miR-34 modulates wing polyphenism in planthopper

Abstract

Polyphenism is a successful strategy adopted by organisms to adapt to environmental changes. Brown planthoppers (BPH, Nilaparvata lugens) develop two wing phenotypes, including long-winged (LW) and short-winged (SW) morphs. Though insulin receptor (InR) and juvenile hormone (JH) have been known to regulate wing polyphenism in BPH, the interaction between these regulators remains largely elusive. Here, we discovered that a conserved microRNA, miR-34, modulates a positive autoregulatory feedback loop of JH and insulin/IGF signaling (IIS) pathway to control wing polyphenism in BPH. Nlu-miR-34 is abundant in SW BPHs and suppresses NlInR1 by targeting at two binding sites in the 3'UTR of NlInR1. Overexpressing miR-34 in LW BPHs by injecting agomir-34 induces the development towards SW BPHs, whereas knocking down miR-34 in SW BPHs by injecting antagomir-34 induces more LW BPHs when another NlInR1 suppressor, NlInR2, is also suppressed simultaneously. A cis-response element of Broad Complex (Br-C) is found in the promoter region of Nlu-miR-34, suggesting that 20-hydroxyecdysone (20E) might be involved in wing polyphenism regulation. Topic application of 20E downregulates miR-34 expression but does not change wing morphs. On the other hand, JH application upregulates miR-34 expression and induces more SW BPHs. Moreover, knocking down genes in IIS pathway changes JH titers and miR-34 abundance. In all, we showed that miRNA mediates the cross talk between JH, 20E and IIS pathway by forming a positive feedback loop, uncovering a comprehensive regulation mechanism which integrates almost all known regulators controlling wing polyphenism in insects.

Fig 1. Nlu-miR-34 targets insulin receptor-1 (NlInR1).

(A) Two binding sites were predicted in the 3’UTR of NlInR1 by miRanda, TargetScan, microTar, PITA, and RNAhybrid. (B) Multiple alignment of miR-34-5p in insects and Caenorhabditis elegans. (C) Schematic mutation of two predicted Nlu-miR-34 binding sites within the Luc-NlInR1-3’UTR luciferase reporter. Asterisks means the mutated miRNA binding sites. (D) Dual luciferase reporter assays confirmed the interactions between Nlu-miR-34 and NlInR1 in vitro. The relative luciferase activities were significantly decreased in the presence of agomir-34. Mutating either of the binding sites abolished the repression effect of agomir-34. Data are means ± SEM, p = 4.99e^-12, six replicates. (E) RNA-binding protein immunoprecipitation (RIP) confirmed the interactions between Nlu-miR-34 and NlInR1 in vivo. Transcripts of NlInR1 were significantly enriched by antibody against Ago1 in agomir-34 treated group compared with it in agomir-NC treated group. Data are means ± SEM, p = 0.002, n = 50, two replicates. **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig 2. Nlu-miR-34 is highly expressed in SW nymphs in critical stages of wing fate determination.

Nlu-miR-34 is highly abundant in the second and third instar SW strain nymphs, indicating that Nlu-miR-34 is expressed during critical stages of wing fate determination. The qPCR data are presented as means ± SEM, p = 0.0001 for the second instar, and p = 0.002 for the third instar, four replicates. **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig 3. Overexpression of Nlu-miR-34 in LW strain induced the development towards SW BPHs by suppressing NlInR1.

(A) The agomir-34 was injected into early third instar nymphs in LW strain, significantly increasing the Nlu-miR-34 expression (p = 0.026). (B) Fluorescence in situ hybridization assay (FISH) showed that Nlu-miR-34 was more abundant in wing buds treated with agomir-34, relative to the negative control (agomir-NC). Scale bars:100 μm. (C) Injection of nymphs with agomir-34 induced a strong bias towards SW BPHs compared to the control (p = 0.032, n = 150). (D) The relative abundance of NlInR1 (p = 0.004) was significantly decreased after nymphs were injected with agomir-34. The qPCR and wing rate data are presented as means ± SEM, three replicates. *p < 0.05, **p < 0.01 (Student’s t-test).

Fig 4. Inhibition of Nlu-miR-34 in SW strain by antagomir-34.

