Identification and Prediction of Differentially Expressed MicroRNAs Associated with Detoxification Pathways in Larvae of Spodoptera frugiperda

Abstract

Spodoptera frugiperda poses a severe threat to crops, causing substantial economic losses. The increased use of chemical pesticides has led to resistance in S. frugiperda populations. Micro ribonucleic acids (MicroRNAs or miRNAs) are pivotal in insect growth and development. This study aims to identify miRNAs across different developmental stages of S. frugiperda to explore differential expression and predict target gene functions. High-throughput sequencing of miRNAs was conducted on eggs, 3rd instar larvae, pupae, and adults. Bioinformatics analyses identified differentially expressed miRNAs specifically in larvae, with candidate miRNAs screened to predict target genes, particularly those involved in detoxification pathways. A total of 184 known miRNAs and 209 novel miRNAs were identified across stages. Comparative analysis revealed 54, 15, and 18 miRNAs differentially expressed in larvae, compared to egg, pupa, and adult stages, respectively. Eight miRNAs showed significant differential expression across stages, validated by quantitative reverse transcription PCR (qRT-PCR). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses predicted target genes' functions, identifying eight differentially expressed miRNAs targeting 10 gene families associated with detoxification metabolism, including P450s, glutathione S-transferase (GSTs), ATP-binding cassette (ABC) transporters, and sodium channels. These findings elucidate the species-specific miRNA profiles and regulatory mechanisms of detoxification-related genes in S. frugiperda larvae, offering insights and strategies for effectively managing this pest.

Keywords: S. frugiperda; detoxification; larva; microRNAs; resistance; the fall armyworm.

Figure 1

Length distribution of sRNAs in different stages of S. frugiperda. X-axis indicates sequence length, while Y-axis shows the frequence in percentage.

Figure 2

Composition of sRNAs in S. frugiperda. (A) Annotation of sRNAs. X-axis shows the sample number, whereas Y-axis shows the proportion of the de-duplicated sequences annotated to each sRNA to the total de-duplicated sequences. (B) First base bias of the miRNAs in S. frugiperda. X-axis shows the miRNAs length, and Y-axis shows the frequency in percentage. (C) Base bias of the miRNAs at each position. X-axis shows the nucleotide position, and Y-axis shows the frequency in percentage.

Figure 2

Composition of sRNAs in S. frugiperda. (A) Annotation of sRNAs. X-axis shows the sample number, whereas Y-axis shows the proportion of the de-duplicated sequences annotated to each sRNA to the total de-duplicated sequences. (B) First base bias of the miRNAs in S. frugiperda. X-axis shows the miRNAs length, and Y-axis shows the frequency in percentage. (C) Base bias of the miRNAs at each position. X-axis shows the nucleotide position, and Y-axis shows the frequency in percentage.

Figure 3

Venn diagram of differential expression in known (A) and novel (B) miRNAs of S. frugiperda. Numbers indicate shared miRNAs.

Figure 4

Heatmap of differentially expressed miRNAs in different stages of S. frugiperda. Red indicates upregulation; blue means downregulation.

Figure 5

Relative expression level of selected differentially expressed miRNAs of S. frugiperda between qRT-PCR and RNA-seq in (A) larvae vs. eggs, (B) larvae vs. pupae, and (C) larvae vs. adults.

Figure 6

The top 20 significantly enriched KEGG pathways of S. frugiperda in (A) larvae vs. adults, (B) larvae vs. eggs, and (C) larvae vs. pupae. X-axis shows the enrichment factor, and Y-axis shows the pathway names. Definitions of the pathways are available at https://www.kegg.jp/kegg/pathway.html. The enrichment degree was measured by Rich factor value and the number of miRNA target genes enriched on this pathway. Rich factor refers to the ratio of the number of differential miRNA target genes enriched in this pathway to the number of differential miRNA target genes annotated. The greater the Rich factor, the greater the degree of enrichment.

Figure 6

The top 20 significantly enriched KEGG pathways of S. frugiperda in (A) larvae vs. adults, (B) larvae vs. eggs, and (C) larvae vs. pupae. X-axis shows the enrichment factor, and Y-axis shows the pathway names. Definitions of the pathways are available at https://www.kegg.jp/kegg/pathway.html. The enrichment degree was measured by Rich factor value and the number of miRNA target genes enriched on this pathway. Rich factor refers to the ratio of the number of differential miRNA target genes enriched in this pathway to the number of differential miRNA target genes annotated. The greater the Rich factor, the greater the degree of enrichment.

Figure 7

The top 30 GO terms of the target genes of differentially expressed miRNAs of S. frugiperda in larvae vs. adults. The enrichment degree was measured using the false discovery rate (FDR) value and the number of miRNA target genes enriched on this pathway. FDR generally ranges from 0 to 1, and the closer it is to zero, the more significant the enrichment. The top 30 pathways with the smallest FDR value, that is, the most significant enrichment, were selected for display.

Figure 8

The top 30 GO terms of the target genes of differentially expressed miRNAs of S. frugiperda in larvae vs. pupae. The enrichment degree was measured by FDR value and the number of miRNA target genes enriched on this pathway. FDR generally ranges from 0 to 1, and the closer it is to zero, the more significant the enrichment. The top 30 pathways with the smallest FDR value, that is, the most significant enrichment, were selected for display.

Figure 9

The top 30 GO terms of the target genes of differentially expressed miRNAs of S. frugiperda in larvae vs. eggs. X-axis shows GO terms, and Y-axis shows −log10 (p-value). The enrichment degree was measured by FDR value and the number of miRNA target genes enriched on this pathway. FDR generally ranges from 0 to 1, and the closer it is to zero, the more significant the enrichment. The top 30 pathways with the smallest FDR value, that is, the most significant enrichment, were selected for display.

Figure 10

A miRNA–mRNA network of 8 differentially expressed miRNAs and their target genes related to detoxification metabolism in S. frugiperda. Red triangles represent upregulated miRNAs while blue triangles represent downregulated miRNAs. Other circles represent different target genes related to detoxification metabolism.