Novaseq 6000 platform

First of all, C.lividipennis and A.lucorum were anesthetized using carbon dioxide. Subsequently, the SGs, guts, and other carcasses without SGs and guts from both insects were carefully dissected under a microscope (Olympus, Japan). Thereafter, the total RNA was extracted from each sample using the Trizol RNA Isolater Total RNA Extraction Reagent (Vazyme Biotech, Nanjing, China), in line with the manufacturer’s protocol. Afterwards, the RNA concentration, quantity, and integrity were assessed by the Agilent 2100 bioanalyzer (Agilent Technologies, California, USA). Then, to synthesize cDNA, the mRNA was purified from the total RNA using magnetic beads carrying Oligo(dT). The purified cDNA then experienced end repairing, adenylation of 3’ ends, and ligation of adaptors. AMPure XP beads (Beckman Coulter, Beverly, USA) were used to screen out 370–420 bp cDNA. Next, the sequencing library was generated through PCR, and AMPure XP beads were further used in a second purification step. Finally, RNA-seq was conducted on the Illumina Novaseq 6000 platform (Novogene, Tianjin, China). The quality summary regarding library sequencing is listed in (Supplementary Table 8), with over 40 million clean reads being obtained in each library.

Furthermore, 3-5 × 10⁷ cells were cross-linked with 1% formaldehyde for 10 minutes and neutralized with 1.25 M glycine at room temperature for 5 minutes. Bioruptor (Diagenode, Liège, Belgium) was used to collect, lyse, and sonicate fixed cells. Sonicated chromatin was incubated with an anti-histone H3 (acetyl K27) antibody (cat. No. ab4729; Abcam, Cambridge, UK) overnight at 4°C. DNA was eluted and purified using a QIAquick PCR purification kit (cat. No. 208106; Qiagen, Hilden, Germany). The samples were sequenced on the NovaSeq 6000 platform (Novogene Bioinformatics Technology Co., Ltd. Beijing, China) and a BGISEQ 2000 platform (Beijing Genomics Institute, Shenzhen, China). Raw data of ChIP-seq H3K27ac analysis were aligned to the reference genome (UCSC hg38) using Bowtie2 (v 2.3.5) [17 (link)], with alignment parameters -p 4 -q -x. Peaks were identified using MACS2 (v2.0.9) [18 (link)], with parameters -g hs -n test -B -q 0.01. The bedGraph files generated by MACS2 were converted to bigwig files using the UCSC bedGraphToBigWig tool, and bigwig files were then visualized by Integrative Genomics Viewer (IGV) [19 (link)]. Superenhancers were then identified using the ROSE (Rank Order of Superenhancers) method [20 (link), 21 (link)], with parameters -s 12500 -t 2000.

The total DNA from the rumen content samples was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. 16S rRNA genes were amplified by PCR from the genomic DNA samples using specific primers for the V4 region of the bacterial 16S rRNA. Amplicon libraries for all samples were quantified by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA) and sequenced on an NovaSeq 6000 platform by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) to generate 2 × 250 bp paired-end reads.
The singletons and chimeras were removed, and then tags were clustered into the operational taxonomic unit (OTU) using UPARSE (V 7.0.1001, http://www.drive5.com/uparse/) at 97% similarity (17 (link)). Afterward, the representative sequences of the OTUs were annotated using the SSUrRNA database (SILVA138, http://www.arb-silva.de/) (18 (link)). The alpha and beta diversity were analyzed by QIIME (19 (link)).
Linear discriminant analysis coupled with effect size (LEfSe) was conducted to identify bacterial taxa differentially represented among the groups at various taxonomy levels. An LDA effect size of more than 2.5 was used as threshold for the LEfSe analysis.

