Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria

Accepted Manuscript Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria Xiaoming Zhao, Zhongyu Qin, Weimin Liu, Xiaojian Liu, Bernard Moussian, Enbo Ma, Sheng Li, Jianzhen Zhang PII:

S0965-1748(17)30178-9

DOI:

10.1016/j.ibmb.2017.11.001

Reference:

IB 3006

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 16 July 2017 Revised Date:

24 October 2017

Accepted Date: 1 November 2017

Please cite this article as: Zhao, X., Qin, Z., Liu, W., Liu, X., Moussian, B., Ma, E., Li, S., Zhang, J., Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Nuclear receptor HR3 controls locust molt by regulating chitin

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synthesis and degradation genes of Locusta migratoria

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Xiaoming Zhao1#, Zhongyu Qin1,2#, Weimin Liu1, Xiaojian Liu1, Bernard Moussian3, Enbo Ma1,

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Sheng Li4 and Jianzhen Zhang1* Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China 2

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College of Life Science, Shanxi University, Taiyuan, Shanxi 030006, China

Angewandte Zoologie, TU Dresden, Zellescher Weg 20b, Dresden 01217, Germany; iBV,

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Universit e Nice, Parc Valrose, Nice 06000, France 4

Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Sciences and School of Life Sciences, South China Normal University, Guangzhou

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510631, China

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* Authors for correspondence: [email protected]

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#

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Abstract:

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20-Hydroxyecdysone (20E) regulates the molting process through activation of a series of

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genes including E74, E75 and HR3 by the 20E receptor EcR. Here, we analyzed the function

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of LmHR3 in the migratory locust Locusta migratoria. By sequence comparison, we first

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identified and characterized the putative nuclear receptor protein (LmHR3) based on L.

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migratoria transcriptome data. The full length cDNA is 2272 bp long encoding a protein of

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455 amino acids that contains a DNA binding domain (zinc finger) and a ligand binding

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domain. Phylogenetic analyses showed that LmHR3 has a high homology with the ortholog

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from Blattaria. RT-qPCR results revealed that LmHR3 has a low level expression in the early

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days of 5th instar nymphs, and then increases and peaks at day 6, followed by a decrease to

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low levels before ecdysis. The LmHR3, hence, coincides with the profile of circulating 20E

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levels. Indeed, we show that transcription of LmHR3 is induced by 20E in vivo, and

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significantly suppressed by successfully knocking down expression of LmEcR. After injection

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of dsRNA for LmHR3 (dsLmHR3) at day 2 of earlier instar nymphs (3rd and 4th instar) and

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final instar nymphs (5th instar), none of the nymphs were able to molt normally, and

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eventually died. Chitin staining and ultra-structural analysis showed that both the synthesis of

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the new cuticle and the degradation of the old cuticle were blocked in the dsLmHR3 treated

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nymphs. Especially, chitin synthesis genes (LmUAP1 and LmCHS1) and chitinase genes

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(LmCHT5 and LmCHT10) were significantly down-regulated in the dsLmHR3 treatment

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group. Together, our results suggest that LmHR3 is involved in the control of chitin synthesis

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and degradation during L. migratoria molting.

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Keywords: HR3, RNA interference, chitin synthesis genes, chitinase genes, Locusta

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migratoria

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1. Introduction

growth

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development

of

insects,

the

steroid

hormone

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These authors contributed equally to this work

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ACCEPTED MANUSCRIPT The integument of insects consists of cuticle, epidermal cell and basement membrane.

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The cuticle, which is secreted by epidermal cell, is made up of epicuticle, exocuticle and

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endocuticle, in that order from the outside to the inside (Moussian et al., 2006). As an

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important component of insects, it is vital to protect insects from outside harm, and the cuticle

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has an important role in this physiological function. Insect molting involves two persistent

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parts including apolysis and shedding of the old cuticle. The old cuticle falls off gradually

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with the generation of new cuticle in the development of insects. First, the occurrence of

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apolysis is under the molt fluid. The cortical layer is first formed, then the envelope and

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epicuticle. As the envelope begins to be deposited, the old cuticle is gradually digested at the

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same time and the inner cuticle is synthesized (Simpson and Douglas, 2013).

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20-Hydroxyecdysone (20E) plays an important role in insect molting. During the growth

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and development of insects, 20E regulates behaviors such as embryonic development, molting,

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morphogenesis and reproduction at different developmental stages (Cruz et al., 2007).

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Nuclear receptors are a class of proteins found within cells that are responsible for sensing

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steroid and thyroid hormones and certain other molecules. The structures of nuclear receptors

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are similar including a highly conserved DNA-binding domain (DBD) and a ligand-binding

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domain (LBD) (Tan and Palli, 2008). The function of nuclear receptors is generally the

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transmission of upstream signals, binding to the special areas of the downstream genes and

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turning on or off transcription (Laudet, 1997). 20E coordinates the expression and biological

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reactions of multiple nuclear receptor genes systematically in vivo (Lam et al., 1997). 20E

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binds to a heterodimer of nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle

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(USP), to transmit signals down, directly induces transcription of early 20E-response genes

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such as BR-C, E74A and E75A, and up-regulates an early-late gene HR3 during larval-pupal

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transition (Horner et al., 1995; White et al., 1997). In Drosophila melanogaster, the

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expression of DmHR3 is coincident with the peaks of 20E during the development (Ruaud et

