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:
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.
At the late stage of the final larval instar
Synthesis of ecdysone
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ACCEPTED MANUSCRIPT 1
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 Sun1†, Qing-Wei Meng1†, Qing-Yu Xu1, Pan Deng1, Wen-Chao Guo 2,
Guo-Qing Li 1 *
1. Education Ministry Key Laboratory of Integrated Management of Crop Diseases
and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing
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2. Department of Plant Protection, Xinjiang Academy of Agricultural Sciences;
Urumqi 830091, China
Running Head: Knockdown of Leptinotarsa CncC and Keap1
Qiang-Kun Sun, [email protected]
Qing-Wei Meng, [email protected]
Qing-Yu Xu, [email protected]
Pan Deng, [email protected]
Wen-Chao Guo, [email protected]
Co-first author 1
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Abstract Drosophila cap ‘n’ collar isoform C (CncC) and Kelch-like ECH associated protein
1 (Keap1) regulate metamorphosis by transcriptional control of a subset of genes
involved in ecdysteroidogenesis, 20-hydroxyecdysone (20E) signaling, and juvenile
hormone (JH) degradation. In the present paper, we found that prothoracicotropic
hormone signal was required for the activation of LdCncC and LdKeap1 in
Leptinotarsa decemlineata. Moreover, RNA interference of LdCncC or LdKeap1 in
the fourth-instar larvae delayed development. As a result, the treated larvae obtained
heavier larval and pupal fresh weights and had larger body sizes than the controls.
Furthermore, knockdown of LdCncC or LdKeap1 significantly reduced the mRNA
levels of four ecdysone biosynthetic genes (Ldspo, Ldphm, Lddib and Ldsad), lowered
20E titer and decreased the transcript levels of five 20E response genes (LdEcR,
LdUSP, LdE75, LdHR3 and LdFTZ-F1). However, the expression of two JH epoxide
hydrolase genes and JH contents were not affected in the LdCncC and LdKeap1 RNAi
larvae. Dietary supplementation with 20E shortened the developmental period to
normal length, rescued the larval and pupal body mass rises, and recovered or even
overcompensated the expression levels of the five 20E response genes in either
LdCncC or LdKeap1 RNAi hypomorphs. Therefore, LdCncC/LdKeap1 signaling
regulates several ecdysteroidogenesis genes, and consequently 20E pulse, to modulate
the onset of metamorphosis in L. decemlineata.
Key words: Leptinotarsa decemlineata, cap ‘n’ collar isoform C, Kelch-like ECH
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
level of juvenile hormone (JH) triggers larval-pupal metamorphosis. Ecdysone is
synthesized in insect prothoracic glands (PGs) from cholesterol, under the
catalyzation of a series of cytochrome P450 monooxygenases (CYPs) encoded by
Halloween genes such as spook (spo), phantom (phm), disembodied (dib) and shadow
(sad). Ecdysone is then released from PGs into hemolymph. It is transported to
peripheral tissues, and is converted to 20E by another CYP, the product of a
Halloween gene shade (shd) (Iga and Kataoka, 2012; Niwa and Niwa, 2014). The
expression of these Halloween genes and consequently the timing of pupation are
regulated by prothoracicotropic hormone (PTTH)-Torso receptor-mitogen activated
protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK
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
by two hydrolases, JH epoxide hydrolase (JHEH, EC 22.214.171.124) and JH esterase (JHE,
EC 126.96.36.199) (Gu et al., 2015; Lü et al., 2015). JHEH falls into the microsomal epoxide
hydrolase family (Arand et al., 2005; Morisseau and Hammock, 2005), and JHE
belongs to the carboxylesterase family (Share and Roe, 1988).
