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.