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 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.


Introduction
The phenomenon of polyphenism is that two or more distinct phenotypes are displayed by an organism with the same genotype. This phenomenon is triggered by environmental cues such as population density, host nutrition, and temperature [1]. For example, locusts show densitydependent phenotypic plasticity and have two phases: a low-density "solitarious" phenotype and a high-density "gregarious" phenotype [2]. In beetles, horn polyphenism is a nutritiondependent trait where some male beetles have fully-developed horns, while other males are completely hornless, depending on their nutrition status and body size [3]. Eusocial insects, including members of Hymenoptera, Blattodea (termites), often display caste differentiation, producing multiple types of offspring with different reproductive and morphological features [4]. Based on the physiological state of the mother, aphids produce winged adults in deteriorating environments and flightless morphs when environmental conditions are stable [5].
Brown planthopper (BPH, Nilaparvata lugens) is one of the most notorious planthoppers, migrating from Vietnam and the Philippines to China and Japan in summer during the rice growing season, then flying back to tropical regions during winter after rice crops have been harvested in temperate zones [6]. Wing polyphenism is a key determinant of the success of planthoppers [7]. Long-winged (LW) BPHs are capable of long-distance migration, while short-winged (SW) morphs display higher reproductive capabilities. Prior works showed that insulin/IGF-1 signaling (IIS) pathways involved in regulating wing polyphenism in BPHs [8]. Two insulin receptors (InR), NlInR1 and NlInR2, have been shown to play opposing roles in determining wing fate. NlInR2 inhibits NlInR1, switching long-winged morphs to shortwinged morphs, and vice versa. These two insulin receptors modulate wing polyphenism by regulating insulin/IGF signaling (IIS) pathway [9]. Wounding causes a shift to short wing through stimulation of the forkhead transcription factor (NlFoxO) and its downstream target Nl4EBP [10]. High glucose concentration in the rice host induces long-winged females, indicating that host nutrition quality has a direct impact on wing polyphenism in female BPH. This work shows that the nutrition is a key determination factor in controlling wing polyphenism in BPH and also raises a question of sex difference in wing polyphenism [11]. Silencing the c-Jun NH2-terminal kinase (NlJNK) increased the proportion of short winged female adults, suggesting that JNK signaling involve in regulating wing polyphenism in BPH [12]. In addition, topic application of juvenile hormone (JH) in the BPH nymphs induces SW morphs [13]. Knocking down the juvenile hormone epoxide hydrolase (Nljheh) prevents the degradation of JH and induces a bias towards SW BPHs [14], providing evidence that JH might also involve in regulating wing polyphenism. However, there is a dispute over JH regulation of wing polyphenism in BPH [8,15].
Though various regulators have been reported to involve in modulating phenotypic plasticity of wing polyphenism in BPHs, how these regulators interact with each other remains an important unsolved question. miR-34 is an important modulator involved in the cross talk between the IIS pathway and hormone in C. elegans and Drosophila. In C. elegans, miR-34 expression is regulated by IIS via a negative feedback loop between miR-34 and DAF-16 (the sole ortholog of FoxO in nematodes), providing robustness to environmental stress [16]. miR-34 is maternally inherited in Drosophila [17], and is activated by JH but is suppressed by 20E through Broad Complex (Br-C) [18]. Here, we found a microRNA (miRNA), Nlu-miR-34, modulates the crosstalk between IIS, JH and 20-hydroxyecdysone (20E) pathways to control wing polyphenism. Nlu-miR-34 suppresses NlInR1, inducing the development towards SW morph. Further, Nlu-miR-34 is activated by JH through Br-C. Moreover, silencing NlInR1 induces the expression of Nlu-miR-34, suggesting that Nlu-miR-34, IIS, and JH form an autoregulatory positive feedback loop to control wing polyphenism in BPHs.