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al., 2010). DmHR3, an early-late gene, in contrast to other early 20E-response transcription

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factors in 20E cascade reaction process, is induced in third instar larvae of D. melanogaster

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(Lam et al., 1997). This delay in expression of DmHR3 is necessary in the transition from

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larvae to pre-pupae, which is similar to that of MsHR3 in Manduca sexta (Hiruma et al., 1997;

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Lan et al., 1997; Langelan et al., 2000; Riddiford et al., 2003). In D. melanogaster, DmHR3

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suppresses 20E early response factors Br-C, E74, E75, whereas it induces a late response

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factor βFtz-F1 downstream. The promoter of βFtz-F1 contains a DmHR3 direct binding site

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(Parvy et al., 2014). These findings suggest that DmHR3 translates the transmission of the

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20E signal and activates the transcription of βFtz-F1 (a key factor in prepupal to the pupal

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transition), and finally completes the transition. It has been found that DmHR3 is very

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important for the formation of adult bristles, wings and the epidermis. Mutation of DmHR3

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causes its embryos to die and the formation of the nervous system and muscle is also

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defective (Carney et al., 1997; Xu et al., 2010). In hemimetabolous insects, Blattella

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germanica, the genes, BgE75、BgHR3 and BgFTZ-F1 are also implicated in response to 20E

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by its receptors BgEcR-A/BgRXR-S/BgRXR-L (BgRXR-S/BgRXR-L homologous to USP). The

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silencing of the above genes by RNAi experiments affects normal insect development and

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metamorphosis (Cruz et al., 2007; Guo et al., 2015). The results showed that HR3 plays an

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important role in molting and metamorphosis of insects. Chitin is a polysaccharide composed of N-acetylglucosamine residues linked by β 1, 4

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glycosidic linkages. It is found in a variety of organisms including plants such as algae, fungi,

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and animals such as shrimp, crab, and insects (Vilcinskas, 2013). In insects, chitin is mainly

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distributed in extracellular matrices secreted by ectodermal epithelial tissues, including

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cuticles, trachea, foregut and hindgut, and the peritrophic matrix secreted by midgut epithelial

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cells. It is chiefly located in procuticle of insects integument (Moussian, 2010). Insects

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undergo periodic molting during the process of growth, which is accompanied by the

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degradation of the old cuticular chitin and the synthesis of chitin in the new cuticle (Kramer et

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al., 1993). The chitin synthesis pathway begins with trehalose, and involves at least eight

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enzymes, including UDP-N-Acetylglucosamine pyrophosphorylases (UAP1 and UAP2),

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chitin synthases (CHS1 and CHS2) (Merzendorfer and Zimoch, 2003). Chitin synthesis genes

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were found to be regulated by 20E in the molting of insects, such as Spodoptera exigua (Yao

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et al., 2014), Ostrinia furnacalis (Qu and Yang, 2012). Insect chitinases are involved in the

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degradation of the old cuticle, which belongs to family 18 of glycoside hydrolases (GH18),

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and these genes have unique developmental and tissue expression patterns in different insects

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(Doucet and Retnakaran, 2012). Chitin degrading enzymes play important roles in insect

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molting, extension of wings, digestion, and immunity (Hale et al., 2015). Chitinase5 (CHT5)

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is the first gene discovered in the M. sexta (Kramer et al., 1993). So far, CHT5 homologous

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genes have been identified from at least 15 different insects, including Bombyx mori,

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Hyphantria cunea (Kim et al., 1998), and Tribolium castaneum (Zhu et al., 2008a). Locusta migratoria is a widespread and destructive agricultural pest in the world (Wang

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et al., 2014). In L. migratoria, several chitin synthesis and chitinase genes were identified and

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shown to be involved in chitin metabolism during molting (Li et al., 2015; Liu, 2013; Liu et

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al., 2012; Yang et al., 2016). In this paper, we identified and characterized a putative LmHR3

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gene based on L. migratoria transcriptomic and genomic databases. The transcription of

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LmHR3 can be induced by 20E in vivo. Further studies suggested that LmHR3 could control

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locust molting by regulating chitin synthesis and chitinase genes of L. migratoria.

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2. Materials and methods

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2.1 Insects rearing

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The eggs of L. migratoria were purchased from locust breeding center of Hebei, China.

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The eggs were incubated at 28±1°C, 50% relative humidity with a light/dark (14 h/10 h) in

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our laboratory. After hatching, the nymphs were fed with fresh wheat sprouts under the same

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conditions. The 3rd, 4th and 5th instar nymphs were prepared for total RNA isolation and RNAi 3

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in the study.

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2.2 Bioinformatics analysis of LmHR3

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The cDNA sequence of LmHR3 was obtained from L. migratoria transcriptome database

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(Zhao et al., 2017) and the genome of L. migratoria (Wang et al., 2014). The amino acid

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sequence of LmHR3 was translated from the cDNA sequence by the translation tools at

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ExPASy

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(http://www.sanger.ac.uk/Software/Pfam/) and SMART (http://smart.embl.de/) tools were

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used to determine the deduced protein domains. The signal peptide, molecular weight and

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isoelectric point were predicted in the EXPASY proteomics server (http://www.expasy.org).

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The gene structure was determined by searching the L. migratoria genome with LmHR3

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cDNA sequence as queries by using the NCBI Blast tool. The exon-intron organization was

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graphed using the Adobe Illustrator CS5 software (Adobe, USA). Multiple amino acid

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sequence alignments were performed using GENEDOC software with the default parameters.