For bacterial species determination, the whole-genomes of the seven onion strains were sequenced. For sequencing, cultures for each bacterial strain were prepared by transferring single colonies from two-day-old NA plates into 4 mL of nutrient broth followed by incubation at 28°C overnight in a rotary shaker at 200 rpm. Total microbial genomic DNA was extracted from 1 mL of an overnight culture using a Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, United States) according to the manufacturer’s instructions. The genomic libraries were prepared using a NEBNext Ultra II DNA Library Prep Kit for Illumina and were sequenced using an Illumina Novaseq 6,000 platform by Novogene Co., Ltd. (Beijing, China). The raw sequences were filtered using fastp v 0.20.0 http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, and quality-checked using fastqc v 0.11.9. Processed reads were assembled using SPAdes v 3.14 (--isolate --cov-cutoff auto mode) (Bankevich et al., 2012 (link)) and filtered to a minimum contig size of 500 bp. The assembly files were deposited in the NCBI database under the BioProject PRJNA776088. The final assemblies were also uploaded to the Life Identification Number (LIN) platform (Tian et al., 2020 (link)).

In total, 0.18–0.22 g stool samples were used to extract total bacteria DNA following the protocol of the DNA extraction kit (#DP328, Tiangen Company, Beijing, China). The 16S rRNA V4 regions were performed using specific primers 515 F GTGCCAGCMGCCGCGGTAA and 806 R GGACTACHVGGGTWTCTAAT. Sequencing libraries were generated using the Illumina TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA) with following manufacturer recommendations, and index codes were added. Sequencing was performed in the Illumina Novaseq 6000 platform (Novogene, China).
Microbiome bioinformatics were performed with QIIME2 2021.4.^{47 (link)} Shannon index for alpha diversity and Bray-Curtis for beta diversity measures. Principle Coordinate Analysis (PCoA) were analyzed using the vegan v2.5–7 R package. Taxonomy was assigned to ASVs using the qiime feature-classifier classify-sklearn. Naive Bayes classifiers trained on Silva 138 99% OTUs from 515 F/806 R region of sequences. Gene functions analysis were predicted as previous method.^{48 (link)} Predicted functions were calibrated for all of samples (n = 73) using Meta-Apo^{49 (link)} based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Thirty paired metagenomic samples were used for training.

Sequence libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA). The libraries were sequenced on the Illumina Novaseq 6000 platform (insert size 350 bp, read length 150 bp) at the Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China).
Raw sequence reads were trimmed using Trimmomatic v0.39 to remove adapters and low-quality regions and then removed of contaminating human reads using Bowtie2 v2.4.2 (Reference database: GRCh38).^{50 (link)} The taxonomic composition was profiled using the default parameters of MetaPhlAn3 v3.0.9.^{51 (link)} The functional gene pathway was profiled using the default settings of HUMAnN3 v3.0.0.alpha.3.^{52 (link)} Functional potential profiling of microbial communities was performed by HUMAnN3 using pangenomes annotated with UniRef90 on all species detectable per sample with MetaPhlAn3. Functional annotations rely on the MetaCyc database and gut-brain modules (GBMs) database.

3–5 × 10⁷ cells were crosslinked with 1% formaldehyde for 10 min and neutralized with 1.25 M glycine for 5 min at room temperature. Fixed cells were harvested, lysed, and sonicated using a Bioruptor (Diagenode, Liège, Belgium). Sonicated chromatin was incubated with anti-histone H3 (acetyl K27) antibody (cat. No. ab4729; Abcam, Cambridge, UK) overnight at 4 °C. DNA was eluted and purified using a QIAquick PCR purification kit (cat. No. 208106; Qiagen, Hilden, Germany). Samples were sequenced on a novaseq 6000 platform (Novogene Bioinformatics Technology Co., Ltd. Beijing, China). Raw data of ChIP-Seq H3K27ac analysis for NB4 cell line was aligned to the reference genome (UCSC hg38) using Bowtie2 (v 2.3.5) [34 (link)], with alignment parameters -p 4 -q -x. Peaks were identified using MACS2 (v2.0.9) [35 (link)], with parameters -g hs -n test -B -q 0.01. The bedgraph files generated by MACS2 were converted to bigwig files using the UCSC bedGraphToBigWig tool, and then bigwig files were visualized by Integrative Genomics Viewer (IGV) [36 (link)].