Kelch-like ECH associated protein 1 signaling is critical for the regulation of ecdysteroidogenesis in the larvae

Accepted Manuscript Leptinotarsa cap ‘n’ collar isoform C/Kelch-like ECH associated protein 1 signaling is critical for the regulation of ecdysteroidogenesis in the larvae Qiang-Kun Sun, Qing-Wei Meng, Qing-Yu Xu, Pan Deng, Wen-Chao Guo, Guo-Qing Li PII:

S0965-1748(17)30050-4

DOI:

10.1016/j.ibmb.2017.04.001

Reference:

IB 2940

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 21 November 2016 Revised Date:

27 March 2017

Accepted Date: 7 April 2017

Please cite this article as: Sun, Q.-K., Meng, Q.-W., Xu, Q.-Y., Deng, P., Guo, W.-C., Li, G.-Q., Leptinotarsa cap ‘n’ collar isoform C/Kelch-like ECH associated protein 1 signaling is critical for the regulation of ecdysteroidogenesis in the larvae, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.04.001. 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.

ACCEPTED MANUSCRIPT

At the late stage of the final larval instar

Synthesis of ecdysone

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Halloween genes

Ecdysone

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Shade

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PTTH

CncC Keap1

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prothoracic gland

20-Hydroxyecdysone

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Leptinotarsa cap ‘n’ collar isoform C/Kelch-like ECH associated

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protein 1 signaling is critical for the regulation of ecdysteroidogenesis

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in the larvae

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Qiang-Kun Sun1†, Qing-Wei Meng1†, Qing-Yu Xu1, Pan Deng1, Wen-Chao Guo 2,

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Guo-Qing Li 1 *

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1. Education Ministry Key Laboratory of Integrated Management of Crop Diseases

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and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing

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210095, China

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2. Department of Plant Protection, Xinjiang Academy of Agricultural Sciences;

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Urumqi 830091, China

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Running Head: Knockdown of Leptinotarsa CncC and Keap1

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Qiang-Kun Sun, [email protected]

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Qing-Wei Meng, [email protected]

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Qing-Yu Xu, [email protected]

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Pan Deng, [email protected]

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Wen-Chao Guo, [email protected]

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*Correspondence

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+86-25-84395248

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†

to

Guo-Qing

Li,

Co-first author 1

Email:

[email protected]

Tel/Fax:

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Abstract Drosophila cap ‘n’ collar isoform C (CncC) and Kelch-like ECH associated protein

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1 (Keap1) regulate metamorphosis by transcriptional control of a subset of genes

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involved in ecdysteroidogenesis, 20-hydroxyecdysone (20E) signaling, and juvenile

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hormone (JH) degradation. In the present paper, we found that prothoracicotropic

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hormone signal was required for the activation of LdCncC and LdKeap1 in

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Leptinotarsa decemlineata. Moreover, RNA interference of LdCncC or LdKeap1 in

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the fourth-instar larvae delayed development. As a result, the treated larvae obtained

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heavier larval and pupal fresh weights and had larger body sizes than the controls.

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Furthermore, knockdown of LdCncC or LdKeap1 significantly reduced the mRNA

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levels of four ecdysone biosynthetic genes (Ldspo, Ldphm, Lddib and Ldsad), lowered

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20E titer and decreased the transcript levels of five 20E response genes (LdEcR,

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LdUSP, LdE75, LdHR3 and LdFTZ-F1). However, the expression of two JH epoxide

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hydrolase genes and JH contents were not affected in the LdCncC and LdKeap1 RNAi

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larvae. Dietary supplementation with 20E shortened the developmental period to

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normal length, rescued the larval and pupal body mass rises, and recovered or even

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overcompensated the expression levels of the five 20E response genes in either

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LdCncC or LdKeap1 RNAi hypomorphs. Therefore, LdCncC/LdKeap1 signaling

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regulates several ecdysteroidogenesis genes, and consequently 20E pulse, to modulate

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the onset of metamorphosis in L. decemlineata.

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Key words: Leptinotarsa decemlineata, cap ‘n’ collar isoform C, Kelch-like ECH

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associated protein 1, 20-hydroxyecdysone, metamorphosis

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1. Introduction In insects, the combination of a high titer of 20-hydroxyecdysone (20E) and a low

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level of juvenile hormone (JH) triggers larval-pupal metamorphosis. Ecdysone is

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synthesized in insect prothoracic glands (PGs) from cholesterol, under the

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catalyzation of a series of cytochrome P450 monooxygenases (CYPs) encoded by

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Halloween genes such as spook (spo), phantom (phm), disembodied (dib) and shadow

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(sad). Ecdysone is then released from PGs into hemolymph. It is transported to

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peripheral tissues, and is converted to 20E by another CYP, the product of a

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Halloween gene shade (shd) (Iga and Kataoka, 2012; Niwa and Niwa, 2014). The

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expression of these Halloween genes and consequently the timing of pupation are

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regulated by prothoracicotropic hormone (PTTH)-Torso receptor-mitogen activated

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protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK

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and ERK) (McBrayer et al., 2007; Rewitz et al., 2009).

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A timely decrease in JH is also crucial for metamorphosis. JH is degraded mainly

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by two hydrolases, JH epoxide hydrolase (JHEH, EC 3.3.2.3) and JH esterase (JHE,

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EC 3.1.1.1) (Gu et al., 2015; Lü et al., 2015). JHEH falls into the microsomal epoxide

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hydrolase family (Arand et al., 2005; Morisseau and Hammock, 2005), and JHE

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belongs to the carboxylesterase family (Share and Roe, 1988).

