential for false positives(Feng et al., 2023; Neuhäuser, 2015). This methodology was adopted to assess whether significant interspecies diversity exists among groups.