In Drosophila melanogaster, cap ‘n’ collar isoform C (CncC) and Kelch-like ECH
associated protein 1 (Keap1), the homologs of mammalian nuclear factor erythroid 2
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,
2014). Up to now, CncC and Keap1 homologs have been found in other insect species
in both holometabolans and hemimetabolans (Deng and Kerppola, 2013; Grimberg et
al., 2011; Kalsi and Palli, 2015; Karim et al., 2015; Misra et al., 2011; Misra et al.,
2013; Peng et al., 2016; Sykiotis and Bohmann, 2008). An interesting question then
arises: are CncC and Keap1 the conserved transcription activators of both
ecdysteroidogenesis and JH hydrolyzation genes among insect species?
Upon biosynthesis and release, 20E, acting through its cognate receptor, a dimer of
ecdysone receptor (EcR)/ultraspiracle (USP), triggers a conserved transcriptional
cascade including early genes such as Broad-Complex (BR-C), Ecdysone-induced
protein 75 (E75) and E74, early-late genes such as hormone receptor 3 (HR3), and
late genes such as Fushi tarazu factor 1 (FTZ-F1), to stimulate metamorphosis (Iga
and Kataoka, 2012; Luan et al., 2013). In Drosophila, CncC/Keap1 signaling has been
proven to activate several early ecdysone-response genes in the salivary glands (Deng,
2014; Deng and Kerppola, 2013, 2014). Is CncC/Keap1 signaling involved in the
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
interference (RNAi) response to double stranded RNAs (dsRNAs) (Guo et al., 2015,
2016; Kong et al., 2014; Liu et al., 2014; Shi et al., 2016a; Shi et al., 2016b; Shi et al.,
2016c; Zhu et al., 2015). Using in vivo RNAi, we previously demonstrated that the
ecdysteroidogenesis and 20E signaling genes were conserved in L. decemlineata (Guo
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
larval-pupal metamorphosis in L. decemlineata.
2. Materials and methods
2.1. Experimental animal
The L. decemlineata beetles were kept in an insectary according to a previously
described method (Shi et al., 2013), with potato foliage at the vegetative growth or
young tuber stages in order to assure sufficient nutrition. At this feeding protocol, the
larvae progressed through the first, second, third, and fourth instars at an approximate
period of 2, 2, 2 and 4 days, respectively. Upon reaching full size, the fourth-instar
larvae stopped feeding, dropped to the ground, burrowed to the soil and entered the
prepupae stage. The prepupae spent an approximately 3 days to pupate. The pupae
lasted about 5 days and the adults emerged.
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)
transcriptome data (Shi et al., 2013). The correctness of the sequences was
substantiated by polymerase chain reaction (PCR) using primers in Table S1. The
full-length cDNAs were obtained by 5'- and/or 3'-RACE, using SMARTer RACE kit
(Takara Bio.), with specific primers listed in Table S1. After obtaining full-length
cDNAs, primer pairs (Table S1) were designed to verify the complete open reading
frames. All of the sequenced cDNAs were submitted to GenBank (accession numbers:
LdCncA, KY458169; LdCncB, KY458170; LdCncC1, KY458171; LdCncC2,
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KY458172; LdKeap1A, KY458173; LdKeap1B, KY458174).
2.3. Preparation of dsRNAs The same method as previously described (Zhou et al., 2013) was used to express
dsPTTH (214 bp), dsTorso (302 bp), dsRas (327 bp), dsphm (345 bp), dsshd (438 bp),
dsEcR (344 bp), dsE75 (361 bp), dsCncC-1 (200 bp), dsCncC-2 (411 bp), dsKeap1-1
(373 bp), dsKeap1-2 (302 bp) and dsegfp (a 414 bp fragment of enhanced green
fluorescent protein gene). The twelve dsRNAs were individually transcribed with
specific primers in Table S1, using Escherichia coli HT115 (DE3) competent cells
lacking RNase III. Individual colonies were inoculated, and grown until cultures
reached an OD600 value of 1.0. The colonies were then induced to express dsRNA by
addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.1 mM.