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In Drosophila melanogaster, cap ‘n’ collar isoform C (CncC) and Kelch-like ECH

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associated protein 1 (Keap1), the homologs of mammalian nuclear factor erythroid 2

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related factor 2 (Nrf2) and Keap1, act as transcription activators of a subset of 3

ACCEPTED MANUSCRIPT Halloween and JH hydrolyzation genes (Deng, 2014; Deng and Kerppola, 2013,

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2014). Up to now, CncC and Keap1 homologs have been found in other insect species

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in both holometabolans and hemimetabolans (Deng and Kerppola, 2013; Grimberg et

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al., 2011; Kalsi and Palli, 2015; Karim et al., 2015; Misra et al., 2011; Misra et al.,

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2013; Peng et al., 2016; Sykiotis and Bohmann, 2008). An interesting question then

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arises: are CncC and Keap1 the conserved transcription activators of both

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ecdysteroidogenesis and JH hydrolyzation genes among insect species?

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Upon biosynthesis and release, 20E, acting through its cognate receptor, a dimer of

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ecdysone receptor (EcR)/ultraspiracle (USP), triggers a conserved transcriptional

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cascade including early genes such as Broad-Complex (BR-C), Ecdysone-induced

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protein 75 (E75) and E74, early-late genes such as hormone receptor 3 (HR3), and

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late genes such as Fushi tarazu factor 1 (FTZ-F1), to stimulate metamorphosis (Iga

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and Kataoka, 2012; Luan et al., 2013). In Drosophila, CncC/Keap1 signaling has been

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proven to activate several early ecdysone-response genes in the salivary glands (Deng,

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2014; Deng and Kerppola, 2013, 2014). Is CncC/Keap1 signaling involved in the

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activation of these early ecdysone-response genes in other insects?

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The Colorado potato beetle Leptinotarsa decemlineata (Say) has a robust RNA

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interference (RNAi) response to double stranded RNAs (dsRNAs) (Guo et al., 2015,

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2016; Kong et al., 2014; Liu et al., 2014; Shi et al., 2016a; Shi et al., 2016b; Shi et al.,

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2016c; Zhu et al., 2015). Using in vivo RNAi, we previously demonstrated that the

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ecdysteroidogenesis and 20E signaling genes were conserved in L. decemlineata (Guo

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et al., 2015, 2016; Kong et al., 2014; Liu et al., 2014; Zhu et al., 2015). In the work 4

ACCEPTED MANUSCRIPT presented here, we knocked down either LdCncC or LdKeap1 to study its roles in the

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larval-pupal metamorphosis in L. decemlineata.

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2. Materials and methods

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2.1. Experimental animal

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The L. decemlineata beetles were kept in an insectary according to a previously

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described method (Shi et al., 2013), with potato foliage at the vegetative growth or

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young tuber stages in order to assure sufficient nutrition. At this feeding protocol, the

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larvae progressed through the first, second, third, and fourth instars at an approximate

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period of 2, 2, 2 and 4 days, respectively. Upon reaching full size, the fourth-instar

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larvae stopped feeding, dropped to the ground, burrowed to the soil and entered the

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prepupae stage. The prepupae spent an approximately 3 days to pupate. The pupae

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lasted about 5 days and the adults emerged.

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2.2. Molecular cloning

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The putative LdCncC and LdKeap1 isoforms were obtained from the genome (https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project)

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transcriptome data (Shi et al., 2013). The correctness of the sequences was

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substantiated by polymerase chain reaction (PCR) using primers in Table S1. The

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full-length cDNAs were obtained by 5'- and/or 3'-RACE, using SMARTer RACE kit

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(Takara Bio.), with specific primers listed in Table S1. After obtaining full-length

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cDNAs, primer pairs (Table S1) were designed to verify the complete open reading

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frames. All of the sequenced cDNAs were submitted to GenBank (accession numbers:

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LdCncA, KY458169; LdCncB, KY458170; LdCncC1, KY458171; LdCncC2,

and

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KY458172; LdKeap1A, KY458173; LdKeap1B, KY458174).

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2.3. Preparation of dsRNAs The same method as previously described (Zhou et al., 2013) was used to express

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dsPTTH (214 bp), dsTorso (302 bp), dsRas (327 bp), dsphm (345 bp), dsshd (438 bp),

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dsEcR (344 bp), dsE75 (361 bp), dsCncC-1 (200 bp), dsCncC-2 (411 bp), dsKeap1-1

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(373 bp), dsKeap1-2 (302 bp) and dsegfp (a 414 bp fragment of enhanced green

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fluorescent protein gene). The twelve dsRNAs were individually transcribed with

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specific primers in Table S1, using Escherichia coli HT115 (DE3) competent cells

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lacking RNase III. Individual colonies were inoculated, and grown until cultures

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reached an OD600 value of 1.0. The colonies were then induced to express dsRNA by

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addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.1 mM.

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The expressed dsRNA was extracted and confirmed by electrophoresis on 1% agarose

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gel (data not shown). Bacteria cells were centrifuged at 5000 ×g for 10 min, and

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resuspended in an equal original culture volume of 0.05 M phosphate buffered saline

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(PBS, pH 7.4). The bacterial solutions (at a dsRNA concentration of about 0.5 µg/ml)

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were used for experiment.

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2.4. Dietary introduction of dsRNA

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The same method as previously reported (Fu et al., 2015) was used to introduce

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dsRNA into larvae. The newly-ecdysed fourth-instar larvae were allowed to feed

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foliage immersed with bacterial suspension containing dsPTTH, dsTorso, dsRas,

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dsphm, dsshd, dsEcR, dsE75, or each of the two dsRNAs of LdCncC (dsCncC-1 and

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dsCncC-2) and LdKeap1 (dsKeap1-1 and dsKeap1-2) for 3 days (replaced with 6

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freshly treated ones each day). The PBS- and dsegfp-dipped foliage were used as

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controls. The larvae were then transferred to untreated foliage if necessary. The beetles were weighed twice during trial period. The adult emergence was

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recorded during a 2-week trial period. The samples on day 3 after the initiation of the

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experiments were collected. The effects of gene silencing, the levels of five

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Halloween genes (Ldspo, Ldphm, Lddib, Ldsad, and Ldshd), five 20E response genes

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(LdEcR, LdUSP, LdE75, LdHR3 and LdFTZ-F1), a total of 14 LdGST transcripts and

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two Jheh genes, and 20E and JH titers were determined.

