Genome-Wide Screening and Functional Analysis Reveal That the Specific microRNA nlu-miR-173 Regulates Molting by Targeting Ftz-F1 in Nilaparvata lugens

Abstract

Background: Molting is a crucial physiological behavior during arthropod growth. In the past few years, molting as well as chitin biosynthesis triggered by molting, is subject to regulation by miRNAs. However, how many miRNAs are involved in insect molting at the genome-wide level remains unknown. Results: We deeply sequenced four samples obtained from nymphs at the 2nd-3rd and 4th-5th instars, and then identified 61 miRNAs conserved in the Arthropoda and 326 putative novel miRNAs in the brown planthopper Nilaparvata lugens, a fearful pest of rice. A total of 36 mature miRNAs with significant different expression levels at the genome scale during molting, including 19 conserved and 17 putative novel miRNAs were identified. After comparing the expression profiles, we found that most of the targets of 36 miRNAs showing significantly differential expression were involved in energy and hormone pathways. One of the 17 putative novel miRNAs, nlu-miR-173 was chosen for functional study. nlu-miR-173 acts in 20-hydroxyecdysone signaling through its direct target, N. lugens Ftz-F1(NlFtz-F1), a transcription factor. Furthermore, we found that the transcription of nlu-miR-173 was promoted by Broad-Complex (BR-C), suggesting that its involvement in the 20-hydroxyecdysone pathway contributes to proper molting function. Conclusion: We provided a comprehensive resource of miRNAs associated with insect molting and identified a novel miRNA as a potential target for pest control.

Keywords: Nilaparvata lugens; NlFtz-F1; molting; nlu-miR-173; pest control.

Figure 1

The numbers of putative novel miRNAs in four samples of N. lugens. E2nd instar, the miRNA numbers acquired from the sample at the end of the 2nd instar; B3rd instar, the miRNA numbers acquired from the sample at the beginning of the 3rd instar; E4th instar, the miRNA numbers acquired from the sample at the end of the 4th instar; B5th instar, the miRNA numbers acquired from the sample at the beginning of the 5th instar. The overlapping numbers indicate the common miRNAs in different samples.

Figure 2

Validation of expression changes of miRNAs during molting. The left heatmap indicates the sequencing data of miRNA expression ratios. The bar represents the scale of the ratios of expression levels (log 2). The right electrophoretograms indicate the semi-quantitative PCR of miRNAs, using U6 as an internal control. E2nd/B3rd, the expression level in the sample at the end of the 2nd instar/the expression level in the sample at the beginning of the 3rd instar; E4th/B5th, the expression level in the sample at the end of the 4th instar/the expression level in the sample at the beginning of the 5th instar. The empty and red boxes indicate false positive results.

Figure 3

Expression profiles of miRNAs and their targets during molting. The bar represents the scale of ratios of expression levels (log 2). E2nd/B3rd, the expression level in the sample at the end of the 2nd instar/the expression level in the sample at the beginning of the 3rd instar; E4th/B5th, the expression level in the sample at the end of the 4th instar/the expression level in the sample at the beginning of the 5th instar. The genes behind the miRNA indicate that these are the potential targets of this miRNA. A single asterisk indicates a significant difference at the p < 0.05 level. A double asterisk indicates a significant difference at the p < 0.01 level.

Figure 4

NlFtz-F1 is a target of nlu-miR-173 in response to 20-hydroxyecdysone signaling. (A) Co-transfection of nlu-miR-173 and its target NlFtz-F1. NC represents an unrelated small RNA sequence, and null represents a reporter without the 3′ UTR sequence. A single asterisk indicates a significant differences between the control group and the treated group at the p < 0.05 level. Each point represents the mean ± SEM from three independent experiments. (B) Transcription analysis. All mRNA levels are shown relative to the level of β-actin. A single asterisk indicates a significant difference between the two groups at the p < 0.05 level. Each point represents the mean ± SEM (n = 3). (C) Western blot. Protein extracts (5 μg) from the injected nymphs were loaded onto 12% SDS-PAGE gels. The gels were immunostained with anti-Ftz-F1 serum. β-Actin was used as a control. (D) Expression levels of nlu-miR-173 and NlFtz-F1. 4L1D: Nymphs on the 1st day of the 4th instar. All miRNA levels are shown relative to the U6 level, all mRNA levels are expressed relative to the level of β-actin, and the expression level of nlu-miR-173 is normalized to 5L2D, and that of NlFtz-F1 is normalized to 5L3D. (E) 20E content. (F) Luciferase assays used to test the BR-C binding sites in the promoters of nlu-miR-173. Black boxes means binding sites for BR-C. Each point represents the mean ± SEM from three independent experiments. A double asterisk indicates a significant difference between the 2 days at the p < 0.01 level. The highest luciferase level of a segment is designated as 100%.

Figure 5

Nlu-miR-173 is required for molting and chitin biosynthesis through NlFtz-F1. (A) Survival rates with feeding of the miRNA mimics or the inhibitor. Each point represents the mean ± SEM from three independent experiments. Different letters indicate significant differences (p < 0.05, LSR, SPSS). (B) Mimics or an inhibitor of nlu-miR-173 changed the chitin content of N. lugens. A single asterisk indicates a significant difference between the two groups at the p < 0.05 level. Each point represents the mean ± SEM (n = 3).

Figure 6

Phenotypes effected by feeding on nymphs. (A) Normal adults. Individuals in the mimic-feeding group died with phenotypes of molting failure (B), abdominal defects (C) and wing defects (D) at rates of 23.70, 28.15, and 14.07%, respectively, whereas individuals in the inhibitor-feeding group died with those phenotypes at rates of 10.37, 27.50, and 10.12%, respectively.

Figure 7

20E signaling regulates nlu-miR-173 through BR-C and E74. (A,B) The expression levels of nlu-miR-173 and Ftz-F1 after 20E injection. (C–H) The expression levels of nlu-miR-173, Ftz-F1, BR-C and E74 after 20E and dsRNA injection. All expression levels are shown relative to the levels of U6 (nlu-miR-173) or β-actin (BR-C, E74, and Ftz-F1), and the expression levels are normalized to those of the dsGFP, dsBR-C, or dsE74 group injected with ethanol. A single asterisk indicates a significant difference between the two groups at the p < 0.05 level. Each point represents the mean ± SEM (n = 3).

Figure 8

Hormone-miRNA-gene crosstalk in insect chitin biosynthesis. Solid arrows in the picture represent the experimental interactions and broken arrows represent the supposed interactions.