The expressed dsRNA was extracted and confirmed by electrophoresis on 1% agarose
gel (data not shown). Bacteria cells were centrifuged at 5000 ×g for 10 min, and
resuspended in an equal original culture volume of 0.05 M phosphate buffered saline
(PBS, pH 7.4). The bacterial solutions (at a dsRNA concentration of about 0.5 µg/ml)
were used for experiment.
2.4. Dietary introduction of dsRNA
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The same method as previously reported (Fu et al., 2015) was used to introduce
dsRNA into larvae. The newly-ecdysed fourth-instar larvae were allowed to feed
foliage immersed with bacterial suspension containing dsPTTH, dsTorso, dsRas,
dsphm, dsshd, dsEcR, dsE75, or each of the two dsRNAs of LdCncC (dsCncC-1 and
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
controls. The larvae were then transferred to untreated foliage if necessary. The beetles were weighed twice during trial period. The adult emergence was
recorded during a 2-week trial period. The samples on day 3 after the initiation of the
experiments were collected. The effects of gene silencing, the levels of five
Halloween genes (Ldspo, Ldphm, Lddib, Ldsad, and Ldshd), five 20E response genes
(LdEcR, LdUSP, LdE75, LdHR3 and LdFTZ-F1), a total of 14 LdGST transcripts and
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
experiments were performed using foliage dipped with dsCncC-1, dsCncC-1+10-6 M
20E, or dsKeap1-1, dsKeap1-1+10-6 M 20E, a concentration used in our previous
research (Guo et al., 2016). The PBS- and dsegfp-immersed leaves were used as
For above expriments, three biological replicates were carried out. 2.5. Real-time quantitative PCR (qRT-PCR)
Each sample contained 5-10 individuals and repeated three times. The RNA was
extracted using SV Total RNA Isolation System Kit (Promega). Purified RNA was
subjected to DNase I to remove any residual DNA according to the manufacturer’s
instructions. Quantitative mRNA measurements were performed by qRT-PCR in
technical triplicate, using internal control genes (the primers listed in Table S1)
according to our published results (Shi et al., 2013). An RT negative control (without
reverse transcriptase) and a non-template negative control were included for each
ACCEPTED MANUSCRIPT primer set to confirm the absence of genomic DNA and to check for primer-dimer or
contamination in the reactions, respectively. Data were analyzed by the 2-∆∆CT method,
using the geometric mean of internal control genes for normalization. All methods
and data were confirmed to follow the MIQE (Minimum Information for publication
of Quantitative real time PCR Experiments) guidelines (Bustin et al., 2009).
2.6. Quantitative determination of 20E and JH
20E was extracted according to a ultrasonic-assisted extraction method (Liu et al.,
2014), and its titer (ng per g body weight) was analyzed by a liquid chromatography
tandem mass spectrometry-mass spectrometry (LC-MS/MS) system using a protocol
the same as described (Zhou et al., 2011).
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Hemolymph was collected and JH was extracted following the methods
described previously (Zhou et al., 2013). An LC-MS was used to quantify JH titers
(ng per ml hemolymph) (Cornette et al., 2008).
2.7. Data analysis
The data were given as means ± SE, and were analyzed by ANOVA followed by the
Tukey-Kramer test, using SPSS for Windows (SPSS, Chicago, IL, USA). A repeated
measures ANOVA was used to test the effects of dsRNAs on larval development.
Since no significant differences between dsRNAs targeting two different regions of
either LdCncC or LdKeap1 (dsCncC-1 and dsCncC-2, dsKeap1-1 and dsKeap1-2)
were found, the data of each gene were combined.
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
found that LdCnc had four splicing isoforms in L. decemlineata. LdCncA and LdCncB
were truncated forms. LdCncC1 and LdCncC2 differed in the first exon but shared the
following 10 exons. The four isoforms shared exon 8, 9 and 10 (Fig. S1A). LdKeap1
gene possessed two splicing isoforms (LdKeap1A and LdKeap1B) which differed in
the first exon (Fig. S2A). Phylogenetic analyses revealed that both CncC- and
Keap1-like proteins formed order based separate clades. Obviously, LdCncC and
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,
we determined their temporal expression patterns. Both genes were expressed
throughout all larval developmental stages. Within the first, second and third larval
instars, their expression levels were higher just before and right after the molt, and
were lower in the intermediate instar. In the fourth larval instar, the two genes reached
their highest expression levels between 24 and 60 hours after ecdysis (Fig. 1).