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A neighbor-joining tree was constructed using MEGA6 software with statistical analysis by

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the bootstrap method, using 1000 repetitions. The GenBank accession numbers are listed in

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Table 1.

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2.3 Tissue-specific and developmental expression analysis of LmHR3

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(http://www.expasy.org/tools/dna.html).

For tissue-specific expression analysis of LmHR3, nine different tissues including

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integument, wing pads, Malpighian tubule, foregut, midgut, hindgut, gonads, and fat body

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were dissected from day 6 of fifth instar nymphs. For the developmental expression analysis,

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abdominal cuticles were carefully separated every day during the fifth-instar stage (ranging

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from N5D1 to N5D8). Total RNA extraction was performed using RNAiso Plus (TaKaRa,

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Tokyo, Japan) according to the manufacturer’s protocol. Four nymphs were collected for

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tissue dissection in one replicate, and four independent biological replications were applied.

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The quality and quantity of total RNA were evaluated on 1.5% agarose gel and NanoDrop

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2000 (Thermo, USA). One µg of total RNA was used to synthesize first-strand cDNA by

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using M-MLV reverse transcriptase (TaKaRa, Japan). Each cDNA sample was diluted 10-fold

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for reverse-transcription quantitative PCR (RT-qPCR) analysis. RT-qPCR analysis was

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performed using SYBR® Premix ExTaq™ II (TaKaRa, Tokyo, Japan) and ABI7300

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Real-Time PCR System (Applied Biosystems, CA, USA). The RT-qPCR reactions contained

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10 µl of 2×SYBR® Premix EX Taq™ (TaKaRa, Japan), 0.4 µl of 50×ROX Reference Dye

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(TaKaRa, Japan) and 2 µl of specific primers (2 µM), and consisted of initial step at 95°C for

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30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s. A melting curve was

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determined for each sample to detect the gene-specific peak and check for the absence of

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primer-dimers. Relative mRNA levels of target genes were calculated with the 2-∆Ct method,

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and the target gene expression level was normalized to the expression of the internal marker

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gene RPL-32 (ribosomal protein L32) that exhibited the most stable expression at different

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stages and in different tissues (Yang et al., 2014). The primer information for each gene is

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listed in Table 2.

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2.4 20-Hydroxyecdysone treatment and RNAi of ecdysone receptor gene (LmEcR) The day 2 of 5th instar nymphs were used for 20-Hydroxyecdysone (20E) treatment.

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According to the results of Li et al., (Li et al., 2015), each nymph in treatment group was

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injected with 10 µg 20E (Sigma, St. Louis, MO, USA) dissolved in 10% ethanol with the final

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concentration of 1 µg/µl, whereas in the control group, each nymph was injected with an

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equal volume of 10% ethanol. All the treated nymphs were reared in beakers as described

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above. For transcriptional analysis of LmHR3, the abdominal segments of nymphs were

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dissected at 1, 3, 6 and 12 h after the treatment, and four nymphs were used as a biological

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replication. To further confirm whether LmHR3 was induced by ecdysone signal, we

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performed RNAi experiment with double-stranded RNA (dsRNA) of ecdysone receptor gene

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(LmEcR). DsLmEcR and dsGFP (green fluorescent protein, GFP) were prepared in vitro using

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T7 RiboMAXTM Express RNAi System (Promega, USA). The primers used for dsRNA

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synthesis are shown in Table S2. For RNAi, 10 µg dsRNA of LmEcR (dsLmEcR) and GFP

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(dsGFP) were injected into the hemocoel between the second and third abdominal segments

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of each 5th instar nymph (2-day) by using a microinjector (Ningbo, China). After 48 h, the

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abdominal segments were collected for expression analyses for LmHR3. Total RNA was

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isolated and RT-qPCR was carried out for gene expression analysis following the above steps.

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2.5 Functional analysis of LmHR3 by RNAi

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To explore the biological roles of LmHR3 in locust development, dsRNA of LmHR3 was

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synthesized using T7 RiboMAXTM Express RNAi System (Promega, USA) as described

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above. 10 µg dsLmHR3 was injected into the hemocoel between the second and third

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abdominal segments of each 3rd, 4th and 5th instar nymph (2-day) by using a microinjector

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(Ningbo, China). dsGFP was injected as control group. Three biological replicates were

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applied for dsGFP and dsLmHR3 injection, and each with 10 or 12 nymphs. The nymphs

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treated with dsRNA were reared under the same conditions as described above for observing

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their phenotypes. To determine silencing efficiency, relative transcript levels of LmHR3 in

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both dsGFP and dsLmHR3 injection nymphs were measured by RT-qPCR as described above.

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2.6 Microsection and Chitin staining

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To further explore the effects of LmHR3 RNAi on cuticle development, microsection and

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chitin staining were performed as described (Song et al., 2016). In brief, paraffin sections (5

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µm) of the third abdominal cuticles from nymphs at day 6 after injection of dsLmHR3 or

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dsGFP in the 2-day 5th instar nymphs were prepared. Locust apolysis began at day 6 of fifth

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instar nymphs (Liu et al., 2016), the sectioned cuticle samples were collected before ecdysis,

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and contained both the newly formed cuticle and the old cuticle. Then tissues were incubated 5

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(Toprak et al., 2010). After washing the tissues three times with PBS, propidium iodide was

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added to label nuclei for 5 min. After washing three times with PBS, the stained samples were

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imaged using a LSM 880 confocal laser-scanning microscope (Zeiss, Germany) at 60

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magnifications. All the images in each staining were collected under the same conditions.

