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Two insulin receptors determine alternative wing morphs in planthoppers

Abstract

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects1. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs1,2,3. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues1,2,4,5,6,7,8, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved4,8,9,10. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)–protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1–PI(3)K–Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration—to our knowledge—of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.

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Figure 1: Knockdown of NlInRs in BPHs.
Figure 2: NlInR2 negatively regulates NlInR1 signalling, and NlFoxo relays the insulin signalling.
Figure 3: NlIlp3 triggers the wing morph switch.
Figure 4: Model of the molecular regulation of wing polyphenism in planthoppers.

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Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The cDNA sequences of NlInR1 and NlInR2 have been deposited in GenBank under accession numbers KF974333 and KF974334, respectively. Gene sequences used for dsRNA synthesis have been deposited in GenBank under the following accession numbers: KF974335 (NlChico), KF974336 (NlLnk), KF974337 (NlAkt), KF974338 (NlPten), KF974339 (NlFoxo), KF974340KF974343 (NlIlp1–4), KF974348 (NlErk), KF974349 (NlRaf), KF974350 (NlTor), KM099280 (NlRaptor), KM099281 (NlRheb), KF974344 (SfInR1), KF974345 (SfInR2), KF974346 (LsInR1) and KF974347 (LsInR2).

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Acknowledgements

We thank R.-Z. Zhang for help with BPH imaging (Fig. 1a). This work was supported by the National Basic Research Program of China (973 Program, no. 2010CB126205) and by the National Science Foundation of China (no. 31201509 and no. 31471765).

Author information

Authors and Affiliations

Authors

Contributions

H.-J.X. conceived and designed the study, wrote the paper, helped perform experiments and analysed the data. J.X. performed most experiments and helped with data analysis. B.L., Y.-Q.J., Q.L., S.-F.H. and J.-Y.X. helped perform experiments and antibody preparation. X.-C.Z. and J.-C.Z. performed gene cloning and immunoprecipitation. X.-F.M. performed RACE experiments. H.-W.F., Y.-X.Y. and P.-L.P. performed qRT–PCR. Y.-Y.B. and H.F.N. discussed data and revised the manuscript. C.-X.Z. organized and directed the project.

Corresponding authors

Correspondence to Hai-Jun Xu or Chuan-Xi Zhang.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 cDNA sequences of NlInR1 and NlInR2.

a, b, NlInR1 (a) and NlInR2 (b) share highly similar domain architectures. The signal peptide (SP) indicated by a vertical arrow is produced via a cleavage site at the amino terminus. Two ligand-binding loops (L1 and L2) and the furin-like cysteine-rich (Fu) region are underlined with solid lines. Three fibronectin type 3 (FN3) domains are labelled with dashed underlines. A single transmembrane (TM) region is highlighted with a box. An ‘NPXY’ motif is shown in green. The highly conserved tyrosine kinase domain (TyrKc) is indicated in blue. A triple tyrosine cluster (YXXXYY), is indicated in red. c, The sequence identity (%) of each domain in the InR receptors compared to its counterpart in the human insulin receptor (HsInR). Dm, D. melanogaster. d, The alignment of the N-terminal part of the Fu domains of NlInR1, NlInR2, DmInR and HsInR. The four cysteine residues that are absent in NlInR2 are indicated by asterisks.

Extended Data Figure 2 Spatio-temporal expression analyses of NlInR1 and NlInR2.

a, Quantitative polymerase chain reaction with reverse transcription (qRT–PCR) analysis was performed on various tissues dissected from 4th-instar nymphs (n = 100), 5th-instar nymphs (n = 60) and adult females (n = 30). ac, NlInR1 was widely expressed in all tissue samples collected from 4th-instar nymphs (a), 5th-instar nymphs (b) or adult females (c). df, NlInR2 was predominately expressed in wing buds in 4th-instar nymphs (d) and 5th-instar nymphs (e), but was widely expressed in adult tissues (f). af, Error bars represent the s.e.m of three technical replicates. g, Relative expression levels of NlInR1 and NlInR2 across development. First-instar nymphs (n = 60), 2nd-instar nymphs (n = 60), 3rd-instar nymphs (n = 20), 4th-instar nymphs (n = 20), 5th-instar (n = 20) nymphs and adults (n = 20) were used for RNA extraction. A relatively high expression of NlInR2 was observed in the 4th- and 5th-instar nymphs. Mean ± s.e.m. from three experiments, P values are indicated (Student’s t-test). SWF, short-winged females; LWF, long-winged females; SWM, short-winged males; LWM, long-winged males.

