Characterization and comparative profiling of MicroRNAs in a sexual dimorphism insect, Eupolyphaga sinensis Walker

Abstract

Background: MicroRNAs are now recognized as key post-transcriptional regulators in animal ontogenesis and phenotypic diversity. Eupolyphaga sinensis Walker (Blattaria) is a sexually dimorphic insect, which is also an important source of material used in traditional Chinese medicine. The male E. sinensis have shorter lifecycles and go through fewer instars than the female. Furthermore, the males have forewings, while the females are totally wingless.

Results: We used the Illumina/Solexa deep sequencing technology to sequence small RNA libraries prepared from the fourth-instar larvae of male and female E. sinensis. 19,097,799 raw reads were yielded in total: 7,817,445 reads from the female library and 11,280,354 from the male, respectively. As a result, we identified 168 known miRNAs belonging to 55 families as well as 204 novel miRNAs. Moreover, 45 miRNAs showed significantly different expression between the female and the male fourth-instar larvae, and we validated 10 of them by Stem-loop qRT-PCR. Some of these differentially expressed miRNAs are related to metamorphosis, development and phenotypic diversity.

Conclusions/significance: This is the first comprehensive description of miRNAs in E. sinensis. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs. These findings not only enrich miRNAs for hemimetabolans but also lay the foundation for the study of post-transcriptional regulation on the phenomena of sexual dimorphism.

Figure 1. Statistics of small RNA sequences from the female (A) and male (B) E. sinensis libraries.

The raw reads were classified into five separate categories, Mappable sequences states the raw reads were passed through a series of the digital filters by Illumina's Genome Analyzer Pipeline software and ACGT101-miR program, and used in the further miRNA identification.

Figure 2. Size distribution of mappable reads related to miRNA in the female (A) and male (B) libraries of E. sinensis.

The total represents the number of all mappable sequences were marked blue, and the unique represents the number of unique sequences were marked red. nt, nucleotides.

Figure 3. The most 20 abundant miRNAs identified in E. sinensis.

The left panel was Relative expression levels between female (black) and male (light gray). The right panel was the normalized reads number.

Figure 4. Comparison of miRNAs between the female and male E. sinensis.

The Venn diagram displays the distribution of 372 unique miRNAs between the male (left, green circle) and female (right, pink circle) libraries. The dashed circles indicate the miRNAs that were significantly differentially expressed (p<0.0001, Bonferroni corrected) in the two samples.

Figure 5. Comparison of the differentially co-expressed miRNAs between the female and male E. sinensis.

45 miRNAs showed significant differential expression between the female (black) and male (light gray) libraries (p-value <0.0001). The frequency of miRNAs in two libraries were normalized.

Figure 6. Relative expression levels of ten in the female and male E. sinensis by Quantitative real-time PCR.

“*” and “**” means a statistically significant difference at level p<0.05 and p<0.001, respectively, for this miRNA in the female and male E. sinensis. The error-bars show standard deviation for three biological replicates.