miR-34 modulates wing polyphenism in planthopper

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 BPH. 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. Author summary Polyphenism is a fascinating phenomenon which significantly improves the ability of a species to explore various environmental resources. Brown planthopper (Nilaparvata lugens, BPH) is a notorious insect pest which causes huge damages to rice. BPH have two wing phenotypes, long-winged (LW) and short-winged (SW) morphologies. LW morphs are capable of long-distance migration, while SW morphs have high reproductive capabilities. Juvenile hormone (JH) and insulin/IGF signaling (IIS) pathway have been known to participate in regulating polyphenism in various organisms. However, how these regulators interact with each other remains largely unknown. Here, we show that a conserved microRNA, miR-34, mediates the crosstalk between JH, 20E and IIS pathway to modulate wing polyphenism in BPH. miR-34 suppresses insulin receptor-1(InR1), increases JH titer, and induces SW morphs. On the other hand, JH increases the expression of miR-34 and induces SW morphs, while 20E decreases miR-34 but does not change proportion of wing morphs. Knocking down genes in IIS pathway changes JH titer and miR-34 abundance. Therefore, miRNA, JH, 20E and IIS form an autoregulatory feedback loop to control wing polyphenism in BPH. Our work presents a comprehensive mechanism of wing polyphenism by integrating various regulators.