Extended Data Figure 3 Effects of knockdown of components of the IIS pathway.

a, Knockdown of NlInR2, NlFoxo or NlPten. b, Knockdown of NlInR1, NlChico or NlAkt. Forewing veins are patterned normally. c, The proportion of short-winged (SW) BPHs, from three experiments (mean ± s.e.m.). dsgfp (n = 142 females, 161 males), dsNlChico (n = 86 females, 98 males), dsNlLnk (n = 100 females, 117 males), dsNlPten (n = 89 females, 78 males), dsNlAkt (n = 106 females, 89 males) and dsNlFoxo (n = 78 females, 82 males). P < 0.05 and P ≤ 0.001 (Student’s t-test), difference from dsgfp. Source data are provided in Supplementary Data 4.

Source data

Extended Data Figure 4 Validation of RNAi effect using the second dsRNA molecules, and knockdown of NlInRs in BPHs at 3rd- and 4th-instar nymphs.

a, The proportion of short-winged (SW) BPHs after treatment of 2nd-instar nymphs with the second dsRNAs targeting NlInRs (dsNlInR1_B or dsNlInR2_B). dsgfp (n = 124 females, 100 males), dsNlInR1_B (n = 90 females, 92 males) and dsNlInR2_B (n = 103 females, 116 males). b, Knockdown of NlInRs in 3rd-instar nymphs. dsgfp (n = 117 females, 114 males), dsNlInR1 (n = 109 females, 130 males) and dsNlInR2 (n = 90 females, 100 males). c, Knockdown of NlInRs in 4th-instar nymphs. dsgfp (n = 101 females, 126, males), dsNlInR1 (n = 90 females, 117 males) and dsNlInR2 (n = 94 females, 100 males). d, The 3rd-instar nymphs were treated with a second dsRNA targeting several key components in the canonical insulin signalling pathway. dsgfp (n = 96 females, 100 males), dsNlChico_B (n = 72 females, 78 males), dsNlAkt_B (n = 86 females, 78 males), dsNlPten_B (n = 111 females, 88 males) and dsNlFoxo_B (n = 95 females, 91 males). ad, Mean ± s.e.m. from three experiments. P < 0.05 and P ≤ 0.001 (Student’s t-test), difference from the ratio obtained for dsgfp. Source data are provided in Supplementary Data 5.

Source data

Extended Data Figure 5 Knockdown of components of the TORC1 and the Ras/MAPK signalling cascades have no effect on wing morph switch.

a, Third-instar nymphs were treated with dsRNAs targeting NlTor and NlRaptor genes in the TORC1 complex, and targeting NlErk and NlRaf in the Ras/MAPK signalling pathway. dsgfp (n = 134 females, 153 males), dsNlTor (n = 122 females, 130 males), dsNlRaptor (n = 103 females, 95 males), dsNlErk (n = 138 females, 148 males) and dsNlRaf (n = 148 females, 143 males). Mean ± s.e.m. from three experiments, no significant change from dsgfp (Student’s t-test). b, Knockdown of NlRheb, an activator for TORC1 signalling activity. Third-instar nymphs were used for dsNlRheb treatment. Seventy-eight individuals (74%) failed to extricate themselves from the old cuticle, and died without completing ecdysis. Twenty-eight individuals finished nymph–adult transformation, of which twenty-five individuals (24%) were unable to stretch their wings correctly, and only three individuals (3%) showed normal morphology. Source data are provided in Supplementary Data 6.

Source data

Extended Data Figure 6 NlInR2 regulates wing morph switch in a tissue-specific way.

an, Second-instar nymphs were treated with dsRNAs targeting gfp, NlInR1, NlInR2, NlChico or NlFoxo, and 4th-instar nymphs were used for the dsNlAkt treatment. a, The short-winged BPHs (dsgfp-SW, n = 20) and long-winged BPHs (dsgfp-LW, n = 20) had a similar length of hind tibiae. The BPHs treated either with dsNlInR2 (n = 20) or with double RNAi (dsNlInR2;dsNlFoxo, n = 20) had a similar length of hind tibiae as the dsgfp-LW (n = 20). b, The BPHs treated with dsNlInR2 (n = 20) or dsNlInR2;dsNlFoxo (n = 20) possessed forewings of the same size or only slightly smaller than those of dsgfp-BPHs (dsgfp-LW, n = 20). c, d, Knockdown of NlInR1 (n = 20), NlChico (n = 20) or NlAkt (n = 20) further reduced the hind tibiae length (c) and forewing size (d) compared to short-winged BPHs treated with dsgfp (dsgfp-SW, n = 20). e, Knockdown of NlInR1 (n = 50) or NlChico (n = 44) but not NlInR2 (n = 66) delayed nymphal development. f, Knockdown of NlInR1 but not NlInR2 resulted in body weight loss in 5th-instar nymphs. dsgfp (n = 107; 110, 24 h; 72 h), dsNlInR1 (n = 117; 82, 24 h; 72 h), and dsNlInR2 (n = 111; 103, 24 h; 72 h). ‘24 h’ and ‘72 h’ represent 24 h and 72 h after ecdysis, respectively. gl, Knockdown of NlInR1 but not NlInR2 reduced levels of glycogen, trehalose and glucose both in nymphs (gi) and adult females (jl). m, n, Knockdown of NlInR1 but not NlInR2 increased levels of triglycerides in both nymphs (m) and adult females (n). an, Mean ± s.e.m. from three experiments. Tukey’s test in a, b, and Student’s t-test in cn, difference from dsgfp (P < 0.05 and P ≤ 0.001). Source data are provided in Supplementary Data 7.

