Molecular characterization and functional analysis of USP-1 by RNA interference in the Asian gypsy moth Lymantria dispar

Abstract

Ecdysteroids play an important role in regulating diverse physiological processes in arthropods, such as molting, metamorphosis, reproduction and diapause. Ecdysteroids mediate the response by binding to a heterodimeric complex of two nuclear receptors: the ecdysone receptor and the ultraspiracle (USP). To investigate the role of USP in development of the Asian gypsy moth (Lymantria dispar), a USP cDNA was obtained from the transcriptome of L. dispar and verified by PCR. In-depth profiling of transcript levels of L. dispar USP-1 (LdUSP-1) at different developmental stages and over time in third-instar larvae and different tissues isolated during the third-instar stage of L. dispar was then carried out. Transcript levels of LdUSP-1 were relatively high before 72 h in the third-instar larvae after ecdysis and in the adult male. The function of LdUSP-1 in molting was analyzed by knockdown of LdUSP-1 in third instar larvae using RNA interference. Silencing of LdUSP-1 significantly downregulated the transcript level of E75, an ecdysone-inducible gene, and of Sad, a Halloween gene. In addition, the duration of the third-instar stage was slightly shortened and larval mortality increased after the LdUSP-1 knockdown.

Keywords

Lymantria dispar / Ultraspiracle gene / Ecdysone / RNA interference / Development

References

[1] Buszczak M, Segraves WA. Insect metamorphosis: out with the old, in with the new. Curr Biol, 2000, 10: 830-833.

[2] Cao JJ, Liu YC, Yang YH, Zhang HJ, Li Z, Yang QP, Zhang SD, Zhang QW, Liu XX. Molecular characterization and functional analysis of the ultraspiracle (USP) in the oriental fruit moth Grapholita molesta (Lepidoptera: Olethreutidae). Comp Biochem Physiol B, 2015, 190: 54-62.

[3] Forman BM, Samuels HH. Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model. Mol Endocrinol, 1990, 4: 1293-1301.

[4] Giraudo M, Audant P, Feyereisen R, Goff GL. Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics. J Insect Physiol, 2013, 59: 560-568.

[5] Hall BL, Thummel CS. The RXR homolog ultraspiracle is an essential component of the Drosophila ecdysone receptor. Development, 1998, 125: 4709-4717.

[6] Hannan GN, Hill RJ. Lcusp, an ultraspiracle gene from the sheep blowfly, Lucilia cuprina: cDNA cloning, developmental expression of RNA and confirmation of function. Insect Biochem Mol Biol, 2001, 31: 771-781.

[7] Jindra M, Huang JY, Malone F, Asahina M, Riddiford LM. Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol, 1997, 6: 41-53.

[8] Kapitskaya M, Wang S, Cress DE, Dhadialla TS, Raikhel AS. The mosquito ultraspiracle homologue, a partner of ecdysteroid receptor heterodimer: cloning and characterization of isoforms expressed during vitellogenesis. Mol Cell Endocrinol, 1996, 12: 119-132.

[9] Kumar MB, Fujimoto T, Potter DW, Deng Q, Palli SR. A single point mutation in ecdysone receptor leads to increased ligand specificity: implications for genes witch applications. Proc Natl Acad Sci USA, 2002, 99: 14710-14715.

[10] Lenaerts C, Wielendaele PV, Peeters P, Broeck JV, Marchal E. Ecdysteroid signalling components in metamorphosis and development of the desert locust, Schistocerca gregaria. Insect Biochem Mol Biol, 2016, 75: 10-23.

[11] Marchal E, Vandersmissen HP, Badisco L, Van de Velde S, Verlinden H, Iga M, Wielendaele PV, Huybrechts R, Simonet G, Smagghe G, Broeck J. Control of ecdysteroidogenesis in prothoracic glands of insects: a review. Peptides, 2010, 31: 506-519.

[12] Martin D, Maestro O, Cruz J, Mané-Padrós D, Bellés X. RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica. J Insect Physiol, 2006, 52: 410-416.

[13] Moeller ME, Danielsen ET, Herder R, O’Connor MB, Rewitz KF. Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila. Development, 2013, 140: 4730-4739.

[14] Nakagawa Y, Henrich VC. Arthropod nuclear receptors and their role in molting. Special Issue Proteases Proteolysis Health Dis, 2009, 276: 6128-6157.

[15] Parvy JP, Wang P, Garrido D, Maria A, Blais C, Poidevin M, Montagne J. Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster. Development, 2014, 141: 3955-3965.

[16] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 2001, 29: e45.

[17] Raikhel AS, Brown MR, Belles X. Hormonal control of reproductive processes. Compr Mol Insect Sci, 2005, 3: 433-491.

[18] Renaud JP, Moras D. Structural studies on nuclear receptors. Cell Mol Life Sci CMLS, 2000, 57: 1748-1769.

[19] Sakurai S. Feedback regulation of prothoracic gland activity. Compr Mol Insect Sci, 2005, 3: 409-431.

[20] Song QS, Gilbert LL. Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland. Insect Biochem Mol Biol, 1998, 28: 849-860.

[21] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol, 2007, 24: 1596-1599.

[22] Tavva VS, Dinkins RD, Palli SR, Collins GB. Development of a methoxyfenozide-responsive genes witch for applications in plants. Plant J, 2006, 45: 457-469.

[23] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997, 25: 4876-4882.

[24] Tsai MJ, O’Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem, 1994, 63: 451-486.

[25] Verma P, Tapadia MG. Early gene broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster. Mol Immunol, 2015, 66: 325-339.

[26] Vogtli M, Imhof MO, Brown NE, Rauch P, Spindler-Barth M, Lezzi M, Henrich VC. Functional characterization of two ultraspiracle forms (CtUSP-1 and CtUSP-2) from Chironomus tentans. Insect Biochem Mol Biol, 1999, 29: 931-942.

[27] Wang SF, Li C, Zhu J, Miura K, Miksicek RJ, Raikhel AS. Differential expression and regulation by 20-hydroxyecdysone of mosquito ultraspiracle isoforms. Dev Biol, 2000, 218: 99-113.

[28] Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H. A canonical structure for the ligand-binding domain of the nuclear receptors. Nat Struct Biol, 1996, 3: 87-94.

[29] Xu QY, Deng P, Li A, Zhang Q, Mu LL, Fu KY, Guo WC, Li GQ. Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay. Insect Mol Biol, 2019

[30] Yan T, Chen H, Sun Y, Yu X, Xia L. RNA Interference of the ecdysone receptor genes EcR and USP in grain aphid (Sitobion avenae F). Affects its survival and fecundity upon feeding on wheat plants. Int J Mol Sci, 2016 17 12 2098

[31] Yao TP, Forman BM, Jiang ZY, Cherbas L, Chen JD, McKeown M, Cherbas P, Evans RM. Functional ecdysone receptor is the product of EcR and ultraspiracle genes. Nature, 1993, 366: 476-479.

[32] Zheng WW, Yang DT, Wang JX, Song QS, Gilbert LI, Zhao XF. Hsc70 binds to ultraspiracle resulting in the upregulation of 20-hydroxyecdsone-responsive genes in Helicoverpa armigera. Mol Cell Endocrinol, 2010, 315: 282-291.