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. 2025 Feb 19;16(2):234.
doi: 10.3390/genes16020234.

Developmental Expression Patterns of miRNA in Mythimna separata Walker (Lepidotera: Noctuidae)

Yuhan Liu  1 , Huiman Tian  1 , Shaoqiu Ren  1 , Xiulin Chen  1 , Kun Luo  1 , Guangwei Li  1 , Boliao Li  1
Affiliations

Developmental Expression Patterns of miRNA in Mythimna separata Walker (Lepidotera: Noctuidae)

Yuhan Liu et al. Genes (Basel). .

Abstract

Background/objectives: miRNAs are a family of single-stranded non-coding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs) for suppression, with an average length of 22 nt. The oriental armyworm, Mythimna separata Walker, is a pest insect with long-distance migratory capability, which causes severe loss of grains and pastures in Eastern Asia, Southeastern Asia, and Oceania. This study aims to elucidate the post-transcriptional regulatory mechanisms of miRNAs in the development of this pest.

Methods: We carried out small RNA sequencing on samples from eggs, third instar larvae, pre-pupae, pupae, and adults.

Results: A total of 400 miRNAs were identified, among which 40 were known and 360 were novel miRNAs. Dynamic trend analysis of miRNAs revealed that 199 miRNAs were highly expressed in eggs (profile 12), while 173 miRNAs were highly expressed in both eggs and pupae (profile 13). The results of differential expression analysis of miRNAs (DEmiR) revealed that 75 miRNAs were significantly more abundant in eggs compared to other developmental stages. Furthermore, more up-regulated miRNAs were observed than down-regulated miRNAs in adults relative to 3rd instar larvae, pre-pupae, and pupae. The core genes for miRNA biosynthesis-Pasha, Dicer1, and Ago1-were highly expressed in eggs but poorly expressed in 3rd instar larvae. KEGG enrichment analyses indicated that several genes in the pentose and glucuronate interconversion pathway, as well as the fructose and mannose metabolism pathway, were regulated by DEmiRs.

Conclusions: DEmiRNAs targeted most genes of M. separata, resulting in a complex miRNA-mRNA regulation mode.

Keywords: armyworm; development; migratory insect; small RNA sequencing.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
The repeatability of miRNA samples from five developmental stages of M. separata. (a) Correlation analysis of miRNA read counts from 15 samples after mRNA, rRNA, snRNA, snoRNA, tRNA, and repeat sequences were filtered. The color spectrum, ranging from purple to orange, represents Pearson correlation coefficients. (b) PCA (principal component analysis of 15 miRNA samples among five groups. Eg, Egg; La, 3rd instar larvae; PP, Pre-pupae; Pu, Pupae; Ad, adult.
Figure 2
Figure 2
Length distribution and abundance of miRNAs from M. separata in deferent developmental stages. Eg, Egg; La, 3rd instar larvae; PP, Pre-pupae; Pu, Pupae; Ad, adult.
Figure 3
Figure 3
Dynamic trend analysis of miRNAs from M. separata. The significant clustered profiles were colored.
Figure 4
Figure 4
Volcano plots of differentially expressed (DE) miRNAs between any two of five developmental stages of M. separata (|log2FoldChange| > 1 and padj < 0.01). Purple dots indicate significant down-regulated miRNAs, orange dots indicate up-regulated miRNAs, and grey dots indicate miRNAs with no significant difference.
Figure 5
Figure 5
Relative expression of miRNAs from M. separata in different developmental stages. The yellow vertical bars represent the log2-transformed fold change in miRNA expression for each stage compared to the mean expression level of the lowest expressed stage based on small RNA sequencing. Bars represent the means ± SE (n = 3). Different lowercase letters above the sky blue vertical bars represent significantly different expression among different developmental stages that were analyzed by Tukey’s multiple comparison based on RT-qPCR (p < 0.05). The relative expression of each miRNA was normalized to U6.
Figure 6
Figure 6
Relative expression of miRNA biosynthesis core genes in M. separata in different developmental stages. Bars represent the means ± SE (n = 3). Different lowercase letters above the vertical bar represent significantly different expression among different developmental stages that were analyzed by Tukey’s multiple comparison. The relative expression of each gene was normalized to the geometrical mean of ribosomal protein S13 (rpS13) and elongation factor 1α (EF-1α).
Figure 7
Figure 7
KEGG pathway enrichment analyses for target genes for differently expressed miRNAs from M. separata in various developmental stages. (a) 3rd instar larvae vs. eggs; (b) Pre-pupae vs. 3rd instar larvae; (c) Pupae vs. pre-pupae; (d) Adults vs. pupae.
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