Source data

Extended Data Figure 7 Subcellular localization of NlFoxo in wing buds and fat body.

a, b, Localization of NlFoxo in wing buds (a) and fat body (b). Third-instar nymphs were treated with dsRNAs, and wing buds and fat body were dissected from 5th-instar nymphs (48 h after ecdysis). For LY294002 treatment, wing buds and fat body dissected from untreated 5th-instar nymphs (48 h after ecdysis) were used for immuno-staining. Red, anti-NlFoxo. Blue (DAPI), cell nucleus. c, Proportion of cells with DAPI/Foxo co-localization in wing buds and fat body treated with dsNlInR2 or dsNlPten. Error bars represent mean ± s.e.m. from cells in three images. P ≤ 0.001 (Student’s t-test). d, Knockdown of NlInR2 did not increase P-NlAkt levels in the fat body. Fat body treated with dsgfp, dsNlInR1, dsNlInR2 or dsNlPten was probed with various antibodies.

Extended Data Figure 8 Tissue distribution of NlIlp1–4 in nymphs and adult females.

al, Various tissues dissected from 4th-instar nymphs (n = 100), 5th-instar nymphs (n = 60) and adult females (n = 30) were exposed to qRT–PCR assays to detect NlIlp1 (ac), NlIlp2 (df), NlIlp3 (gi) and NlIlp4 (jl) transcripts. The fold changes for transcripts in the head versus the fat body are indicated. Mean ± s.e.m. from three technical replicates.

Extended Data Figure 9 Knockdown of two insulin receptors in planthoppers, Sogatella furcifera and Laodelphax striatellus.

a, The wing morphs of S. furcifera planthoppers (SFPs). b, Knockdown of S. furcifera (Sf)InR1 resulted in the short-winged morph. dsgfp (n = 110 females, 89 males), dsSfInR1 (n = 101 females, 135 males) and dsSfInR2 (n = 132 females, 143 males). c, The wing morphs of L. striatellus planthoppers (LSPs). d, Knockdown of L. striatellus (Ls)InR2 resulted in the long-winged morph. dsgfp (n = 107 females, 123 males), dsLsInR1 (n = 42 females, 128 males) and dsLsInR2 (n = 113 females, 107 males). e, Third-instar nymphs were treated with dsRNAs targeting gfp (n = 200), LsInR1 (n = 180) or LsInR2 (n = 190). The survival rate of LSPs was monitored every day before metamorphosis. Mean ± s.e.m. from three experiments, no significant change from dsgfp (Student’s t-test). f, The metamorphosis rate was monitored every day at nine days after dsRNAs treatments. dsgfp (n = 200), dsLsInR1 (n = 180) and dsLsInR2 (n = 190). Most dsLsInR1-treated nymphs (>70%) died before adult eclosion, and thus yielded a significantly low metamorphosis rate. bf, Mean ± s.e.m. from three experiments. P < 0.05 and P ≤ 0.001 (Student’s t-test), difference from dsgfp. Source data are provided in Supplementary Data 8.

Source data

Extended Data Figure 10 Examination of RNAi efficiency by qRT–PCR.

av, Individual nymphs were pooled (n = 20) to extract total RNA after dsRNAs treatments, and cDNA was synthesized with random primers. The relative expression of each gene was normalized to the expression level of ribosomal 18S rRNA. Mean ± s.e.m. from three experiments.

Supplementary information

Supplementary information

This file contains Supplementary notes 1-9 and Supplementary References. (PDF 800 kb)

Supplementary Data

This file contains Supplementary Table 1 which shows the sex ratio of BPHs following dsNlInRs treatments. (XLSX 9 kb)

Supplementary Data

This file contains Supplementary Table 2, a list of the main primers used in this study. (XLS 40 kb)

An immunofluorescence assay of NlILP3 in brains of 5th-instar nymphs

The stained nerve cords (red) were crossed over a short distance from the medial neurosecretory cells to extend backwards in an arc through the brain. The cell nucleus is stained with DAPI (blue). (MOV 1542 kb)

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Xu, HJ., Xue, J., Lu, B. et al. Two insulin receptors determine alternative wing morphs in planthoppers. Nature 519, 464–467 (2015). https://doi.org/10.1038/nature14286

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