Nlu-miR-34 controls NlInR1 in BPHs
Insulin receptors in BPH (NlInRs) were reported to determine the wing polyphenism in BPH [9]. To investigate whether miRNA may play a role in regulating wing polyphenism, we carried out a bioinformatics analysis to identify miRNAs targeting at NlInR1 or NlInR2. The results showed that only Nlu-miR-34 can target NlInR1 with two putative binding sites in the 3 ' untranslated region (UTR) predicted by all five target prediction algorithms (Fig 1A, see  methods). miR-34 is highly conserved in insects and nematodes (Fig 1B, see methods) and is predicted to have 19 target genes in BPH (S1 Table). To confirm the interaction between Nlu-miR-34 and NlInR1, we introduced the 3'UTR sequence of NlInR1 at the downstream of the firefly luciferase gene in the pMIR-REPORT vector. Constructs with or without (negative control) the 3'UTR of NlInR1 were both transfected into human embryonic kidney 293T (HEK293T) cells. The luciferase activity was significantly reduced to 30% relative to the negative control in the presence of agomir-34 (the mimics of Nlu-miR-34) (Fig 1C and 1D, Student's t-test, p = 4.99e -12 , n = 300, six replicates). Mutating any of the binding sites abolished the suppression effect of agomir-34 on the reporter activity of the NlInR1 target sites (Fig 1D), indicating that both two binding sites are essential for the interaction between Nlu-miR-34 and NlInR1. Next, we performed RNA-binding protein immunoprecipitation (RIP) assay with the antibody-Ago1 (Argonaute 1) in wing buds of BPH nymphs. The transcripts of NlInR1 was significantly enriched in the Ago1-immunoprecipitated RNAs from agomir-34-injected BPHs compared with those from the control samples (Fig 1E, 9.30-fold, Student's t-test, p = 0.002, n = 150, two replicates), showing that Nlu-miR-34 directly interacted with NlInR1 in the wing buds in vivo. Then, we used fluorescence in situ hybridization (FISH) assay to examine whether Nlu-miR-34 co-localize with NlInR1 in wing buds. Indeed, both of Nlu-miR-34 and NlInR1 were detected in wing buds of the fourth instar BPHs (S1

Nlu-miR-34 is highly expressed in SW nymphs in critical stages of wing fate determination
The critical stages of wing fate determination in BPH is the second to fourth instar of nymph [19]. However, the wing morphs cannot be discriminated at nymph stages. To examine the expression of Nlu-miR-34 in nymphs with different wing fate, we first developed two BPH strains by continually selecting SW or LW adults for crossing for more than 50 generations (SW♀ x SW♂ for SW strain, and LW♀ x LW♂ for LW strain) [14]. Almost all adults in SW strain emerge as short-winged adults while 80% of LW strain individuals become long-winged adult morphs. Because these two strains were selected from the same population, SW and LW strain should have the same genetic background. Each generation was purified in the adult stage using a previously described method [20]. We measured the expression of Nlu-miR-34 in the nymph stages of both SW and LW strain. The results show that Nlu-miR-34 was significantly highly expressed in the second and third instars nymphs in SW strain (Fig 2, Student's t-test, p = 0.0001 for the second instar, and p = 0.002 for the third instar, four replicates), suggesting that Nlu-miR-34 might be essential to develop SW adults. However, we found that NlInR1 was also highly expressed in the second and third instar SW nymphs when using the whole body for experiments (S2

Overexpressing Nlu-miR-34 induces SW morphs by repressing NlInR1
To decipher the roles of Nlu-miR-34 in controlling wing polyphenism, we increased its abundance by injecting agomir-34 into the third instar nymphs at 24 h post molt in LW strain. The abundance of Nlu-miR-34 was significantly increased by 3.06-fold compared to the negative control, which was treated with agomir-NC (a synthesized small RNA with a randomly shuffled sequence of Nlu-miR-34) (Fig 3A, Student's t-test, p = 0.026, three replicates). Fluorescence in situ hybridization (FISH) assay showed that the abundance of Nlu-miR-34 increased in the wing bud cells at 24 h post injection (Fig 3B). Next, the agomir-34 and agomir-NC treated nymphs were kept for wing observation until emergence into adults. The results showed that overexpression of Nlu-miR-34 induced the development of SW morphs  51.08 ± 6.16% in a significantly higher proportion than those in the control group 25.78 ± 4.78% (Fig 3C, Student's t-test, p = 0.032, n = 150, three replicates). In addition, overexpression of Nlu-miR-34 downregulated the expression of its target gene NlInR1 (Fig 3D, Student's t-test, p = 0.005, three replicates).