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To test the rescuing effects of 20E in larval development, two additional

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experiments were performed using foliage dipped with dsCncC-1, dsCncC-1+10-6 M

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20E, or dsKeap1-1, dsKeap1-1+10-6 M 20E, a concentration used in our previous

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research (Guo et al., 2016). The PBS- and dsegfp-immersed leaves were used as

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controls.

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For above expriments, three biological replicates were carried out. 2.5. Real-time quantitative PCR (qRT-PCR)

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Each sample contained 5-10 individuals and repeated three times. The RNA was

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extracted using SV Total RNA Isolation System Kit (Promega). Purified RNA was

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subjected to DNase I to remove any residual DNA according to the manufacturer’s

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instructions. Quantitative mRNA measurements were performed by qRT-PCR in

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technical triplicate, using internal control genes (the primers listed in Table S1)

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according to our published results (Shi et al., 2013). An RT negative control (without

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reverse transcriptase) and a non-template negative control were included for each

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contamination in the reactions, respectively. Data were analyzed by the 2-∆∆CT method,

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using the geometric mean of internal control genes for normalization. All methods

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and data were confirmed to follow the MIQE (Minimum Information for publication

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of Quantitative real time PCR Experiments) guidelines (Bustin et al., 2009).

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2.6. Quantitative determination of 20E and JH

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20E was extracted according to a ultrasonic-assisted extraction method (Liu et al.,

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2014), and its titer (ng per g body weight) was analyzed by a liquid chromatography

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tandem mass spectrometry-mass spectrometry (LC-MS/MS) system using a protocol

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the same as described (Zhou et al., 2011).

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Hemolymph was collected and JH was extracted following the methods

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described previously (Zhou et al., 2013). An LC-MS was used to quantify JH titers

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(ng per ml hemolymph) (Cornette et al., 2008).

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2.7. Data analysis

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The data were given as means ± SE, and were analyzed by ANOVA followed by the

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Tukey-Kramer test, using SPSS for Windows (SPSS, Chicago, IL, USA). A repeated

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measures ANOVA was used to test the effects of dsRNAs on larval development.

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Since no significant differences between dsRNAs targeting two different regions of

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either LdCncC or LdKeap1 (dsCncC-1 and dsCncC-2, dsKeap1-1 and dsKeap1-2)

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were found, the data of each gene were combined.

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3. Results 8

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3.1. The expression of LdCncC and LdKeap1 By mining the genome and transcriptome data and performing RT-PCR, we

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found that LdCnc had four splicing isoforms in L. decemlineata. LdCncA and LdCncB

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were truncated forms. LdCncC1 and LdCncC2 differed in the first exon but shared the

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following 10 exons. The four isoforms shared exon 8, 9 and 10 (Fig. S1A). LdKeap1

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gene possessed two splicing isoforms (LdKeap1A and LdKeap1B) which differed in

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the first exon (Fig. S2A). Phylogenetic analyses revealed that both CncC- and

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Keap1-like proteins formed order based separate clades. Obviously, LdCncC and

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LdKeap1 belonged to coleopteran clade (Fig. S1B and Fig. S2B).

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Using primers from the common sequences of LdCncC and LdKeap1 isoforms,

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we determined their temporal expression patterns. Both genes were expressed

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throughout all larval developmental stages. Within the first, second and third larval

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instars, their expression levels were higher just before and right after the molt, and

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were lower in the intermediate instar. In the fourth larval instar, the two genes reached

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their highest expression levels between 24 and 60 hours after ecdysis (Fig. 1).

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In D. melanogaster, CncC/Keap1 pathway is necessary and sufficient for

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xenobiotic-induced transcription of a wide range of detoxification genes including

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CYPs (e.g. DmCYP6A2, DmCYP6A8) and glutathione S-transferases (such as

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DmGSTd1, DmGSTe1) (Karim et al., 2015; Misra et al., 2011; Misra et al., 2013).

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Here we tested the expression of all LdGSTd genes that were identified previously in

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L. decemlineata (Han et al., 2016). We found that the three LdGSTd genes exhibited

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similar temporal expression patterns to LdCncC and LdKeap1 (Fig. S3).

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ACCEPTED MANUSCRIPT The tissue expression profiles of LdCncC and LdKeap1 genes were also tested.

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Their mRNAs were easily detectable in all tested tissues including brain-corpora

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cardiaca-corpora allata complex, prothoracic gland, foregut, midgut, hindgut,

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Malpighian tubules, epidermis, fat body and hemocyte of the day 2 fourth-instar

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larvae. The templates were also present in female ovary and male testis of the adults.

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LdCncC was highly expressed in larval midgut and prothoracic gland, and adult ovary,

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whereas LdKeap1 was transcribed at the highest level in the larval brain-corpora

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cardiaca-corpora allata complex, and at higher levels in the larval foregut and

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prothoracic gland, and the adult ovary (Fig. 2A, 2B).

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LdCncC

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cardiaca-corpora allata complex, prothoracic gland and fat body through the fourth

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instar stage were also tested. As expected, both LdCncC and LdKeap1 were highly

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expressed between 24 and 60 hours after ecdysis. Moreover, both genes were highly

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transcribed in brain-corpora cardiaca-corpora allata complex and prothoracic gland

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(Fig. 2C, 2D).

