Changes in the gut bacterial community affect miRNA profiles in Riptortus pedestris under different rearing conditions

The ncRNA-mediated regulatory networks of defensins and lysozymes in Riptortus pedestris: involvement in response to gut bacterial disturbances

“…Although there are accumulating reports on lncRNAs and circRNAs that involved in controlling the transcription of immune genes by interaction with various signaling factors in insects (Zhou et al, 2021a,b;Yin et al, 2021;Zhou et al, 2022a,b;Lin et al, 2023), there is little evidence on the ncRNA-mediated networks of AMPs in R. pedestris under environmental changes. In the current study, we obtained the different number of ncRNAs to explore the regulatory networks of two AMPs in R. pedestris at post-transcriptional level, combined with our previous work that reported miRNA profiles of bean bugs in response to feeding conditional changes (Ren et al, 2023). First, we construct a interaction network of miRNAdefensin/lysozyme in R. pedestris by target prediction, including twenty-nine miRNA-defensin and seventy-four miRNA-lysozyme pairs.…”

“…The newly hatched eggs from above collected samples were cultured in Petri dishes, and then newly 2nd-instar nymphs were divided into three experimental groups until to emerging adults. In brief, the groups were set as follows: (i) feeding soybean seeds and ascorbic acid (DWA) water (Blank group, B); (ii) feeding potted soybean plants and DWA water (Control group, C); (iii) alternatively feeding with soybean seeds and 0.05% (w/v) DWA or only distilled water containing 50 μg/mL tetracycline (Antibiotic group, A) on the following day (Ren et al, 2023). Later, the whole guts of these above adults and field-collected adults (Field group, F) were dissected (starved after 2 days), and ultimately, three guts from the same group were surface-sterilized in 70% ethanol for 1 min and randomly pooled as one biological replicate, with three biological replicates prepared for each experimental group, and then all these samples were flash-frozen in liquid nitrogen and stored at −80°C until further extraction.…”

“…In brief, we selected lncRNAs, circRNAs, miRNAs and mRNAs to build the ceRNA network based on the same expression trends of lncRNAs or circRNAs and mRNAs and opposite expression of those with miRNAs, and the p value and |Pearson correlation coefficients| of lncRNA-miRNA, circRNA-miRNA, and miRNA-mRNA pairs were less than 0.05 and more than 0.6, respectively. In addition, the prediction and annotation of the miRNA-mRNA pairs were from our previous report (Ren et al, 2023).…”

“…To detect the associations between DE defensin, DE lysozymes and related DE ncRNAs, and most changes in the gut bacterial microbiota at the genus level among the six comparison groups, the heatmaps were produced using R software (version 2.15.3) based on Pearson correlation coefficients and the levels of statistically significant difference at *p < 0.05, **p < 0.01, and *** p < 0.001 (Ren et al, 2023).…”

“…The quantitative real-time PCR (qRT-PCR) assay was executed with a Green qPCR SuperMix Kit (TransGen, Beijing, China) on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) following the manufacturer's instructions. The PCR procedures were performed as described previously (Fu et al, 2021;Ren et al, 2023). The EF-1α gene was used as an internal control for normalization of the expression levels, and specific primers of selected DE AMPs and ncRNAs were designed by Primer Premier 5 software (Supplementary Table S5).…”