In D. melanogaster, CncC/Keap1 pathway is necessary and sufficient for
xenobiotic-induced transcription of a wide range of detoxification genes including
CYPs (e.g. DmCYP6A2, DmCYP6A8) and glutathione S-transferases (such as
DmGSTd1, DmGSTe1) (Karim et al., 2015; Misra et al., 2011; Misra et al., 2013).
Here we tested the expression of all LdGSTd genes that were identified previously in
L. decemlineata (Han et al., 2016). We found that the three LdGSTd genes exhibited
similar temporal expression patterns to LdCncC and LdKeap1 (Fig. S3).
ACCEPTED MANUSCRIPT The tissue expression profiles of LdCncC and LdKeap1 genes were also tested.
Their mRNAs were easily detectable in all tested tissues including brain-corpora
cardiaca-corpora allata complex, prothoracic gland, foregut, midgut, hindgut,
Malpighian tubules, epidermis, fat body and hemocyte of the day 2 fourth-instar
larvae. The templates were also present in female ovary and male testis of the adults.
LdCncC was highly expressed in larval midgut and prothoracic gland, and adult ovary,
whereas LdKeap1 was transcribed at the highest level in the larval brain-corpora
cardiaca-corpora allata complex, and at higher levels in the larval foregut and
prothoracic gland, and the adult ovary (Fig. 2A, 2B).
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cardiaca-corpora allata complex, prothoracic gland and fat body through the fourth
instar stage were also tested. As expected, both LdCncC and LdKeap1 were highly
expressed between 24 and 60 hours after ecdysis. Moreover, both genes were highly
transcribed in brain-corpora cardiaca-corpora allata complex and prothoracic gland
(Fig. 2C, 2D).
3.2. PTTH-Torso signaling is required for the expression of LdCncC and LdKeap1
The temporal transcription patterns of LdCncC and LdKeap1 reminded us of the
expression fluctuation of LdTorso (Zhu et al., 2015). To test whether LdPTTH
regulates LdTorso transcription in vivo, we examined the transcript levels of LdPTTH
and LdTorso throughout the fourth-instar larvae in brain-corpora cardiaca-corpora
allata complex, prothoracic gland and fat body. As expected, LdPTTH exhibited a
similar expression pattern to LdTorso in the three representative tissues (Fig. S4). 10
ACCEPTED MANUSCRIPT To determine whether PTTH-Torso signaling is required for the activation of
LdCncC and LdKeap1 in vivo, LdCncC and LdKeap1 mRNA levels in the LdPTTH,
LdTorso and LdRas RNAi larvae (Fig. S5) were tested. Compared with those in the
control specimens, LdCncC and LdKeap1 transcription levels were dramatically
decreased in these RNAi hypomorphs
Our previous results showed that RNAi of the Halloween genes reduced 20E
titers in L. decemlineata (Kong et al., 2014). In this study, we knocked down Ldphm
and Ldshd to lower 20E titers (Fig. S6), and found that LdCncC and LdKeap1 mRNA
levels were significantly increased in the Ldphm and Ldshd RNAi larvae, compared
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
identified (Guo et al., 2015, 2016; Liu et al., 2014; Ogura et al., 2005). In this survey,
we knocked down LdEcR-A and LdE75 (Fig. S7), and found that inhibition of 20E
signaling in the LdEcR-A and LdE75 RNAi larvae had no influence or upregulated the
expression of LdCncC and LdKeap1, compared with those in the control specimens
(Fig. 3F, 3G).