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3.2. PTTH-Torso signaling is required for the expression of LdCncC and LdKeap1

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The temporal transcription patterns of LdCncC and LdKeap1 reminded us of the

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expression fluctuation of LdTorso (Zhu et al., 2015). To test whether LdPTTH

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regulates LdTorso transcription in vivo, we examined the transcript levels of LdPTTH

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and LdTorso throughout the fourth-instar larvae in brain-corpora cardiaca-corpora

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allata complex, prothoracic gland and fat body. As expected, LdPTTH exhibited a

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similar expression pattern to LdTorso in the three representative tissues (Fig. S4). 10

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LdCncC and LdKeap1 in vivo, LdCncC and LdKeap1 mRNA levels in the LdPTTH,

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LdTorso and LdRas RNAi larvae (Fig. S5) were tested. Compared with those in the

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control specimens, LdCncC and LdKeap1 transcription levels were dramatically

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decreased in these RNAi hypomorphs

(Fig. 3A-3C).

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Our previous results showed that RNAi of the Halloween genes reduced 20E

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titers in L. decemlineata (Kong et al., 2014). In this study, we knocked down Ldphm

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and Ldshd to lower 20E titers (Fig. S6), and found that LdCncC and LdKeap1 mRNA

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levels were significantly increased in the Ldphm and Ldshd RNAi larvae, compared

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with those in the controls (Fig. 3D, 3E).

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In L. decemlineata, LdEcR-A, LdUSP, LdE75, LdHR3 and LdFTZ-F1 have been

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identified (Guo et al., 2015, 2016; Liu et al., 2014; Ogura et al., 2005). In this survey,

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we knocked down LdEcR-A and LdE75 (Fig. S7), and found that inhibition of 20E

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signaling in the LdEcR-A and LdE75 RNAi larvae had no influence or upregulated the

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expression of LdCncC and LdKeap1, compared with those in the control specimens

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(Fig. 3F, 3G).

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3.3. Knockdown of LdCncC or LdKeap1 delays larval development

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To dissect the physiological roles of LdCncC and LdKeap1 in larval development,

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we dietarily introduced each of the two dsRNAs derived from the common sequences

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of either LdCncC or LdKeap1 into the newly-molted fourth-instar larvae. Combined

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data revealed that continuous ingestion of a dsCncC or a dsKeap1 for 3 days

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significantly downregulated its target gene (Fig. 4A, 4D). 11

ACCEPTED MANUSCRIPT In D. melanogaster, depletion of DmCncC reduced whereas knockout of

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DmKeap1 increased the expression of DmGSTd1 and DmGSTe1 (Karim et al., 2015;

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Misra et al., 2011; Misra et al., 2013). In order to evaluate whether ingestion of a

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dsCncC or a dsKeap1 successfully knocks down its target gene, we also tested the

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expression levels of all LdGSTd and LdGSTe members in the resultant larvae. Out of

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the 14 transcripts, the expression of 11 ones was significantly suppressed in the

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dsCncC-fed larvae, whereas the expression of 4 was significantly activated and 5 was

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dramatically repressed in the dsKeap1-fed larvae. Interestingly, the expression of

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LdGSTd1 and LdGSTe1 mimicked that of their Drosophila partners in the DmCncC-

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and DmKeap1-depleted flies (Fig. S8, Fig. S9).

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The dsCncC-fed larvae spent a longer period of time to develop (Fig. 4B).

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Moreover, fully-grown larvae and pupae obtained heavier fresh weights and had

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larger body sizes than the controls (Fig. 4C, 4G and 4H).

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Knockdown of LdTKeap1 completely simulated the negative effects observed in the LdCncC hypomorphs (Fig. 4).

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3.4. Effects of dsCncC and dsKeap1 on PTTH, 20E and JH signaling pathways

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The expression levels of LdPTTH, LdTorso and LdRas were measured in the

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LdCncC and LdTKeap1 RNAi larvae. All the three genes showed similar transcription

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levels in the LdCncC and LdKeap1 RNAi larvae to those in the controls (Fig. S10).

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In D. melanogaster, CncC and Keap1 have been documented to regulate

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Halloween genes (Deng, 2014; Deng and Kerppola, 2013). In L. decemlineata, five

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Halloween genes (spo, phm, dib, sad and shd) have been cloned (Kong et al., 2014; 12

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Wan et al., 2013). We determined their expression levels in the LdCncC and LdKeap1

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RNAi larvae. Ingestion of dsCncC and dsKeap1 significantly downregulated the expression of

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Ldspo, Ldphm, Lddib and Ldsad, but did not affect the transcription of Ldshd (Fig. 5A

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and 5B). As a result, 20E titers in the treated larvae were significantly lowered (Fig.

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5C). Moreover, consumption of dsCncC and dsKeap1 significantly reduced the

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transcript levels of five 20E response genes (LdEcR-A, LdUSP, LdE75, LdHR3 and

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LdFTZ-F1) (Fig. 6).

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In D. melanogaster, endogenous Keap1 and CncC activates transcription of

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DmJheh genes (DmJheh1, DmJheh2 and DmJheh3) (Deng and Kerppola, 2014). In L.

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decemlineata, two Jheh genes (LdJheh1 and LdJheh2) have been cloned (Lü et al.,

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2015). We found the expression of LdJheh1 and LdJheh2, and the JH titers were not

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significantly affected in the LdCncC and LdKeap1 RNAi larvae (Fig. S11).

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3.5. Rescuing effect of 20E in the LdCncC and LdKeap1 RNAi larvae

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Ingestion of 20E by the LdCncC and LdKeap1 RNAi larvae did not affect the

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expression of their respective genes (Fig. 7A and 7D). However, feeding of 20E

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recovered the developmental period to the normal length (Fig. 7B and 7E). At the

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same time, it rescued the larval and pupal body mass rises (Fig. 7C and 7F). Moreover,

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consumption of 20E rescued or even overcompensated the expression levels of

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LdEcR-A, LdUSP, LdE75, LdHR3 and LdFTZ-F1 in the LdCncC and LdKeap1 RNAi

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hypomorphs (Fig. S12).

