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. 2021 Feb 9;17(2):e1009312.
doi: 10.1371/journal.pgen.1009312. eCollection 2021 Feb.

Vestigial mediates the effect of insulin signaling pathway on wing-morph switching in planthoppers

Jin-Li Zhang  1 , Sheng-Jie Fu  1 , Sun-Jie Chen  1 , Hao-Hao Chen  1 , Yi-Lai Liu  1 , Xin-Yang Liu  1 , Hai-Jun Xu  1   2   3
Affiliations

Vestigial mediates the effect of insulin signaling pathway on wing-morph switching in planthoppers

Jin-Li Zhang et al. PLoS Genet. .

Abstract

Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a 'master signal' that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS-FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS-FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig 1
Fig 1. Wing-morph switching during the wing-morph decision stage.
(A) Wild-type LW and SW BPH adults. (B) Schematic diagram of regulation of NlInR1, NlInR2, and NlFoxO on LW and SW BPHs. NlInR1 and NlInR2 have opposite roles in wing-morph development, and depletion of NlInR1 and NlInR2 leads to SW and LW, respectively. NlFoxO negatively regulates LW development, and depletion of Nlfoxo leads to LW morphs. (C) Female adults with different wing morphs previously treated with dsNlInR2 or dsgfp. Forewings at the left side were removed. (D) Numbers of female adults with different wing morphs after dsNlInR2 treatment. Fifth-instar nymphs collected at designed time (6, 12, 18, 24, 30, 36, 42, and 48 hAE) were microinjected with dsNlInR2 or dsgfp. hAE, hours after ecdysis. (E) Numbers of female adults with different wing morphs after double-gene knockdown. Fifth-instar nymphs at 2 hAE were microinjected with dsNlInR2, and then microinjected with dsNlInR1 or dsgfp at 24 and 48 hAE. SW, short-winged. IMW, intermediate-size wings. LW, long-winged. Arrowheads, forewings. Arrows, hindwings. Non-significant (n.s.) and significant (***P < 0.001, Pearson’s χ2 test) differences from the control group (dsgfp) are indicated.
Fig 2
Fig 2. Regulation of Nlvg by the IIS pathway.
(A) Schematic depiction of the notum tissue used for RNA-seq. Fifth-instar nymphs at 2 h after ecdysis (hAE) were microinjected with dsNlInR2 or dsgfp. At 24- and 48-hAE, notum (indicate in yellow) was dissected for RNA-seq. (B) Numbers of up-regulated and down-regulated genes in dsNlInR2-treated notum compared to dsgfp treatment. The Nlvg expression level is indicated by an arrow. (C) Quantitative real-time PCR (qRT-PCR) analysis of Nlvg expression in fourth- and fifth-instar nymphs previously treated with dsNlInR2 or dsNlfoxo. (D) Temporal expression of Nlvg in the thorax of wild-type (wt) SW and LW BPH strains. (E) Tissue distribution of Nlvg in fifth-instar nymphs of the wt SW-BPH strain. (F) Nlvg expression in dsNlInR1-treated (SW-destined) and dsgfp-treated (LW-destined) nymphs. Fifth-instar nymphs (2 hAE) were microinjected with dsNlInR1, and then collected at 24, 48, and 72 hAE for quantification of Nlvg transcripts via qRT-PCR. Bar represents mean ± s.e.m. derived from three independent biological replicates. Statistical comparisons between two groups were performed using a two-tailed Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 3
Fig 3. Knockdown of Nlvg reverses the dsNlfoxo effect.