3.3. Knockdown of LdCncC or LdKeap1 delays larval development
To dissect the physiological roles of LdCncC and LdKeap1 in larval development,
we dietarily introduced each of the two dsRNAs derived from the common sequences
of either LdCncC or LdKeap1 into the newly-molted fourth-instar larvae. Combined
data revealed that continuous ingestion of a dsCncC or a dsKeap1 for 3 days
significantly downregulated its target gene (Fig. 4A, 4D). 11
ACCEPTED MANUSCRIPT In D. melanogaster, depletion of DmCncC reduced whereas knockout of
DmKeap1 increased the expression of DmGSTd1 and DmGSTe1 (Karim et al., 2015;
Misra et al., 2011; Misra et al., 2013). In order to evaluate whether ingestion of a
dsCncC or a dsKeap1 successfully knocks down its target gene, we also tested the
expression levels of all LdGSTd and LdGSTe members in the resultant larvae. Out of
the 14 transcripts, the expression of 11 ones was significantly suppressed in the
dsCncC-fed larvae, whereas the expression of 4 was significantly activated and 5 was
dramatically repressed in the dsKeap1-fed larvae. Interestingly, the expression of
LdGSTd1 and LdGSTe1 mimicked that of their Drosophila partners in the DmCncC-
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).
Moreover, fully-grown larvae and pupae obtained heavier fresh weights and had
larger body sizes than the controls (Fig. 4C, 4G and 4H).
Knockdown of LdTKeap1 completely simulated the negative effects observed in the LdCncC hypomorphs (Fig. 4).
3.4. Effects of dsCncC and dsKeap1 on PTTH, 20E and JH signaling pathways
The expression levels of LdPTTH, LdTorso and LdRas were measured in the
LdCncC and LdTKeap1 RNAi larvae. All the three genes showed similar transcription
levels in the LdCncC and LdKeap1 RNAi larvae to those in the controls (Fig. S10).
In D. melanogaster, CncC and Keap1 have been documented to regulate
Halloween genes (Deng, 2014; Deng and Kerppola, 2013). In L. decemlineata, five
Halloween genes (spo, phm, dib, sad and shd) have been cloned (Kong et al., 2014; 12
ACCEPTED MANUSCRIPT 267
Wan et al., 2013). We determined their expression levels in the LdCncC and LdKeap1
RNAi larvae. Ingestion of dsCncC and dsKeap1 significantly downregulated the expression of
Ldspo, Ldphm, Lddib and Ldsad, but did not affect the transcription of Ldshd (Fig. 5A
and 5B). As a result, 20E titers in the treated larvae were significantly lowered (Fig.
5C). Moreover, consumption of dsCncC and dsKeap1 significantly reduced the
transcript levels of five 20E response genes (LdEcR-A, LdUSP, LdE75, LdHR3 and
LdFTZ-F1) (Fig. 6).
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In D. melanogaster, endogenous Keap1 and CncC activates transcription of
DmJheh genes (DmJheh1, DmJheh2 and DmJheh3) (Deng and Kerppola, 2014). In L.
decemlineata, two Jheh genes (LdJheh1 and LdJheh2) have been cloned (Lü et al.,
2015). We found the expression of LdJheh1 and LdJheh2, and the JH titers were not
significantly affected in the LdCncC and LdKeap1 RNAi larvae (Fig. S11).
3.5. Rescuing effect of 20E in the LdCncC and LdKeap1 RNAi larvae
Ingestion of 20E by the LdCncC and LdKeap1 RNAi larvae did not affect the
expression of their respective genes (Fig. 7A and 7D). However, feeding of 20E
recovered the developmental period to the normal length (Fig. 7B and 7E). At the
same time, it rescued the larval and pupal body mass rises (Fig. 7C and 7F). Moreover,
consumption of 20E rescued or even overcompensated the expression levels of
LdEcR-A, LdUSP, LdE75, LdHR3 and LdFTZ-F1 in the LdCncC and LdKeap1 RNAi
hypomorphs (Fig. S12).