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2.7 Transmission electron microscopy

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To further observe the structure of cuticle after treated with dsLmHR3 or dsGFP,

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transmission electron microscopy (TEM) was performed as described (Liu et al., 2009). The

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third abdominal cuticles from each of three locusts at day 6 after treatment with dsLmHR3 or

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dsGFP in the 2-day 5th instar nymphs were dissected, and fixed with 3% glutaraldehyde in 0.2

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M phosphate buffer (pH 7.2) for 48 h at 4°C. The samples were then rinsed 3 times with the

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phosphate buffer followed by post-fixation in 1% osmium tetroxide for 3 h at 4°C. The

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samples were washed twice, each for 10 min, and put into a series of ascending concentration

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of acetone (50, 70, 80, 90 and 100%) for dehydration. They were embedded in Epon 812 at

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room temperature for 2 h. Then the samples were trimmed to prepare ultrathin sections.

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Sections were collected on copper grids, and images were captured with a

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JEM-1200EX transmission electron microscope (TEM, JEOL, Japan).

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2.8 Expression of chitin synthesis and chitinase genes after RNAi of LmHR3

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To determine the expression of chitin synthesis genes (LmUAP1 and LmCHS1) and

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chitinase genes (LmCHT5 and LmCHT10), the integuments were collected from the locusts

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treated with dsLmHR3 and dsGFP for 48 h at day 2 of 3rd, 4th and 5th instar nymphs,

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respectively. RT-qPCR was used to analyze the expression of LmUAP1, LmCHS1, LmCHT5

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and LmCHT10 as described above. Three independent biological replicates were performed.

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2.9 Statistical Analysis

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All of the data were statistically analyzed by independent sample student t-test. Asterisks

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indicate significant differences (*, P<0.05; **, P<0.01; ***, P<0.001).

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3. Results

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3.1 Identification and characterization of LmHR3

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LmHR3 was identified from the L. migratoria transcriptome and genome database.

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Comparing the cDNA sequence with the locust genomic sequence, the cDNA of LmHR3 is

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2272 bp containing ten exons (Fig. 1A). The open reading frame is 1368 bp encoding a

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protein of 455 amino acids containing a zinc finger domain (amino acids 11-82) and a ligand

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binding domain (amino acids 261-424), with a theoretical molecular weight and pI for the

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protein of 51.2 kDa and pH 7.10, respectively (Fig. 1B), which is similar to HR3 of D. 6

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sequence alignment analysis showed a high conservation in DNA binding domain and ligand

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binding domain with other insects (Fig. 1C). Phylogenetic analyses using full length sequence

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revealed that LmHR3 is in the same cluster with Blattaria HR3 and has a high similarity with

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HR3 orthologs from different insect species (Fig. 1D).

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3.2 Tissue-specific and developmental expression of LmHR3

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To investigate the expression characteristics of LmHR3 in L. migratoria, RT-qPCR was

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used to analyze the expression of LmHR3 in different tissues and developmental stages of 5th

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instar nymphs. The results showed that LmHR3 was distributed in all the tested tissues on day

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6 of 5th instar nymphs, and had a high expression level in integument, foregut, hindgut and fat

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body (Fig. 2A). The expression of LmHR3 during the 5th instar nymphal stage showed that

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LmHR3 mRNA was expressed at a low level in the early nymphal days (N5D1-N5D3), then

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increased gradually starting from N5D4, reached a peak on N5D6, followed by a decrease to

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low level from N5D7 (Fig. 2B). The expression pattern of LmHR3 is coincident with the 20E

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titer (Liu et al., 2016), implying that it may be induced by 20E.

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3.3 Expression of LmHR3 in response to 20E

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To test whether LmHR3 is induced by 20E, day 2 of 5th instar nymphs were treated with

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20E in solvent 10% ethanol or 10% ethanol (control) for 1, 3, 6 and 12 h. The abdominal

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segments of nymphs after treatment were dissected to detect expression of LmHR3 by

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RT-qPCR. The results showed that the expression of LmHR3 was significantly up-regulated

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after treatment with 20E compared to that of control at 1, 3, 6 and 12 h, and its expression

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level increased gradually from 1 h, peaking at 3 h, and then decreased slightly from 6 h to 12

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h (Fig. 3A). To further confirm whether LmHR3 is regulated by ecdysone signaling pathway,

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LmEcR was knocked down by dsRNA mediated RNAi on day 2 of 5th instar nymphs, and

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LmHR3 mRNA was determined at 48 h after RNAi by RT-qPCR. The results showed that the

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mRNA level of LmHR3 was significantly down-regulated after injection of dsLmEcR

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compared to that of control insects (Fig. 3B).

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3.4 Functional analysis of LmHR3

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In order to investigate the role of LmHR3 in the locust molting process, dsRNAs for

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LmHR3 (dsLmHR3) and GFP (dsGFP, control) were synthesized in vitro and injected into the

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hemocoel between the second and third abdominal segments of each 5th instar nymph (2-day).