(A) Examination of RNAi efficiency by qRT-PCR. Third-instar nymphs were microinjected with dsNlvg or dsgfp, and then fifth-instar nymphs (n = 5) were collected for qRT-PCR analysis. The relative expression level of Nlvg was normalized to that of the Nl18s gene. Bar represents mean ± s.e.m. derived from three independent biological replicates. Statistical comparisons between two groups were performed using a two-tailed Student’s t-test (***P < 0.001). (B) Female adults with Nlvg or gfp knockdown. LW, long wings. IMW, intermediate-size wings. (C) Numbers of BPH adults with different wing morphs treated with dsNlvg or dsgfp. ***, P <0.001 (Pearson χ2 test: χ2 = 29.474, df = 1). (D) The relative wing size in BPHs treated with dsNlvg (n = 20) or dsgfp (n = 20). (E) Female adults with double-gene knockdown. Third-instar nymphs were microinjected with either dsNlfoxo;dsNlvg or dsNlfoxo;dsgfp. Forewings at the right side were removed. Arrow and arrow heads represent hindwings and forewings, respectively. (F) Numbers of BPH adults with different wing morphs treated with dsNlfoxo;dsgfp or dsNlfoxo;dsNlvg. ***, P <0.001 (Pearson χ2 test: χ2 = 254.06, df = 1). (G) Morphology of forewings in BPHs with double-gene knockdown. (H) Relative wing size in BPHs treated with dsNlfoxo;dsNlvg (n = 20) or dsNlfoxo;dsgfp (n = 20). Each dot represents the wing size and tibia length derived from an individual female.
Fig 4
Fig 4. Knockdown of S. furcifera vestigial homologue (Sfvg).
(A) Wild-type LW and SW S. furcifera adults. (B) Knockdown of Sfvg led to an adult with intermediate-size wings (n = 20). (C) Morphology of forewings treated with dsSfvg or dsgfp. (D) Relative wing size in S. furcifera treated with dsSfvg (n = 20) or dsgfp (n = 20). Each dot represents the wing size and tibia length derived from an individual female. LW, long-winged. SW, short-winged. IMW, intermediate-size wings.
Fig 5
Fig 5. Electrophoretic mobility shift assay (EMSA).
(A) Schematic depiction of the NlFoxO domains and sequence alignment. NlFoxO contains a DNA binding domain (DBD) and a transactivation domain (TAD). The NlFoxO DBD was aligned with its orthologs from D. melanogaster (dFoxO), C. elegans (DAF16), and H. sapiens (hFoxO3). (B) Schematic diagram of the distribution of the FoxO recognition element (FRE) in Nlvg. The FoxO consensus binding site (TGTTTAC or its complementary sequence, in red) and its flanking sequences are indicated in FRE1–5. The FRE1_m was generated by randomly mutating the FoxO consensus binding site (TGTTTAC) of FRE1 to ‘CCATTAC’ (in green). (C) EMSA of MBP/DBD binding to Alexa Fluor 680-labeled FRE1-5. (D) EMSA of the MBP/DBD binding to Alexa fluor 680-labeled FRE1, unlabeled FRE1, and mutant FRE1 (FRE1_m). (E) EMAS of the MBP/DBD binding to Alexa fluor 680-labeled FRE1 containing substitutions within the FoxO consensus sequences. The protein-DNA complex is indicated by arrowheads. The relative binding affinity is shown at the bottom. (F) Binding of MBP/DBD to FRE1–5 in a yeast one-hybrid assay. Vectors containing p53 and mutant FRE1 (FRE1_m) served as positive and negative controls, respectively. AbA, Aureobasidin A antibiotic. Experiments were performed three times with similar results (1st-, 2nd-, and 3rd-rep.).
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References

    1. Harrison RG. Dispersal polymorphisms in insects. Annu Rev Ecol Syst. 1980;11:95–118. 10.1146/annurev.es.11.110180.000523 - DOI
    1. Roff DA. Life-history consequences of bioenergetic and biomechanical constraints on migration. Am Zool. 1991;31:205–15.
    1. Denno RF, Roderick GK. Density-related dispersal in planthoppers-Effects of interspecific crowding. Ecology. 1992;73:1323–34. 10.2307/1940679 - DOI
    1. Roff DA. The evolution of wing dimorphism in insects. Evolution. 1986;40:1009–20. 10.1111/j.1558-5646.1986.tb00568.x . - DOI - PubMed
    1. Roff DA, Fairbairn DJ. Wing dimorphisms and the evolution of migratory polymorphisms among the insecta. Am Zool. 1991;31:243–51.

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HJX was supported by grants from National Natural Science Foundation of China (http://www.nsfc.gov.cn/) (Grants 31522047, 31772158, and 31972261). The funders have no role in the study design, data collection and analysis, decidson to publish, or preparation of the manuscript.

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