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Ecdysone Signaling Underlies the Pea Aphid Transgenerational Wing Polyphenism

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Ecdysone Signaling Underlies the Pea Aphid Transgenerational Wing Polyphenism Ecdysone signaling underlies the pea aphid transgenerational wing polyphenism Neetha Nanoth Vellichirammala,1, Purba Guptab, Tannice A. Hallc, and Jennifer A. Brissonb,2 aSchool of Biological Sciences, University of Nebraska–Lincoln, Lincoln, NE 68588; bDepartment of Biology, University of Rochester, Rochester, NY 14627; and cDepartment of Life Sciences, University of the West Indies, Mona, Kingston 7, Jamaica Edited by David L. Denlinger, Ohio State University, Columbus, OH, and approved December 21, 2016 (received for review October 24, 2016) The wing polyphenism of pea aphids is a compelling laboratory particular aphid genotype likely depends on a balance between the model with which to study the molecular mechanisms underlying high fecundity of wingless morphs and the superior dispersal phenotypic plasticity. In this polyphenism, environmental stressors ability of the winged morph. Wing morph determination in pea such as high aphid density cause asexual, viviparous adult female aphids occurs prenatally, during embryogenesis (11). Only during aphids to alter the developmental fate of their embryos from this sensitive period can embryos receive the maternal signal that wingless to winged morphs. This polyphenism is transgenerational, determines the wing phenotype. Once born, a nymph’s de- in that the pea aphid mother experiences the environmental velopmental trajectory, and thus its adult phenotype, is set. signals, but it is her offspring that are affected. Previous research We previously performed a genome-wide transcriptional pro- suggested that the steroid hormone ecdysone may play a role in filing study on pea aphids (Acyrthosiphon pisum) during prenatal this polyphenism. Here, we analyzed ecdysone-related gene ex- wing determination to identify the key molecular pathways in- pression patterns and found that they were consistent with a volved in this process (12). We found that the genes differentially down-regulation of the ecdysone pathway being involved in the expressed between aphid mothers producing winged or wingless production of winged offspring. We therefore predicted that progeny suggested that ecdysone signaling may play a role in reduced ecdysone signaling would result in more winged offspring. controlling wing morph determination. For example, the pro- Experimental injections of ecdysone or its analog resulted in a thoracicostatic peptide gene, whose gene product inhibits ecdy- decreased production of winged offspring. Conversely, interfering sone synthesis (13), was at higher levels in females producing with ecdysone signaling using an ecdysone receptor antagonist or winged offspring. This indicated that ecdysone levels were lower knocking down the ecdysone receptor gene with RNAi resulted in these females. Consistent with this inference, genes that are ac- in an increased production of winged offspring. Our results tivated in response to dropping ecdysone levels [eclosion hormone, are therefore consistent with the idea that ecdysone plays a causative role in the regulation of the proportion of winged crustacean cardioactive peptide, bursicon (14)] were at higher levels offspring produced in response to crowding in this polyphenism. in female adults producing winged offspring. In contrast, genes that Our results also show that an environmentally regulated maternal are ecdysone inducible, or are coactivators of the ecdysone receptor – hormone can mediate phenotype production in the next genera- (15 17), were at higher levels in adult females producing wingless tion, as well as provide significant insight into the molecular mecha- offspring. These include the two genes whose products form the nisms underlying the functioning of transgenerational phenotypic heterodimeric ecdysone receptor (EcR and ultraspiracle), along plasticity. with other well-known ecdysone-inducible genes such as broad and ftz-f1. Combined, these data showed that genes associated with wing polyphenism | phenotypic plasticity | pea aphid | ecdysone signaling were differentially expressed in aphid mothers Acyrthosiphon pisum | ecdysone signaling Significance ne of the clearest examples of the effects of organism– Oenvironment interactions is phenotypic plasticity, where a Phenotypic plasticity is common in nature, yet the mechanisms single genotype can give rise to multiple phenotypes, depending by which environmental inputs are detected and alter de- on environmental cues (1). Polyphenism is the most extreme form velopmental programs are not well understood. Even less of phenotypic plasticity, with only a few discontinuous phenotypes explored are the mechanisms underlying transgenerational produced. Despite