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Compared to the control, the expression of LmHR3 significantly decreased at 48 h after

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injection with dsLmHR3 at the 2-day 5th instar nymphs (Fig. 4). In the control group, all the

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nymphs molted normally to adults during nymphal-adult transition (Fig. 4A), whereas the

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nymphs with injection of dsLmHR3 were unable to molt, retained the nymph form, and

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eventually died (Fig. 4B). In order to detect effects of dsLmHR3 injection on 7

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nymphal-nymphal transition, we performed RNAi experiments at day 2 of 3rd and 4th instar

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nymphs as described above. Similarly, nymphal-nymphal molt was also disrupted after

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injection of dsLmHR3 (Fig. S1A’-B’ lower) when compared to those of control insects (Fig.S1

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A’-B’ upper). These results suggested that LmHR3 is involved in the nymphal-nymphal and

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nymphal-adult transition in L. migratoria. In order to observe the structure of the cuticle affected by dsLmHR3, we performed

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microsection and chitin staining of nymphs at day 6 after treatment with dsLmHR3 at day 2 of

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5th instar nymphs. We observed that apolysis occurred before ecdysis after treated with

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dsLmHR3 or dsGFP (Fig. 5A). However, we found that the newly formed cuticle of nymphs

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with dsLmHR3 treatment was thinner than that of the control nymphs treated with dsGFP. In

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contrast, the old cuticle was thicker in insects with reduced LmHR3 expression than in control

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insects (Fig. 5). The thickness of the newly formed cuticle and the old cuticle were measured

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using a light microscope. We found that the thickness of both the new cuticle and the old

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cuticle between dsGFP and dsLmHR3 treatments was significantly different (Fig. 5B). To

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further observe the ultra-structural changes in the cuticle after interfering with LmHR3

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expression, we performed TEM analysis. As shown in Fig. 5C, the old cuticle was degraded

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distinctly in the control insects, whereas there was obvious lamellar structure in the old cuticle

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of insects treated with dsLmHR3 (Fig. 5C, upper). In contrast, the new cuticle was normally

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formed with lamellar structure in the control case, but not in the dsLmHR3 treated locust (Fig.

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5C, lower). In addition, we also observed pathogens in the apical site of the old cuticle and

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between the new cuticle and the old cuticle in the dsLmHR3 treated locusts, which were

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maintained under the same conditions as the control animals (Fig. 5C). As reported, insect

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cuticle prevents fungal pathogens to infect, penetrate and degrade the cuticle through a

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number of proteases and chitin degrading enzymes (Evison et al., 2017). Thus, we speculate

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that suppressing LmHR3 blocks the formation of the new cuticle and the degradation of the

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old cuticle, which finally led to the declined immunity of locusts and infection by pathogens.

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3.5 Effects of LmHR3 RNAi on transcripts of chitin synthesis and degradation genes

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How did LmHR3 affect the formation of the new cuticle and the degradation of the old

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After RNAi with dsLmHR3 or dsGFP at 3rd, 4th and 5th instar nymph (2-day), we

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cuticle?

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examined the expression of chitin synthesis genes (LmUAP1 and LmCHS1) and chitinase

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genes (LmCHT5 and LmCHT10) which are involved in the synthesis and degradation of

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cuticular chitin, respectively. The results of RT-qPCR showed that the transcript levels of

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LmUAP1 and LmCHS1, LmCHT5 and LmCHT10 were significantly down-regulated in

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nymphs treated with dsLmHR3 compared to those of control insects (Fig. 6, and Fig.S2),

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suggesting that LmHR3 is involved in the regulation of genes involved in chitin synthesis and

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degradation during nymphal-nymphal and nymphal-adult molt. Thus, we propose a

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hypothesis that LmHR3 controls the molting of locusts by directly or indirectly regulating the

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expression of chitin synthase and chitinase in L. migratoria. 8

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Discussion Molts are triggered and regulated by invertebrate steroid hormones secreted by

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prothoracic glands which are collectively referred to as ecdysteroids (Rees, 1989). Ecdysis

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depends on fast fluctuations of circulating 20-hydroxyecdysone (20E) occurring when the

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levels peak and then decline after the molt, which regulate the synthesis and release of

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transcription factors governing the behavior associated with ecdysis (Riddiford, 1985). In D.

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melanogaster, it was found that HR3, an ecdysone-inducible early-late gene, is required for

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the prepupal-pupal transition and differentiation of adult structures during metamorphosis

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(Carney et al., 1997; Lam et al., 1999). After silencing HR3, the larvae of Leptinotarsa

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decemlineata are unable to enter the pupal stage and retain the nymph form (Guo et al., 2015).

305

Similarly, the RNAi experiment of HR3 in B. germanica also indicated that HR3 is involved

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in its molting process (Cruz et al., 2007). In Caenorhabditis elegans, interference with

307

expression of HR3, the homologue of Drosophila HR3, led to molting arrest, and CeHR3

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induced expression of dpy-7 in epidermal cells, which is involved in worm molting

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(Kostrouchova et al., 1998, 2001). However, the mechanism how HR3 controls the molting of

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insects remains unclear.