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4. Discussion

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4.1. PTTH/Torso is required for the expression of LdCncC and LdKeap1 In the work present here, we found that: 1) The expression levels of LdCncC and

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LdKeap1 showed clear parallels with the transcripts of LdTorso at the larval stage in L.

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decemlineata (Zhu et al., 2015). Moreover, the expression peaks of LdPTTH and

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LdTorso in the fourth-instar larvae were earlier than those of LdCncC and LdKeap1at

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the fourth-instar stage. 2) Knockdown of LdPTTH, LdTorso or LdRas suppressed the

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expression of LdCncC and LdKeap1, whereas silencing of LdCncC and LdKeap1 did

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not change the expression level of LdPTTH, LdTorso or LdRas. It appears that

298

PTTH/Torso signal is required for the expression of LdCncC and LdKeap1. In

299

agreement with our results, constitutive K-RasG12D expression in mice caused a

300

two-fold increase in the transcript of Nrf2, the mammalian homolog of CncC

301

(DeNicola et al., 2011). Conversely, constitutive RasV12 expression in Drosophila

302

prothoracic gland did not alter the transcription level of either DmCncC or DmKeap1

303

(Deng and Kerppola, 2013).

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Since knockdown of LdPTTH, LdTorso or LdRas significantly decreased 20E titer

305

in the present paper (also see our previously documented data (Zhu et al., 2015)),

306

the underexpression of LdCncC or LdKeap1 in these RNAi hypomorphs may be a

307

direct effect of PTTH-Torso-MAPK signaling, or alternatively, an effect from

308

decreased 20E titer. Thus, we knocked down two Halloween genes Ldphm and Ldshd

309

in L. decemlineata (Kong et al., 2014; Wan et al., 2013) to lower 20E titers. To our

310

surprise, instead of underexpression, LdCncC and LdKeap1 were overexpressed in the

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Ldphm and Ldshd RNAi larvae. Furthermore, we silenced two 20E signaling-involved

312

genes, LdEcR and LdE75 (Guo et al., 2016; Ogura et al., 2005), and found that

313

LdCncC and LdKeap1 were normally or highly transcribed. It can accordingly be hypothesized that PTTH signaling at the late stage of each

315

larval instar activates the transcription of CncC and Keap1 in L. decemlineata. The

316

resultant CncC and Keap1 proteins subsequently mediate PTTH signal, stimulate the

317

expression of a subset of Halloween genes in the PGs, and trigger the biosynthesis

318

and release of ecdysone. The subsequent 20E pulse then suppresses the transcription

319

of CncC and Keap1, and forms a negative feedback circuit, as proposed previously

320

(Moeller et al., 2013). At the early stage of each instar, in contrast, the 20E titer is too

321

low to inhibit the expression of LdCncC and LdKeap1. As a result, the expression

322

peaks of LdCncC and LdKeap1 occur.

323

4.2. LdCncC/LdKeap1 signaling regulates ecdysteroidogenesis

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In the present paper, we provided three lines of experimental evidence to supply

324

that

326

ecdysteroidogenesis in L. decemlineata, like their Drosophila homologs (Deng, 2014;

327

Deng and Kerppola, 2013).

LdCncC

and

LdKeap1

were

involved

in

the

regulation

of

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both

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Firstly, we found that both LdCncC and LdKeap1 in the day 2 fourth-instar L.

329

decemlineata larvae were highly expressed in the PGs. Similarly, marked DmKeap1

330

expression was seen in the PG cells in larval ring gland (Sykiotis and Bohmann,

331

2008). Moreover, both DmCncC and DmKeap1 were present in the nuclei of PG cells

332

(Deng and Kerppola, 2013). 15

ACCEPTED MANUSCRIPT The second line of experimental evidence was that RNAi-aided knockdown of

334

LdCncC or LdKeap1 caused typical 20E deficient phenotypes: the resultant larvae had

335

longer development periods than the controls. Moreover, the fully-grown larvae and

336

pupae possessed heavier fresh weights and larger body sizes. Likewise, the

337

development was arrested in the cncK6/K6 and Keap1EY5/EY5 D. melanogaster mutants

338

and the DmCncC or DmKeap1 depletion larvae. The pupa size formed by larvae that

339

silenced DmCncC in the PG was larger than that of the control (Deng and Kerppola,

340

2013).

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Our data showed that ingestion of either dsCncC or dsKeap1 at the fourth-instar

342

stage significantly reduced the mRNA levels of four Halloween genes (Ldspo, Ldphm,

343

Lddib and Ldsad), and lowered 20E titers. To determine whether LdCncC or LdKeap1

344

knockdown only reduces the ecdysone biosynthetic genes in the PGs, we examined

345

the transcription of another Halloween gene Ldshd expressed in the peripheral tissues

346

(Kong et al., 2014). As expected, its expression level was not downregulated.

347

Consistent with our results, knockdown of DmCncC in the PG cells reduced the levels

348

of five ecdysteroidogenesis gene transcripts (Dmneverland, Dmspo, Dmphm, Dmdib,

349

and Dmsad) that were expressed exclusively in D. melanogaster PG cells, and

350

silencing of DmKeap1 decreased the levels of Dmneverland, Dmspo and Dmphm. In

351

contrast, the level of Dmshd transcript was not diminished by DmCncC or DmKeap1

352

depletion (Deng and Kerppola, 2013).

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353

The third line of experimental evidence was ingestion of 20E by the LdCncC and

354

LdKeap1 RNAi larvae rescued the defective phenotypes. Similarly, supplementation 16

ACCEPTED MANUSCRIPT 355

with 20E restored the development period nearly to that of wild-type larvae in D.