Nlu-miR-34 is upregulated by JH through Br-C
Next, to further uncover the factors regulating Nlu-miR-34 expression, we amplified the 5'UTR of Nlu-miR-34 using rapid amplification of cDNA ends (RACE). The transcription starting site (TSS) was determined by mapping the 5'UTR sequence to the BPH genome (Gen-Bank assembly accession: GCA_000757685.1), showing that the TSS of Nlu-miR-34 is located at position 119,684 in scaffold0000007224(-). We searched for transcription binding sites (TFBS) nearby the TSS of Nlu-miR-34 (-2,000 to +100) using PROMO [21] and Match [22]. Both programs identified a potential cis-response element (CRE) of Broad Complex (Br-C), suggesting that NlBr-C might involve in regulating Nlu-miR-34 (Fig 6A). To confirm this, we knocked down NlBr-C by injecting dsNlBr-C at the third instar nymphs in SW strain (Fig 6B, Student's t-test, p = 3.21e -5 , three replicates), resulted in the increase of Nlu-miR-34 by 1.44-fold (Fig 6C, Student's t-test, p = 0.011, three replicates). Because Br-C is an ecdysone inducible transcription factor [23], we then used 20-hydroxyecdysone (20E) to treat the third instar nymphs in SW strain. Topical application of 20E decreased Nlu-miR-34 significantly (Fig 6D, Student's t-test, p = 0.0001, three replicates). However, similar as the effect of using antagomir-34 alone without knocking down NlInR2, 20E application did not change the proportion of wing morphs.
Since JH and 20E show antagonistic actions through Br-C [24] and JH was known to regulate wing polyphenism in BPH [13,25], we hypothesize that JH might involve in regulating the expression of Nlu-miR-34. To test this mode, we used topical application of JH III analogue at the third instar of BPHs in LW strain. Acetone were used as the negative control. The results showed that increased JH titer led to a strong bias towards SW BPHs (increasing by 23.09 ± 6.16%) (Fig 6E, Student's t-test, p = 0.003, n = 100, three replicates). After JH application, Nlu-miR-34 was significantly increased by 1.91-fold while NlInR1 was decreased by 39.7 ± 14.26%, miR-34 modulates wing polyphenism in planthopper but it didn't change the NlInR2 level (Fig 6F-6H, Student's t-test, p = 6.04e -5 and 0.008, three replicates). Moreover, NlBr-C was also decreased by 25.18 ± 12.13% at 24h post JH treatment (Fig 6I, Student's t-test, p = 0.022, three replicates). These results showed that the upregulation of Nlu-miR-34 by JH and the downregulation of Nlu-miR-34 by 20E are likely mediated by NlBr-C in a transcriptional regulation manner.
To test whether NlInR1 also involve in this autoregulatory loop, we next knocked down NlInR1 by injecting dsNlInR1 in the third instar nymphs in LW strain (Fig 7C). The results showed that knocking down NlInR1 increased Nlu-miR-34 by 2.03-fold (Fig 7E, Student's ttest, p = 0.006, three replicates) and led to a strong bias towards SW morphs (Fig 7D, Student's t-test, p = 2.51e -7 , n = 100, three replicates). Taking together, these results suggested that miR-  (Fig 7F).