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In this paper, we identified a nuclear receptor gene HR3 through the locust

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transcriptomic database, and its mRNA expression pattern in developmental stages of 5th

313

instar nymphs is coincident with that of 20E titer (Liu et al., 2016). We further demonstrated

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that LmHR3 expression is mediated by 20E signaling, the expression is suppressed by

315

silencing LmEcR and induced by 20E in vivo (Fig. 3). In B. germanica, the expression of

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BgHR3 was found to be the highest at 2 days before molting, and directly induced by 20E,

317

which suggested that BgHR3 is involved in ecdysis (Cruz et al., 2007). Similar results were

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obtained in lepidopteran insects such as M. sexta and Helicoverpa armigera (Langelan et al.,

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2000; Zhao et al., 2004). Insect growth and metamorphosis are strictly dependent on the

320

capability to remodel chitin-containing structures. Chitin synthases and chitinases are

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responsible for the synthesis or degradation of chitins, respectively (Merzendorfer, 2003). In

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the process of insect molting, chitinases hydrolyze the old cuticle and peritrophic

323

matrix-associated chitin into soluble sugars, and thus play important roles in insect survival,

324

reproduction, and molting (Arakane and Muthukrishnan, 2010; Merzendorfer, 2003; Zhang et

325

al., 2012; Zhu et al., 2008b). Previous studies have shown that several enzymes are involved

326

in the chitin biosynthetic pathway (Merzendorfer, 2006; Zhu et al., 2016), and that RNAi

327

against these genes leads to molting defects, loss of chitin, and mortality in D. melanogaster

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(Wang et al., 2015), T. castaneum (Arakane and Muthukrishnan, 2010; Zhu et al., 2008b),

329

Haemaphysalis longicornis (Huang et al., 2007), Spodoptera exigua (Zhang et al., 2012), and

330

Nilaparvata lugens (Xi et al., 2015). Our results showed that after interfering with expression

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of LmHR3 by RNAi, the locusts are unable to molt and die during nymphal-nymphal and

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nymphal-adult transition (Fig. 4), suggesting that LmHR3 is also involved in molting of

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locusts. But whether it controls molting of insects by regulating the expression of chitin

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metabolism is yet unclear. In L. migratoria, two chitin biosynthesis genes (LmUAP1 and LmCHS1) and two chitin

336

degradation genes (LmCHT5 and LmCHT10) have been shown to be involved in the synthesis

337

and degradation of chitin, respectively (Li et al., 2015; Liu et al., 2016; Yang et al., 2016). In

338

our studies, we found that LmHR3 RNAi blocked ecdysis of the locust, and they were unable

339

to molt during nymphal-nymphal and nymphal-adult transition after interfering with

340

expression of LmHR3 (Fig. 4 and Fig. S1). Furthermore, we observed that the newly formed

341

cuticle of dsLmHR3-treated insects was thinner than that of control insects. In contrast, the

342

old cuticle was thicker in dsLmHR3 treated insects than in control insects by light microscopy

343

and TEM, indicating that silencing of LmHR3 affected the formation of the new cuticle and

344

degradation of the old cuticle. Finally, we found that RNAi of LmHR3 suppressed the

345

expression of the two chitin biosynthesis genes and two chitinase genes during

346

nymphal-nymphal and nymphal-adult transitions (Fig. 6 and Fig. S2). Taken together, our

347

results suggested that LmHR3 controls locust molting by regulating chitin synthesis and

348

degradation during nymphal-nymphal and nymphal-adult transition. In addition, although 20E

349

induces the expression of chitin synthase and chitinase genes, its roles in the regulation of

350

chitin synthase and chitinase genes remain a matter of controversy. 20E activates the

351

transcription of chitin biosynthesis genes (DmeCHS-1 and DmeCHS-2) during Drosophila

352

metamorphosis (Gagou et al., 2002, Gangishetti et al. 2012), and chitinase genes (LmCHT5

353

and TmCHT5) expression during the molting process of L. migratoria and Tenebrio molitor,

354

respectively (Li et al., 2015; Royer et al., 2002). However, the regulative relations between

355

20E and chitin synthase genes or chitinase genes are still largely unknown. In the present

356

paper, we showed that LmHR3 mediates the 20E signaling to regulate the expression of chitin

357

synthesis and chitinase genes controlling the locust molt.

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Based on our results and previous studies, we propose a hypothesis for the roles of

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LmHR3 in the molting of the locust (Fig. 7). 20E binds to the heterodimer of the ecdysone

360

receptor (EcR and RXR), which activates the expression of early genes, then up-regulates the

361

expression of the early-late gene, LmHR3. Thus, our data underline that in insects, this

362

mechanism is evolutionary conserved. Reduced expression of LmHR3 using RNA

363

interference results in the inhibition of cuticle chitin synthesis and degradation by

364

down-regulating the expression of chitin biosynthesis genes (LmUAP1 and LmCHS1) and

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chitinase genes (LmCHT5 and LmCHT10), and nymphs injected with dsLmHR3 are unable to

366

normally molt and die with the block of cuticle chitin synthesis and degradation.

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Acknowledgments

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This work was supported by National Natural Science Foundation of China (Grant No.

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31640075, 31672364, 31402020), The Natural Science Foundation of Shanxi Province, China

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(201601D021102). We thank Dr. Subbaratnam Muthukrishnan (Department of Biochemistry 10

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& Molecular Biophysics, Kansas State University, Manhattan, KS 66506) for helpful

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comments during preparation of this manuscript. We also acknowledge Dr. Juanjuan Wang

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from the Scientific Instrument Center at Shanxi University for her help with LSM 880

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confocal laser-scanning microscope measurements.

