Identification and Characterization of MicroRNAs in Gonads of Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract

The high fecundity of the most destructive pest Helicoverpa armigera and its great resistance risk to insecticides and Bt crops make the reproductive-destruction-based control of this pest extremely appealing. To find suitable targets for disruption of its reproduction, we observed the testis and ovary development of H. armigera and conducted deep sequencing of the ovary and testis small RNAs of H. armigera and quantitative RT-PCR (RT-qPCR) validation to identify reproduction-related micro RNAs (miRNAs). A total of 7,592,150 and 8,815,237 clean reads were obtained from the testis and ovary tissue, respectively. After further analysis, we obtained 173 novel and 74 known miRNAs from the two libraries. Among the 74 known miRNAs, 60 miRNAs existed in the ovary and 72 existed in the testis. Further RT-qPCR validation of 5 miRNAs from the ovary and 6 miRNAs from the testis confirmed 8 of them were indeed ovary- (miR-989a, miR-263-5p, miR-34) or testis-biased (miR-2763, miR-998, miR-2c, miR-2765, miR-252a-5p). The 8 ovary- or testis-biased miRNAs had a total of 30,172 putative non-redundant target transcripts, as predicted by miRanda and RNAhybrid. Many of these target transcripts are assigned to reproduction-related GO terms (e.g., oocyte maturation, vitellogenesis, spermatogenesis) and are members of multiple reproduction-related KEGG pathways, such as the JAK-STAT signaling pathway, oocyte meiosis, the insulin signaling pathway, and insect hormone biosynthesis. These results suggest that the 8 gonad-biased miRNAs play important roles in reproduction and may be used as the targets for the development of reproductive-destruction-based control of H. armigera and, possibly, other lepidopteran pests.

Keywords: development; miRNAs; ovary; piRNAs; reproductive destruction; sex; testis.

Figure 1

Development of H. armigera testis and ovary. (A–H) The testes of L4, L5, L6, pre-pupa, pupa, and newly emerged (0-day-old) male adults, respectively. (A’–D’) The ovaries of pupa, newly emerged (0-day-old) female adults, 1-day-old female adults, and 2-day-old female adults, respectively. Bar = 1 mm. (I–K) The testis tissue of larvae, late pupal stage, and newly emerged adults, respectively, by H&E staining. Bar = 100 μm. (A–H,A’–D’) Photos taken by a hyperdepth 3D microscope (Smartzoom5 1810070S, Zeiss). Photos of ovaries were stitched by microscope.

Figure 2

The length distribution of small RNAs in the testis of a 0-day-old male adult and the ovary of a 2-day-old female adult of H. armigera.

Figure 3

Composition of different types of small RNAs in the testes (A) and ovaries (B) of 0-day-old males and 2-day-old female adults of H. armigera. Small RNAs were annotated by the Blast search against the Rfam and piRBase databases. The range length for small RNAs is 15–50 bp.

Figure 4

The numbers of the shared and testis- or ovary-specific known (A) and novel (B) miRNAs expressed in the gonads of a 0-day-old male adult and a 2-day-old female adult of H. armigera.

Figure 5

Expression levels of known miRNAs in the testes and ovaries of 0-day-old male adults and 2-day-old female adults of H. armigera. Putatively biased miRNAs (|log₂FC| ≥ 1) between the testis and ovary are labeled with *.

Figure 6

RT-qPCR validation of the expression levels of 11 ovary- and testis-biased candidate miRNAs in the testes and ovaries of 0-day-old male and 2-day-old female adults of H. armigera. U6 snRNA and let-7 were used as reference small RNAs to normalize the expression levels of the 11 gonad-biased candidate miRNAs. The data and error bars represent the means and standard errors of at least three biological replicates. The difference in the expression level of each miRNA between the testes and ovaries of sexually mature adults of H. armigera was analyzed by two sample t-tests. Significant differences at p < 0.05, p < 0.01, and p < 0.001 are depicted with *, ** and *** asterisks, respectively. NS = not significant.

Figure 7

Correlation between the deep sequencing read data and RT-qPCR expression levels of the 11 testis- or ovary-biased candidate miRNAs.

Figure 8

Gene ontology (GO) (A) and KEGG pathway (B) classifications of the 30,172 non-redundant putative target transcripts of the eight validated ovary- or testis-biased miRNAs.