356

melanogaster (Deng and Kerppola, 2013). Therefore, the function of CncC/Keap1 signaling in the regulation of

358

ecdysteroidogenesis is conserved in at lease two insect species, according to our

359

results in the present paper and those from D. melanogaster (Deng and Kerppola,

360

2013).

361

4.3. Does LdCncC/LdKeap1 signaling mediate 20E signaling?

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In response to 20E signal, Drosophila CncC/Keap1 signaling activated several

363

early ecdysone-regulated genes in the salivary glands (Deng, 2014; Deng and

364

Kerppola, 2013).

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In the work present here, we found that knockdown of either LdCncC or LdKeap1

366

decreased the transcripts of five 20E response genes (LdEcR-A, LdUSP, LdE75,

367

LdHR3 and LdFTZ-F1). Dietary supplementation with 20E completely restored or

368

even overcompensated their mRNA levels in the LdCncC and LdKeap1 RNAi larvae.

369

It seems that the activation of LdCncC/LdKeap1 signaling to early ecdysone response

370

genes in the L. decemlineata peripheral tissues, if any, may be secondary or

371

dispensable, in contrast to that in D. melanogaster (Deng and Kerppola, 2013).

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The conclusion was supported by another piece of experimental evidence: the

373

extent of defects differed when CncC/Keap1 was inhibited in D. melanogaster and L.

374

decemlineata. In this survey, we found that silencing of LdCncC or LdKeap1 caused

375

typical 20E deficient phenotypes, but did not kill the larvae. In contrast, loss of

376

function mutations in and RNAi of DmCncC or DmKeap1 not only delayed 17

ACCEPTED MANUSCRIPT 377

development period and resulted in larger body size, but also induced larval lethality

378

in D. melanogaster (Deng and Kerppola, 2013; Sykiotis and Bohmann, 2008; Veraksa

379

et al., 2000). Since DmCncC/DmKeap1 signaling plays more physiological roles in D.

380

melanogaster

381

knockout/knockdown of DmCncC or DmKeap1 caused serious negative effects.

382

4.4. LdCncC/LdKeap1 signaling does not stimulate the expression of JH

pathway

in

L.

decemlineata,

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LdCncC/LdKeap1

degradation genes

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383

than

In Drosophila, endogenous DmKeap1 and DmCncC stimulated transcription of the

385

DmJheh1, DmJheh2 and DmJheh3. Moreover, ectopic DmKeap1 expression

386

increased DmCncC binding at the Jheh gene loci and triggered their transcription

387

(Deng and Kerppola, 2014).

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Our previous results revealed that silencing of either LdJheh1 and LdJheh2, or both

389

genes significantly augmented JH titers (Lü et al., 2015), indicating the two genes

390

encoding functional JH degradation enzymes. Moreover, we found that knockdown of

391

either LdJheh1 or LdJheh2, or both genes also delayed larval development (Lü et al.,

392

2015), a phenotype similar to the LdKeap1 and LdCncC RNAi hypomorphs in the

393

present paper.

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Therefore, we determined the expression levels of LdJheh1 and LdJheh2, and the

395

JH titers in the LdKeap1 and LdCncC RNAi hypomorphs. Surprisingly, our results

396

showed that the expression levels of LdJheh1 and LdJheh2 and the JH titers were not

397

affected in the LdKeap1 and LdCncC RNAi larvae. It appears that LdCncC/LdKeap1

398

signaling may not be involved in the activation of JH degradation genes in L. 18

ACCEPTED MANUSCRIPT 399

decemlineata.

400

4.5. LdCncC and LdKeap1 plays other physiological roles Except the L. decemlineata larval PGs, both LdCncC and LdKeap1 genes were

402

easily detectable in other tested larval tissues such as guts, and adult ovaries and testis.

403

Similarly, DmCncC and DmKeap1 mRNAs were abundantly expressed in the D.

404

melanogaster larval alimentary canal, Malpighian tubules, salivary glands and brain,

405

as well as adult female and male flies (Sykiotis and Bohmann, 2008). Since digestive

406

tract represents the first line of defense to environmental stressors, and the Malpighian

407

tubules are major sites of detoxification, the tissue expression profiles in both L.

408

decemlineata and D. melanogaster larvae are reminiscent of a crucial role of the

409

Keap1/Nrf2 module as a multiorgan protector in mammalians (Itoh et al., 1977;

410

Kensler et al., 2007; Kobayashi and Yamamoto, 2006; Leiser and Miller, 2010;

411

Venugopal and Jaiswal, 1996).

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Moreover, DmCncC/DmKeap1 pathway was necessary and sufficient for

413

xenobiotic-induced transcription of a wide range of detoxification genes in

414

insecticide-resistant D. melanogaster strains (Deng and Kerppola, 2013; Karim et al.,

415

2015; Misra et al., 2011; Misra et al., 2013; Sykiotis and Bohmann, 2008). In Aphis

416

gossypii, in vivo RNAi of AgCncC dramatically suppressed the expression of

417

AgCYP6DA2, and increased the sensitivity to gossypol (Peng et al., 2016). In

418

Tribolium castaneum, TcCncC and V-maf musculoaponeurotic fibrosarcoma oncogene

419

homolog regulated the expression of deltamethrin metabolism gene TcCYP6BQ (Kalsi

420

and Palli, 2015). Similarly, LdCncC and LdMaf are involved in the regulation of four

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19

ACCEPTED MANUSCRIPT cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25 and CYP9Z29) that are

422

required for defense against both natural and synthetic chemicals (Kalsi and Palli,

423

2017). In the present paper, we found that the expression of 11 (out of 14) LdGSTd

424

and LdGSTe members was significantly suppressed in the LdCncC RNAi hypomorphs,

425

whereas the expression of 4 transcripts was significantly activated and 5 transcripts

426

was dramatically repressed in the LdKeap1 RNAi larvae.