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Author contribution XMZ and ZYQ conceived and coordinated the study and wrote the manuscript. JZZ, BM, SL,

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XJL and EBM revised figures and the manuscript. XMZ and ZYQ designed the experiments.

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XMZ, ZYQ and WML performed and analyzed the results of the experiments. All authors

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reviewed the results and approved the final version of the manuscript.

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Conflict of interest

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The authors declare that they have no conflicts of interest with the contents of this article. References

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Figure Legends

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Fig.1 Bioinformatics analysis of LmHR3 in Locusta migratoria. A. Genome structure of LmHR3; B. Schematic diagram of deduced domains of LmHR3.

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ACCEPTED MANUSCRIPT Yellow ovals represent DNA binding domain (zinc finger) and green hexagons represent ligand binding domain; C. Multiple sequence alignments of the deduced HR3 proteins in insect species. The DNA binding domain and ligand binding domain were boxed in black boxes, respectively; D. A phylogenetic tree was constructed with the neighbor-joining method of MEGA 6 using the pairwise deletion of indels. Bootstrap support was based on 1,000 resembled data sets. The GenBank accession numbers are listed in Table 1.

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Fig.2 Expression of LmHR3 in different tissues and stages of 5th instar nymph in L.

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migratoria.

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A. Expression of LmHR3 in different tissues of N5D6 nymphs as detected by RT-qPCR.

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Different tissues are listed. IN: Integument, WP: wing pads, MT: Malpighian tubules, FG:

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foregut, MG: midgut, HG: hindgut, GO: gonad, FB: fat body; B. Expression of HR3 in

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integument of N5D1-N5D8 nymphs as detected by RT-qPCR; RPL-32 was used as the

553

reference control. All data are reported as means ± SE of three independent biological

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replications.

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Fig.3 Relative expression levels of LmHR3 after 20E treatment and RNAi of LmEcR.

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A. Effect of 20E on LmHR3 mRNA expression detected by RT-qPCR. RNA was extracted at 1,

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3, 6 and 12 h after 20E treatment. CK: 10% ethanol injection; 20E: 20E injection; B. The

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expression of LmHR3 in locusts after dsGFP or dsLmEcR injection; RPL-32 was used as the

559

reference control. Data are reported as means ± SE of three independent biological

560

replications, asterisks indicate significant differences, *, P<0.05, **, P<0.01, ***, P<0.001.

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Fig.4 Effects of dsLmHR3 injection in the 5thinstar nymphs on the LmHR3 transcript

562

level and development of L. migratoria.

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A. Relative expression level of LmHR3 in integuments after the dsGFP injection as detected

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by RT-qPCR and the phenotype of the nymphs after dsGFP injection. All 5th instar nymphs

565

developed into adults successfully. B. Relative expression level of LmHR3 in integuments

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after the dsLmHR3 injection as detected by RT-qPCR and the phenotype of the nymphs after

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dsLmHR3 injection. All 5th instar nymphs could not develop into adults successfully, and died

568

before ecdysis.

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Fig.5 Chitin staining and TEM analysis of cuticle after injection with dsLmHR3.

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A. Effect on the formation of cuticle by chitin staining after injection with dsRNA. The

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paraffin sections (5 µm) of the third abdominal cuticles from nymphs at day 6 after injection

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of dsLmHR3 or dsGFP in the 2-day 5th instar nymphs were prepared. Propidium iodide was

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used to label Nuclei (red); Fluorochrome28 was used to stain chitin (blue). Scale bars, 20 µm.

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The old cuticle (old) is detached from the epithelial cells, shed and replaced by a new cuticle

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(new) during the molting process. B. The thickness (unit/mm) of old cuticle and new cuticle

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were measured. Data are reported as means ± SE of three independent biological replications,

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asterisks indicate significant differences, **, P<0.01. C. The ultra-structure of the new formed

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cuticle and the old cuticle was observed after RNAi in 5th instar nymphs through TEM. Pro:

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procuticle, Scale bar=2 µm.

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A-B. The expression of chitinase genes (LmCHT5 and LmCHT10)was examined by RT-qPCR

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after RNAi with dsLmHR3 or dsGFP; C-D. The expression of chitin synthesis genes

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(LmUAP1 and LmCHS1) was examined by RT-qPCR after RNAi with dsLmHR3 or dsGFP.

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RPL-32 was used as the reference control. Data are reported as means ± SE of three

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independent biological replications, asterisks indicate significant differences, *, P<0.05, **,

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P<0.01.

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Fig.7 Schematic description of the hypothesis that LmHR3 controls locust molt.

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20-hydroxyecdysone (20E) binds to the heterodimer of the ecdysone receptor (EcR and RXR),

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which activates the expression of early genes, then up-regulates the expression of early-late

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gene LmHR3. Reduced expression of LmHR3 using RNA interference could result in the

591

inhibition of cuticle chitin synthesis and degradation by down-regulating the expression of

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chitin biosynthesis genes (LmUAP1 and LmCHS1) and chitinase genes (LmCHT5 and

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LmCHT10), and unlike the control insects treated with dsGFP, nymphs injected with

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dsLmHR3 fail to molt normally with the block of cuticle chitin synthesis and degradation, and

595

die.

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Fig. S1 Effects of dsLmHR3 injection on the LmHR3 transcript level and development of

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the 3rd and 4th instar nymphs.