Discussion
Many animals with the same genetic background exhibit polyphenism to adapt to different environment conditions [1]. It has been showed that insulin-like peptides (ILP), the IIS pathway, and JH involve in the regulation of polyphenism in various organisms [26][27][28][29]. However, it remains elusive how these regulators interact with each other. Here, we showed that Nlu-miR-34 is a mediator between the crosstalk between IIS and hormonal signals by forming a positive feedback loop. miR-34 is a conserved miRNA that plays an important regulatory role in a wide range of organisms. It seems the positive feedback loop mediated by miR-34 might be a conserved process because miR-34 is also regulated by IIS via a negative feedback loop between miR-34 and DAF-16 (the sole ortholog of FoxO in nematodes) in C. elegans [16]. We propose a regulation mode of JH -miRNA-IIS feedback loop in BPH (Fig 7F). Through this positive autoregulatory loop, minor changes in environment conditions, above a certain threshold, would be amplified significantly in vivo inducing a phenotype shift. Two pathways involved in miRNA or InR regulation are redundant or at least partially redundant. It seems that InR regulation is more sensitive to nutritive environment whereas miRNA regulation is likely to be hormonally regulated. This discovery extends our understanding of the interplay between nutrition status-based signals and hormone signals in modulating phenotypic plasticity.
Polyphenism is a phenomenon in which a single genotype can arise into two or more discrete phenotypes in response to environmental stimuli; these phenotypes do not segregate in F1 generations produced from parents with distinct phenotypes, suggesting that polyphenotypic changes are not determined by only one or two genes [30]. However, knocking down NlInR1 induces formation of 100% SW BPHs [9], suggesting that the effect of insulin or hormone regulation is normally determinative in most cases. Therefore, other regulators should participate in regulation; and these regulators should be either sensitive to environmental cues or dose dependent. Here, we showed that Nlu-miR-34 mediates the cross talk between JH and IIS, and miRNAs are normally expressed at moderate levels [31]. In contrast to other regulatory molecules, miRNAs are typically very fine-tuned, specifically binding to and regulating their targets, acting as buffers to ensure developmental robustness [32]. These features enable them to act as ideal regulators of phenotype polyphenism, responding to varied environment cues. To the best of our knowledge, we presented the first evidence that miRNAs regulate developmental plasticity by modulating the cross talk between the JH and IIS pathways. It should be noted that the experiments of overexpression of Nlu-miR-34 were repeated independently in three labs in Nanjing, Wuhan and Hangzhou, ensuring the reliability of miRNA modulation of wing polyphenism in BPH (S4 Fig). JH was reported to regulate wing polyphenism in BPH twenty years ago by Tojo and his colleagues but without further evidence [13,25]. A recent report showed that knocking down juvenile hormone epoxide hydrolase (Jheh) enhanced short wing formation [14]. However, Zhang and his colleagues argued that lack of direct evidence of JH regulation of wing polyphenism in BPH [8,15]. Here we present evidence that JH participates in the regulation of wing polyphenism in BPH by upregulating Nlu-miR-34 expression. JH has been reported to regulate miRNA expression during larvae development in D. melanogaster [18]. JH can redirect the development of LW to SW/wingless morphs in BPHs [13,25], aphids [33], and crickets [34], but the relationship between JH and wing polyphenism is inverse with that in ants [35][36][37]. It is interesting to notice that both winged ants and SW BPHs have higher fecundity than other morphs, inferring JH regulation of wing polyphenism varies between different insects and JH might be involved in reproduction regulation [38], which requires further investigations.
Though JH has been well understood in regulating phenotype polyphenism [1,28], the role of 20E is less understood in this field. We showed that miR-34 is activated by JH but is suppressed by 20E in planthopper. A CRE of Br-C was found in the promoter region of Nlu-miR- 34. It has been reported that downregulation of miR-34 requires Br-C in Drosophila [18]. Since we did not find any JH response element (JHRE) in the promoter region of Nlu-miR-34, JH might regulate Nlu-miR-34 in an indirectly manner. JH has been reported to show antagonistic interaction with 20E through Br-C [24], it is likely that JH may activate the expression of Nlu-miR-34 through a cross talk between JH and 20E mediated by Br-C. However, topic application of 20E did not change the proportion of wing morphs. These results open a new question whether 20E regulates wing polyphenism in BPHs, and if so, in which way the 20E may regulate wing polyphenism.
In summary, we showed, for the first time, a comprehensive regulation mechanism of wing polyphenism in BPH by integrating almost all known regulators (miRNA, IIS, JH, and 20E) into a positive autoregulatory feedback loop (Fig 7F). We also presented evidence that 20E might also involve in regulating wing polyphenism by cross talking with JH. These discoveries miR-34 modulates wing polyphenism in planthopper extend our understanding of the mechanism regulating insect polyphenism, and shed new lights on how environmental cues determine which alternative phenotypes are produced by a given genotype.