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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
LdKeap1 showed clear parallels with the transcripts of LdTorso at the larval stage in L.
decemlineata (Zhu et al., 2015). Moreover, the expression peaks of LdPTTH and
LdTorso in the fourth-instar larvae were earlier than those of LdCncC and LdKeap1at
the fourth-instar stage. 2) Knockdown of LdPTTH, LdTorso or LdRas suppressed the
expression of LdCncC and LdKeap1, whereas silencing of LdCncC and LdKeap1 did
not change the expression level of LdPTTH, LdTorso or LdRas. It appears that
PTTH/Torso signal is required for the expression of LdCncC and LdKeap1. In
agreement with our results, constitutive K-RasG12D expression in mice caused a
two-fold increase in the transcript of Nrf2, the mammalian homolog of CncC
(DeNicola et al., 2011). Conversely, constitutive RasV12 expression in Drosophila
prothoracic gland did not alter the transcription level of either DmCncC or DmKeap1
(Deng and Kerppola, 2013).
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Since knockdown of LdPTTH, LdTorso or LdRas significantly decreased 20E titer
in the present paper (also see our previously documented data (Zhu et al., 2015)),
the underexpression of LdCncC or LdKeap1 in these RNAi hypomorphs may be a
direct effect of PTTH-Torso-MAPK signaling, or alternatively, an effect from
decreased 20E titer. Thus, we knocked down two Halloween genes Ldphm and Ldshd
in L. decemlineata (Kong et al., 2014; Wan et al., 2013) to lower 20E titers. To our
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
genes, LdEcR and LdE75 (Guo et al., 2016; Ogura et al., 2005), and found that
LdCncC and LdKeap1 were normally or highly transcribed. It can accordingly be hypothesized that PTTH signaling at the late stage of each
larval instar activates the transcription of CncC and Keap1 in L. decemlineata. The
resultant CncC and Keap1 proteins subsequently mediate PTTH signal, stimulate the
expression of a subset of Halloween genes in the PGs, and trigger the biosynthesis
and release of ecdysone. The subsequent 20E pulse then suppresses the transcription
of CncC and Keap1, and forms a negative feedback circuit, as proposed previously
(Moeller et al., 2013). At the early stage of each instar, in contrast, the 20E titer is too
low to inhibit the expression of LdCncC and LdKeap1. As a result, the expression
peaks of LdCncC and LdKeap1 occur.
4.2. LdCncC/LdKeap1 signaling regulates ecdysteroidogenesis
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In the present paper, we provided three lines of experimental evidence to supply
ecdysteroidogenesis in L. decemlineata, like their Drosophila homologs (Deng, 2014;
Deng and Kerppola, 2013).
Firstly, we found that both LdCncC and LdKeap1 in the day 2 fourth-instar L.
decemlineata larvae were highly expressed in the PGs. Similarly, marked DmKeap1
expression was seen in the PG cells in larval ring gland (Sykiotis and Bohmann,
2008). Moreover, both DmCncC and DmKeap1 were present in the nuclei of PG cells
(Deng and Kerppola, 2013). 15
ACCEPTED MANUSCRIPT The second line of experimental evidence was that RNAi-aided knockdown of
LdCncC or LdKeap1 caused typical 20E deficient phenotypes: the resultant larvae had
longer development periods than the controls. Moreover, the fully-grown larvae and
pupae possessed heavier fresh weights and larger body sizes. Likewise, the
development was arrested in the cncK6/K6 and Keap1EY5/EY5 D. melanogaster mutants
and the DmCncC or DmKeap1 depletion larvae. The pupa size formed by larvae that
silenced DmCncC in the PG was larger than that of the control (Deng and Kerppola,
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Our data showed that ingestion of either dsCncC or dsKeap1 at the fourth-instar
stage significantly reduced the mRNA levels of four Halloween genes (Ldspo, Ldphm,
Lddib and Ldsad), and lowered 20E titers. To determine whether LdCncC or LdKeap1
knockdown only reduces the ecdysone biosynthetic genes in the PGs, we examined
the transcription of another Halloween gene Ldshd expressed in the peripheral tissues
(Kong et al., 2014). As expected, its expression level was not downregulated.