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In D. melanogaster, CncC and Keap1 mediate transcriptional responses to

428

xenobiotic genes and developmental signals using distinct mechanisms. DmKeap1

429

regulates xenobiotic response genes through inhibiting nuclear DmCncC levels. It can

430

interact with DmCncC and trigger its ubiquitination and proteasomal degradation in

431

the cytoplasm. Interference of this interaction in response to stimuli leads to

432

stabilization and nuclear accumulation of DmCncC (Itoh et al., 1999; Kobayashi et al.,

433

2004). In contrast, DmKeap1 regulates some developmental genes through facilitating

434

DmCncC binding to chromatin (Deng, 2014). In the work present here, we found that

435

knockdown of LdCncC resulted in the suppression of both xenobiotic agents and

436

several Halloween genes. In contrast, silencing of LdKeap1 led to the suppression of

437

several Halloween genes and a subset of xenobiotic genes, but resulted in the

438

upregulation of another subset of xenobiotic genes. Moreover, a subset of CYP genes

439

were regulated by CncC/Maf in both T. castaneum (Kalsi and Palli, 2015) and L.

440

decemlineata (Kalsi and Palli, 2017). Therefore, the two distinct mechanisms of CncC

441

signaling to mediate transcriptional responses are conserved in L. decemlineata,

442

although the specific gene subsets regulated are different between the beetle and D.

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melanogaster (Deng, 2014; Itoh et al., 1999; Kobayashi et al., 2004) . In addition, our results showed that the expression levels of LdCncC, LdKeap1 and

445

the three LdGSTd genes were higher right after the molt. It gives the impression that

446

the active LdCncC/LdKeap1 signaling triggers the expression of detoxification genes,

447

such as CYPs and GSTs, during the early and mid instar stages. The resultant enzymes

448

may degrade xenobiotics from food to protect the larvae from poisoning when they

449

are actively feeding.

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451

Acknowledgments

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This research was supported by the National Natural Science Foundation of China

453

(31272047 and 31360442), and the Fundamental Research Funds for the Central

454

Universities (KYTZ201403).

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cooperates with a small Maf subunit to specify pharyngeal development and suppress deformed homeotic function in the Drosophila head. Development 127, 4023-4037. Wan, P.-J., Shi, X.-Q., Kong, Y., Zhou, L.-T., Guo, W.-C., Ahmat, T., Li, G.-Q., 2013. Identification of cytochrome P450 monooxygenase genes and their expression profiles in cyhalothrin-treated Colorado potato beetle, Leptinotarsa decemlineata. Pesticide biochemistry and physiology 107, 360-368. Zhou, J., Qi, Y., Hou, Y., Zhao, J., Li, Y., Xue, X., Wu, L., Zhang, J., Chen, F., 2011. Quantitative determination of juvenile hormone III and 20-hydroxyecdysone in queen larvae and drone pupae of Apis mellifera by ultrasonic-assisted extraction and liquid chromatography with electrospray ionization tandem mass spectrometry. Journal of Chromatography B 879, 2533-2541. Zhou, L.T., Jia, S., Wan, P.J., Kong, Y., Guo, W.C., Ahmat, T., Li, G.Q., 2013. RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say). Journal of Insect Physiology 59, 1049-1056. Zhu, T.-T., Meng, Q.-W., Guo, W.-C., Li, G.-Q., 2015. RNA interference suppression of the receptor tyrosine kinase Torso gene impaired pupation and adult emergence in Leptinotarsa decemlineata. Journal of Insect Physiology 83, 53-64.

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Fig. 1. Temporal expression patterns of LdCncC (A) and LdKeap1 (B) genes in L.

636

decemlineata. The cDNA templates are derived from the day 3 eggs, the whole bodies

637

of the first, second and third larval instars at an interval of one day, and from the

638

fourth larval instars at an interval of twelve hours (D0/H0 indicated newly ecdysed

639

larvae). For each sample, 3 independent pools of 5-10 individuals are measured in

640

technical triplicate using qRT-PCR. The mean ± SE (n=3) is calculated using the

641

2-∆∆Ct method, normalized to the geometrical mean of housekeeping gene expression.

642

The relative transcripts are the ratios of relative copy numbers in individuals at

643

specific developing stages to that in the day 1 third-instar larvae (A) or the eggs (B).

EP

AC C

644

TE D

635

26

Fig. 2. Tissue expression patterns of LdCncC and LdKeap1 genes in L. decemlineata. For A and B, the cDNA templates are derived from brain-corpora cardiaca-corpora allata complex (BCC), prothoracic gland (PG), ventral ganglia (VG), foregut (FG), midgut (MG), hindgut (HG), Malpighian tubules (MT), epidermis (EP), fat body (FB) and hemocyte (HE) of the day 2 fourth-instar larvae. The templates are also from female ovary (OV) and male testis (TE) of the adults. The expression levels in BCC, PG and FB through the fourth instar stage were also tested at an interval of twelve hours (H0 indicated newly ecdysed larvae) (C and D). For each sample, 3 independent pools of 5-10 individuals are measured in technical triplicate using qRT-PCR. The mean ± SE (n=3) is calculated using the 2-∆∆Ct method, normalized to the geometrical mean of housekeeping gene expression. The relative transcripts are the ratios of relative copy numbers in specific tissues to that in the ventral ganglia (A) or the fat body (B), or in tissues at specific developing stages to that in the FB at 72 (C) or 80 (D) hours after moulting.