Insects
Wild BPH populations were obtained from rice fields in Wuhan, Hubei Province, China, and raised on a BPH-susceptible rice variety Taichuang Native 1 (TN1) in the greenhouse. SW and LW strains were selected to propagate for more than 50 generations [14]. The percentage of SW morphs in the generated SW strain was more than 80%, while LW morphs represented approximately 80% of the LW strain. Each generation was purified in the adult stage using a previously described method [18]. BPHs were raised in growth chambers at 26˚C (± 2˚C) under a photoperiod of 16:8h (light: dark) with the relative humidity of 75% (± 5%).

microRNAs targeting NlInR1
To investigate whether miRNA may play a role in regulating wing polyphenism, we carried out a bioinformatics analysis to identify miRNAs targeting at NlInR1 or NlInR2. The results showed that three miRNAs target on NlInR1 and four miRNAs target on NlInR2 (S2 Table). Then, we used luciferase reporter assays to confirm the interaction between miRNA and their targets, and the results confirmed the interactions between NlInRs and three miRNAs, Nlu-miR-34, Nlu-miR-989b and Nlu-miR-989c. We overexpressed these three miRNAs separately to test their effects on wing polyphenism in BPH, showing that only Nlu-miR-34 can significantly change the ratio of wing morphs (Fig 3A and S5 Fig). So, we focused on Nlu-miR-34 in further studies.

In vitro luciferase assays
The 3'UTRs of NlInR1 and NlInR2 were amplified and introduced into the pMIR-REPORT vector (Obio, China) downstream of the firefly luciferase gene. The pRL-CMV vector (Promega, USA), which contains the Renilla luciferase gene, was used as a control luciferase reporter vector. The predicted binding site (5'-CACTAGTGACCGCGTAGTTGCCTGCCG-3') was completely removed for mutant 1, and another binding site (5'-TGACGTTGCCGCCGC CACTGCCG-3') was removed for generation of mutant 2. HEK293T cells (Obio, China) were cultured at 37˚C in 5% CO 2  The activity of the two luciferase enzymes was measured 48 h after transfection following the manufacturer's recommendations (Dual-Luciferase Reporter Assay System, Promega, USA) with Infinite M1000 (Tecan, Switzerland). Three replicates were performed for each experiment. Results are expressed as the means of the ratio of firefly luciferase activity/Renilla luciferase activity. Controls were set to one, and the two-tailed t-test was used for statistical analysis.

RNA-binding protein immunoprecipitation Assay (RIP)
A Magna RIP Kit (Millipore, Germany) was used to perform the RIP assay according to the manufacturer's instructions. Approximately 50 wing buds of the fourth instar nymphs were collected and crushed using a homogenizer in ice-cold RIP lysis buffer. Magnetic beads were incubated with 5 μg of RIPAb+ Ago-1 antibody (Millipore, Germany) or normal mouse IgG (Millipore, Germany). The homogenates in the RIP lysates were centrifuged and the Ago 1-bound mRNAs in supernatants were pulled down by magnetic bead-antibody complex at 4˚C overnight. The immunoprecipitated RNAs were released by digestion with protease K. Finally, the RNAs were purified by which methods and used for cDNA synthesis. The Prime-Script 1 st Strand cDNA Synthesis Kit (Takara, Japan) was used and the abundance of InR1 was quantified by qPCR.