considerable interest, known control mecha- plasticity. Here we address the problem of how asexual aphids nisms of polyphenic development remain limited to a handful of produce winged or wingless offspring in response to external taxa (e.g., refs. 2–6) in which environmental factors act directly cues. We find that gene expression patterns suggest the im- on the individuals that go on to display the corresponding pheno- portance of the hormone ecdysone in this polyphenism. We type. In contrast, aphids exhibit a transgenerational wing poly- show that aphids injected with ecdysone or its analog produce phenism in which external cues act maternally and are transmitted fewer winged offspring, and aphids with inhibited ecdysone to offspring, which are the individuals that develop into the alter- signaling produce more winged offspring. Our results provide native winged and wingless morphs. Wing polyphenism in aphids insight into the hormonal control of alternative aphid morphs, has served as a powerful evolutionary model for the study of trade- which have long been a textbook example of polyphenism, but offs between dispersal and reproduction (7–9), but the molecular whose control mechanisms have long proven elusive. mechanisms underlying the induction of winged vs. wingless aphids Author contributions: N.N.V. and J.A.B. designed research; N.N.V., P.G., and T.A.H. per- remain unknown. More generally, little is known about the mo- formed research; N.N.V., P.G., T.A.H., and J.A.B. analyzed data; and N.N.V. and J.A.B. lecular mechanisms that control transgenerational polyphenism. wrote the paper. The goal of our study was to illuminate these mechanisms. The authors declare no conflict of interest. The aphid wing polyphenism occurs during the asexual portion This article is a PNAS Direct Submission. of the aphid life cycle. Wingless asexual females parthenoge- 1Present address: Department of Genetics, Cell Biology, and Anatomy, College of Medi- netically and viviparously produce genetically identical wingless cine, University of Nebraska Medical Center, Omaha, NE 68198-5805. and winged offspring. Winged aphids are produced in response 2To whom correspondence should be addressed. Email: [email protected]. PHYSIOLOGY to stressful environmental conditions such as crowding, poor food This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. quality, or the presence of predators (10). The overall success of a 1073/pnas.1617640114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1617640114 PNAS | February 7, 2017 | vol. 114 | no. 6 | 1419–1423 Downloaded by guest on October 1, 2021 that were induced to produce winged offspring compared with their we hypothesized that higher levels of ecdysone would result in uninduced counterparts (summarized in Fig. S1;geneidentities lower percentages of winged offspring. To test this, we raised listed in Table S1). aphids at moderate density (20 per plant) so that they would Ecdysone (and its bioactive derivative 20-OH ecdysone) is a produce both winged and wingless offspring. After reaching well-studied insect steroid hormone, primarily known for its role adulthood, aphids were placed on an artificial liquid diet (diet in molting and metamorphosis (18). Ecdysone binds to its nu- “A” of ref. 28) containing 250 μg/mL 20-hydroxyecdysone (20E) clear receptor, the ecdysone receptor, a heterodimer comprising or on a control without 20E for 24 h. As anticipated, 20E-treated the EcR protein and the Ultraspiracle protein (15). Upon as- females produced a significantly lower proportion of winged sociation with its hormone, the receptor binds cis-regulatory offspring compared with control aphids when ingesting 20E [Fig. DNA (19) elements and recruits cofactors to regulate tran- 1A, day 1; Mann–Whitney U test (MW) test, offspring counted scription (20–23). Ecdysone-mediated signaling therefore has the from n = 18 groups of three adult aphids for treatment, n = 19 ability to mediate the expression of the many phenotypes that groups of three aphids for control; P = 0.014]. This effect dis- differ between the two morphs by acting as a transcriptional sipated after the aphids were returned to plants posttreatment; modulator. Ecdysone-mediated control of seasonal morphs has offspring produced in the second 24 h did not have significantly been described before in the butterflies Araschnia levana, Precis different percentages of winged offspring (Fig. 1A, day 2; MW, coenia, and Bicyclus anynana (24–26), and the ecdysone receptor n = 18 groups of three aphids for treatment, n = 19 groups of three gene product (EcR) mediates the quantitative differences in aphids for control; P = 0.197). plasticity between fore and hindwings in Bicyclus anynana (27). To corroborate that ecdysone could cause a shift in the wing Here we demonstrate a causative role for ecdysone signaling in polyphenism
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