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A-A’. Relative expression level of LmHR3 in integuments as detected by RT-qPCR and the

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phenotype of the nymphs after the dsGFP or dsLmHR3 injection at day 2 of 3rd instar nymphs.

601

B-B’. Relative expression level of LmHR3 in integuments as detected by RT-qPCR and the

602

phenotype of the nymphs after the dsGFP or dsLmHR3 injection at day 2 of 4th instar nymphs.

603

All 3rd and 4th instar nymphs developed into next instar successfully after the dsGFP injection.

604

All 3rd and 4th instar nymphs could not molt successfully, and die before ecdysis after the

605

dsLmHR3 injection.

EP

606

TE D

597

Fig.S2 Effects of LmHR3 RNAi on transcripts of chitin synthesis and chitinase genes at

608

3rd and 4th instar nymphs.

609

A-D. The expression of chitinase genes (LmCHT5 and LmCHT10) and chitin synthesis genes

610

(LmUAP1 and LmCHS1) was examined by RT-qPCR after RNAi with dsLmHR3 or dsGFP at

611

day 2 of 3rd instar nymphs; A’-D’. The expression of chitinase genes (LmCHT5 and LmCHT10)

612

and chitin synthesis genes (LmUAP1 and LmCHS1) was examined by RT-qPCR after RNAi

613

with dsLmHR3 or dsGFP at day 2 of 4th instar nymphs.

614

RPL-32 was used as the reference control. Data are reported as means ± SE of three

615

independent biological replications, asterisks indicate significant differences, *, P<0.05, **,

616

P<0.01, ***, P<0.001.

AC C

607

617 618 16

ACCEPTED MANUSCRIPT Table 1 Species and GenBank accession no. for Phylogenetic tree used in this study

Blattaria

Hymenoptera

Hemiptera

Coleoptera

Gene name

GenBank accession no.

Locusta migratoria

LmHR3

KY817189

Zootermopsis nevadensis

ZnHR3

KDR23541.1

Blattella germanica

BgHR3 isoform A

CAJ90621.1

Blattella germanica

BgHR3 isoform B2

CAJ90623.1

Harpegnathos saltator

HsHR3

Cephus cinctus

CcHR3

Apis dorsata

AdHR3

Apis mellifera

AmHR3

Nilaparvata lugens

NlHR3

Cimex lectularius

ClHR3

Leptinotarsa decemlineata

LdHR3

Dendroctonus ponderosae

DpHR3

XP_019758192.1

Anoplophora glabripennis

AgHR3

XP_018562890.1

Tribolium castaneum

TcHR3

XP_015837105.1

AtHR3

XP_019866848.1

DpHR3

ACY56691.1

Daphnia magna

DmHR3

ACY56690.1

Aedes aegypti

AaHR3

AAF36970.1

Drosophila melanogaster

DmHR3

NP_001097256.1

Musca domestica

MdHR3

XP_019890398.1

Stomoxys calcitrans

ScHR3

XP_013103854.1

Aethina tumida

EP

Diptera

Daphnia pulex

TE D

Diplostraca

620

RI PT

Orthoptera

Species

XP_011153943.1 XP_015589102.1 XP_006621131.1 XP_016768650.1

SC

Order

APD25634.1 XP_014245653.1

AKN21733.1

M AN U

619

Table 2 Primer sequences used in this study

AC C

621 Gene

dsLmEcR

dsLmHR3

LmHR3

LmUAP1

LmCHS1

Primer sequences (5′-3′)

Application

Length of product (bp)

F:TAATACGACTCACTATAGGGGCAGCAACGCCGCACCCT R:TAATACGACTCACTATAGGGGCACTGGTACACGGCATTT

F:TAATACGACTCACTATAGGGGTTACTCATATAACAATGAT R:TAATACGACTCACTATAGGGCTGAGCACATTCAAGCCACA

F:GAAGGTGGAGGACGAGGTG R:TGCCGTTGTAGGCGGACTG F:TACGGGACCGTAAGGTGTTGG R:CCACATTCTGCATTTTTGCTTATAC F: CTTGAGCCAATTGGTTTGGT R: TGAGTTCTGTGGATGCAAGG 17

dsRNA

408

dsRNA

663

RT-qPCR

129

RT-qPCR

139

RT-qPCR

121

ACCEPTED MANUSCRIPT LmCHT5

LmCHT10

RPL-32

F:CATCAAAGCGAAGGGCTACGGC R:AGATTAGTGCGTCCTTCGGGCCA F:GCAATTGGTGGTTGGAATGAT R:GGTCTAGTCCTTCAAATCCATACTTTTC F:ACTGGAAGTCTTGATGATGCAG R:CTGAGCCCGTTCTACAATAGC

RT-qPCR

92

RT-qPCR

130

RT-qPCR

97

AC C

EP

TE D

M AN U

SC

RI PT

622

18

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Research Highlights We identified and characterized a nuclear receptor gene LmHR3 from Locusta migratoria transcriptome database. LmHR3 can be induced by 20E and repressed by dsLmEcR injection in vivo.

during nymphal-nymphal and nymphal-adult transition.

RI PT

The locusts failed to molt normally and eventually died after RNAi of LmHR3

RNAi of LmHR3 blocks the synthesis of new cuticle and degradation of old cuticle

of L. migratoria.

SC

LmHR3 controls locust molt by regulating chitin synthesis and degradation genes

AC C

EP

TE D

M AN U

of L. migratoria.