Fluorescence in situ hybridization (FISH)
An antisense nucleic acid detection probe (5'-ACAACCAGCUAACCACACUGCCA -3') designed to detect Nlu-miR-34 was labeled with Cy3. The probe for detecting NlInR1 (5'-GA ACAGCCAGGACAGGCCGAAUCCUCCAUG-3') was labeled with FAM. The random shuffled probe (5'-UUCUCCGAACGUGUCACGUU-3') and the probe (5'-UUGUACUACAAA AGUACUG-3') were used as miRNA and mRNA negative controls, respectively. The wing buds were dissected from the third or fourth instar stage nymphs injected with agomir or antagomir (RiboBio, China). For fluorescence in situ hybridization, wing buds were treated for 24 h, fixed in 4% paraformaldehyde for 2 hours, and then incubated with the miRNA probes at 37˚C for 24 h. The samples were washed in PBS containing Triton X-100 (5% v/v) and then incubated with the DAPI (RiboBio, China) at room temperature for 30 min. Signals were analyzed and images were recorded using a Zeiss LSM 780 confocal microscope (Carl Zeiss SAS, Germany). Figures were prepared using Zeiss LSM ZEN 2010 software (Carl Zeiss SAS, Germany).

5'UTR amplification and promoter analysis of Nlu-miR-34
Total RNA was isolated from ten fourth to fifth instar nymphs of SW BPHs using the TRIzol regent (Invitrogen, USA). The SMARTer RACE cDNA amplification kit (Clontech, USA) was used to obtain the 5'UTR of Nlu-miR-34. 5'RACE nested-PCR was performed using Ex-Taq (Takara, Japan) according to the manufacturer's instructions. The amplified products were separated by agarose gel electrophoresis and purified using a Gel Extraction Kit (Takara, Japan). The purified DNA was ligated into the pMD19-T vector (Takara, Japan), and sent to BGI-Shenzhen for sequencing.
The transcript starting site (TSS) of miR-34 was confirmed by alignment to genomic scaffold sequences (GenBank assembly accession: GCA_000757685.1). Any unknowns "N" in the genomic sequences were confirmed by PCR validation. A 2100 bp flanking sequence (-2000 to +100) upstream of the TSS was used as the promoter region for analysis by Promoter Scan web server (http://www.cbs.dtu.dk/services/Promoter/). A putative TATA-box was identified at position -1707. To identify putative transcription factor binding sites (TFBS) in the promoter miR-34 modulates wing polyphenism in planthopper region, two algorithms, including PROMO 3.0 [18] and MATACH 1.0 [19] based on TRANS-FAC database (http://gene-regulation.com/pub/databases.html), were computationally analyzed using default parameters. A Br-C Z4 biding site (TTTTGTTTAAATT) at position -699 was predicted by both programs. The Nlu-miR-34 sequence, including the intact 5'UTR, was deposited in GenBank (MG894367).

Injection of agomir-34 and antagomir-34
BPH nymphs were collected for microinjection on the first day of the third instar. 15 nl of agomir-34 (40 ng, 15 nl) or antagomir-34 (40 ng, 15 nl) were microinjected into the conjunctive of each nymph between the prothorax and mesothorax using a Nanoliter 2000 injector (WPI, USA). 150 nymphs were used for each experiment. Ten nymphs were transferred into glass tubes 24 h post injection and placed in a growth chamber. Agomir or antagomir of random shuffled sequences was injected with equivalent volume as negative control (RiboBio, China). All experiments were performed in triplicate.

RNA interference (RNAi)
In order to knock down NlInR1 and NlBr-C expression, dsRNA was synthesized using the T7 RiboMAXTM Express RNAi System (Promega, USA) following the manufacturer's instructions. Each nymph, at the first day of the third instar, was injected with dsRNA (250 ng, 15 nl) using a Nanoliter 2000 injector (WPI). Equivalent volumes of dsGFP were injected as negative controls. 100 nymphs were used for each treatment, and all experiments were carried out in triplicate.