(A) The expression of Nlu-miR-34 was significantly decreased by injecting antagomir-34 (p = 0.004). (B) Fluorescence in situ hybridization assay (FISH) showed that Nlu-miR-34 was less abundant in the wing bud treated with antagomir-34, relative to the negative control (antagomir-NC). Scale bars:100 μm. (C) Inhibition of Nlu-miR-34 by antagomir-34 does not induce changes in wing morphology (n = 50). The qPCR and wing rate data are presented as means ± SEM, three replicates. **p < 0.01 (Student’s t-test).

Fig 5. Simultaneously inhibition Nlu-miR-34 and NlInR2 in SW strain induced the decrease of SW BPHs.

(A) Antagomir-34 (with two different quantities) and dsNlInR2 (0.84 ng) were injected into the third instar SW strain nymphs, showed that injection of antagomir-34 decreased the ratio of SW type BPH (Chi-square test, 40ng: χ² = 5.8, df = 1, p = 0.003; 60ng: χ² = 7.9, df = 1, p = 0.042). (B) The antagomir-34 (40 ng) and dsNlInR2 (with three different quantities) were simultaneously injected into the third instar SW strain nymphs. The results showed that effect of low level of dsNlInR2 (0.42 ng) with antagomir-34 is familiar with control, while higher level of dsNlInR2 (0.84 and 3.38 ng) with antagomir-34 induced a bias towards the development of LW BPHs compared to the control. Injection of antagomir-34 (40 ng) and dsNlInR2 (0.84 ng) decreased ratio of SW BPH to around 20% (Chi-square test, χ² = 8.4, df = 1, p = 0.004). High level of dsInR2 (3.38 ng) with antagomir-34 decreased the ratio of SW BPH (Chi-square test, χ² = 3.3, df = 1, p = 0.068). 200 insects were used for each experiment. Data are means ± SEM, three replicates. *p < 0.05, **p < 0.01.

Fig 6. Nlu-miR-34 is upregulated by JH through Br-C.

(A) A cis-response element specific to NlBr-C Z4 activity was identified in the promoter region of Nlu-miR-34. (B) Expression of NlBr-C was successfully knocked down in LW strain after treating with dsNlBr-C (p = 3.21e^-5). (C) Knockdown of NlBr-C leads to the increase of Nlu-miR-34 transcripts (p = 0.011). (D) 20E application in SW strain results in the decreased of Nlu-miR-34 (p = 0.0001, four replicates). (E) Application of JH in LW-strain induced a strong bias towards development of SW BPHs (p = 0.003, n = 100). (F) Increased expression of Nlu-miR-34 (p = 6.04e^-5), (G) significantly decrease of NlInR1 (p = 0.008), (H) No effect on the expression of NlInR2. (I) JH application results in decreased abundance of NlBr-C transcripts (p = 0.022). The qPCR and wing rate data are presented as means ± SEM, three replicates. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig 7. Nlu-miR-34, JH, 20E and NlInR1 form a positive autoregulatory loop.

(A) JH titers were increased significantly in BPHs after treated with agomir-34 in LW strain. ND: not detected. More than 300 insects were used for each experiment. (B) Injection of agomir-34 in LW strain resulted in decreased abundance of NlBr-C transcripts (p = 6.72e^-5). (C) Knockdown of NlInR1 significantly decreased the expression of NlInR1 (p = 0.001). (D) Knocking down NlInR1 in LW strain induced a strong bias towards SW BPHs (p = 2.51e^-7, n = 100). (E) Higher transcriptional level of Nlu-miR-34 in the knockdown SW BPHs (p = 0.006). The qPCR and wing rate data are presented as means ± SEM, three replicates. *p < 0.05, **p < 0.01, *** p < 0.001 (Student’s t-test). (F) Schematic model of the interplay between the IIS pathway, JH, 20E and miRNA during wing determination in BPHs. In LW BPHs, low transcriptional level of Nlu-miR-34 fails to inhibit the expression of NlInR1, which activates NlAkt, inactivates NlFoxO, and reduces JH titer activates the activity of NlBr-C, which will maintain the low transcriptional level of Nlu-miR-34. In SW BPHs, high transcriptional level of Nlu-miR-34 inhibits the expression of NlInR1, fails to inactivate NlFoxO by NlAkt, stimulating to a high JH titer and repression of NlBr-C, which will maintain the high transcriptional level of Nlu-miR-34. The components that are less active or inactive are shown in grey.