AC C

645 646 647 648 649 650 651 652 653 654 655 656 657 658 659

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

27

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

660 661

TE D

EP

670

Fig. 3. Induction of the expression of LdCncC and LdKeap1 by PTTH-Torso signaling in L. decemlineata. The newly-ecdysed fourth-instar larvae have ingested dsPTTH (A)-, dsTorso (B)-, dsRas (C)-, dsphm (D)-, dsshd (E)-, dsEcR (F)- or dsE75 (G)-dipped leaves for 3 days. The PBS (CK)- and dsegfp-immersed leaves are used as controls. The mean ± SE (n=3) is calculated using the 2-∆∆Ct method, normalized to the geometrical mean of housekeeping gene expression. The relative transcripts are the ratios of relative copy numbers in dsRNA-ingested individuals to PBS-fed ones (CK). Different letters above the bars indicate significant difference at P value < 0.05.

AC C

662 663 664 665 666 667 668 669

28

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

671

Fig. 4. Effects of knockdown of LdCncC (A) and LdKeap1 (B) genes in L.

673

decemlineata fourth-instar larvae. The newly-ecdysed fourth-instar larvae have

674

been allowed to ingest PBS (CK)-, dsegfp- and dsCncC-immersed leaves, or PBS

675

(CK)-, dsegfp- and dsKeap1-dipped leaves for 3 days, and normal foliage for an

676

additional 2 days. The relative transcripts (A, D) are measured on the 3 days after the

677

initiation of experiment. The mean ± SE (n=3) is calculated using the 2-∆∆Ct method,

678

normalized to the geometrical mean of housekeeping gene expression. The relative

679

transcripts are the ratios of relative copy numbers in dsRNA-ingested individuals to

680

PBS-fed ones (CK). The emergence rates in the same days after dsRNA exposure are

681

compared (B, E). Knockdown either gene causes development delay. The larvae and

682

pupae are weighed on the 5 and 10 days after the initiation of experiment (C, F).

683

Different letters above the bars indicate significant difference at P value < 0.05. The

684

larval and pupal sizes of dsCncC-, and dsKeap1-fed beetles are shown (G, H).

AC C

EP

672

29

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

685

686 687

Fig. 5. Knockdown of LdCncC and LdKeap1 genes in L. decemlineata

689

fourth-instar larvae affecting ecdysteroidogenesis. The newly-ecdysed fourth-instar

690

larvae have been confined in dishes containing PBS (CK)-, dsegfp- and

691

dsCncC-immersed leaves, or PBS (CK)-, dsegfp- and dsKeap1-dipped leaves for 3

692

days. The relative transcripts of five Halloween genes (Ldspo, Ldphm, Lddib, Ldsad

693

and Ldshd) (A, B) and the 20-hydroxyecdysone (20E) titer (C) are determined. The

694

mean ± SE (n=3) is calculated using the 2-∆∆Ct method, normalized to the geometrical

695

mean of housekeeping gene expression. The relative transcripts are the ratios of

696

relative copy numbers in dsRNA-ingested individuals to PBS-fed ones (CK).

697

Different letters above the bars indicate significant difference at P value < 0.05.

AC C

EP

TE D

688

698 699 700 701 30

RI PT

ACCEPTED MANUSCRIPT

SC

702 703

Fig. 6. Knockdown of LdCncC (A) and LdKeap1 (B) genes in L. decemlineata

705

fourth-instar

706

fourth-instar larvae have ingested dsCncC-, or dsKeap1-dipped leaves for 3 days. The

707

PBS (CK)- and dsegfp-immersed leaves are used as controls. The relative transcripts

708

of five 20-hydroxyecdysone response genes (LdEcR-A, LdUSP, LdE75, LdHR3 and

709

LdFTZ-F1) are quantified. The mean ± SE (n=3) is calculated using the 2-∆∆Ct method,

710

normalized to the geometrical mean of housekeeping gene expression. The relative

711

transcripts are the ratios of relative copy numbers in dsRNA-ingested individuals to

712

PBS-fed ones (CK). Different letters above the bars indicate significant difference at

713

P value < 0.05.

716 717 718

TE D

ecdysone

EP

715

disturbing

AC C

714

larvae

M AN U

704

719 720 721 722 723 31

signaling.

The

newly-ecdysed

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

724

725 726 727

Fig. 7. Rescuing effect of 20-hydroxyecdysone (20E) on the phenotypes of the

729

LdCncC and LdKeap1 knockdown larvae in L. decemlineata. The newly-ecdysed

730

fourth-instar larvae have ingested foliage immersed with dsCncC or dsCncC+20E

731

(A-C), or dsKeap1 or dsKeap1+20E (D-F) for 3 days. The dsegfp-immersed leaves

732

are used as control. The relative transcripts of LdCncC and LdKeap1 (A, D) are tested.

733

The mean ± SE (n=3) is calculated using the 2-∆∆Ct method, normalized to the

734

geometrical mean of housekeeping gene expression. The relative transcripts are the

735

ratios of relative copy numbers in dsCncC or dsKeap1-ingested individuals to

736

dsegfp-fed ones. The emergence rates in the same days after dsRNA exposure are

737

compared (B, E). The fresh larval weights (C, F) are measured.

738

above the bars indicate significant difference at P value < 0.05. Ingestion of 20E

739

completely restores the development delay and overweight phenotypes in the LdCncC

740

and LdKeap1 knockdown larvae.

AC C

EP

TE D

728

741 742

32

Different letters

ACCEPTED MANUSCRIPT Highlights PTTH signaling stimulates expression of cap'n'collar isoform C (CncC) and Kelch-like ECH associated protein 1 (Keap1) genes in Leptinotarsa.

RI PT

Knockdown of either LdCncC or LdKeap1 reduced expression of steroidogenic genes and caused a 20E deficiency phenotype.

Dietary 20E suppressed the defective phenotypes in LdCncC and LdKeap1

AC C

EP

TE D

M AN U

SC

RNAi larvae.