Co-injection of dsNlInR2 and antagomir
BPH nymphs were collected for microinjection on the first day of the third instar. The dsNlInR2, the concentration of which were divided into three groups, 0.42 ng, 0.84 ng and 3.38 ng, was injected into nymphs respectively with 40 ng antagomir-34. Either 40 ng or 60 ng antagomir-34 was injected into nymphs with 0.84 ng dsNlInR2. The volumes of mixtures were 15 nl. All of microinjections were done using a Nanoliter 2000 injector (WPI, USA). Approximately 200 nymphs were used for each experiment. Control nymphs were injected with equivalent volumes of the mixtures of dsNlInR2 and antagomir-NC. All experiments were performed in triplicate.

Topical application of JH
Juvenile hormone III (JH III, 96%) was purchased from Toronto Research Chemicals (Sigma, USA). 200 ng of JH III was dissolved in acetone and dropped 40 nl onto the mesonotum of the third instar BPH nymphs using a Nanoliter 2000 injector (WPI, USA), after anaesthetizing with carbon dioxide [14]. Equivalent volumes of acetone were used as negative controls. The rearing condition and methods of observation were same as the method of RNAi and injection of agomir-34 and antagomir-34. 100 nymphs were used for each experiment, all experiments were performed in triplicate.

Quantitative real-time PCR
Total RNA, enriched for miRNAs and mRNA, was extracted using the miRNeasy Mini kit (Qiagen, Germany) and TransZol Up Plus RNA kit (TransGen, China) from whole insect bodies (n = 30), respectively. The miScript II RT kit (QIAGEN) and the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen) were used to prepare cDNA. Quantitative real-time PCR (qPCR) reactions were conducted with an ABI Prism 7300 miR-34 modulates wing polyphenism in planthopper (Applied Biosystems, USA) using miScript SYBR Green (Qiagen, Germany) and SYBR1 Premix Ex Taq II RR820A (Takara, Japan), according to the manufacturer's instructions. The data was analyzed using the 2 -44Ct method. Actin1 (GenBank accession No. EU179846.1) was used as the endogenous control. This gene has been studied to show it is suitable to be used as a reference gene [46]. All treatments were carried out in triplicate. Independent reactions were performed in quadruplicate for each RNA sample, and the signal intensity of the target gene is presented as the average value. The qPCR primers used in this study are listed in S3 Table. Juvenile hormone estimation by LC-MS/MS About 300 BPHs were collected and pooled together for analysis. Extraction of JH III was performed according to a previously described method with some modifications [47,48]. JH III dilutions of 20, 10, 1, 0.5, 0.25, 0.125, and 0.0625 ng/ml were prepared in 70% methanol and used for calculation of the standard curve. The LC-MS/MS analyses were performed using an Agilent 6460 triple quadrupole mass spectrometer (Agilent, USA) equipped with an electrospray ionization (ESI) source, operated in the positive ion multiple-reaction monitoring (MRM) mode. Agilent Mass Hunter Workstation software was used for system operation, data acquisition, and data analysis. Chromatographic separation was carried out using a Zorbax SB-Aq column (100 mm × 2.1 mm, 3.5 μm) (Agilent, USA). JH III was separated using a binary gradient. Briefly, mobile phase A consisted of 0.1% formic acid in water and mobile phase B was comprised of 0.1% formic acid in acetonitrile. The gradient program was as follows: 0-5 min, 60%-85% of B; 5-8 min, 85%-95% of B. The flow rate of the mobile phase and the column temperature were set at 0.3 mL/min and 30˚C, respectively, and the injection volume was 5 μl. The retention time of JH III was 3.82 min and the total run time was 10 min. Mass spectrometric detection was completed by use of an electrospray ionization (ESI) source in positive ion multiple-reaction monitoring (MRM) mode using the following parameters: precursor (m/z), 267; ion product (m/z), 235.2; fragmentor voltage (V), 80; collision Energy (eV), 1. SPSS 17.0 software (IBM SPSS Inc. USA) was used for statistical analysis. The differences between treatments were compared using the Student's t-test. The effects of antagomir on distribution of wing types in BPHs were analyzed using the Chi-square test. p < 0.05 was considered statistically significant. All results are expressed as means ± SEM, three replicates.