Consistent with our results, knockdown of DmCncC in the PG cells reduced the levels
of five ecdysteroidogenesis gene transcripts (Dmneverland, Dmspo, Dmphm, Dmdib,
and Dmsad) that were expressed exclusively in D. melanogaster PG cells, and
silencing of DmKeap1 decreased the levels of Dmneverland, Dmspo and Dmphm. In
contrast, the level of Dmshd transcript was not diminished by DmCncC or DmKeap1
depletion (Deng and Kerppola, 2013).
The third line of experimental evidence was ingestion of 20E by the LdCncC and
LdKeap1 RNAi larvae rescued the defective phenotypes. Similarly, supplementation 16
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with 20E restored the development period nearly to that of wild-type larvae in D.
melanogaster (Deng and Kerppola, 2013). Therefore, the function of CncC/Keap1 signaling in the regulation of
ecdysteroidogenesis is conserved in at lease two insect species, according to our
results in the present paper and those from D. melanogaster (Deng and Kerppola,
4.3. Does LdCncC/LdKeap1 signaling mediate 20E signaling?
In response to 20E signal, Drosophila CncC/Keap1 signaling activated several
early ecdysone-regulated genes in the salivary glands (Deng, 2014; Deng and
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In the work present here, we found that knockdown of either LdCncC or LdKeap1
decreased the transcripts of five 20E response genes (LdEcR-A, LdUSP, LdE75,
LdHR3 and LdFTZ-F1). Dietary supplementation with 20E completely restored or
even overcompensated their mRNA levels in the LdCncC and LdKeap1 RNAi larvae.
It seems that the activation of LdCncC/LdKeap1 signaling to early ecdysone response
genes in the L. decemlineata peripheral tissues, if any, may be secondary or
dispensable, in contrast to that in D. melanogaster (Deng and Kerppola, 2013).
The conclusion was supported by another piece of experimental evidence: the
extent of defects differed when CncC/Keap1 was inhibited in D. melanogaster and L.
decemlineata. In this survey, we found that silencing of LdCncC or LdKeap1 caused
typical 20E deficient phenotypes, but did not kill the larvae. In contrast, loss of
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
in D. melanogaster (Deng and Kerppola, 2013; Sykiotis and Bohmann, 2008; Veraksa
et al., 2000). Since DmCncC/DmKeap1 signaling plays more physiological roles in D.
knockout/knockdown of DmCncC or DmKeap1 caused serious negative effects.
4.4. LdCncC/LdKeap1 signaling does not stimulate the expression of JH
In Drosophila, endogenous DmKeap1 and DmCncC stimulated transcription of the
DmJheh1, DmJheh2 and DmJheh3. Moreover, ectopic DmKeap1 expression
increased DmCncC binding at the Jheh gene loci and triggered their transcription
(Deng and Kerppola, 2014).
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Our previous results revealed that silencing of either LdJheh1 and LdJheh2, or both
genes significantly augmented JH titers (Lü et al., 2015), indicating the two genes
encoding functional JH degradation enzymes. Moreover, we found that knockdown of
either LdJheh1 or LdJheh2, or both genes also delayed larval development (Lü et al.,
2015), a phenotype similar to the LdKeap1 and LdCncC RNAi hypomorphs in the
Therefore, we determined the expression levels of LdJheh1 and LdJheh2, and the
JH titers in the LdKeap1 and LdCncC RNAi hypomorphs. Surprisingly, our results
showed that the expression levels of LdJheh1 and LdJheh2 and the JH titers were not
affected in the LdKeap1 and LdCncC RNAi larvae. It appears that LdCncC/LdKeap1
signaling may not be involved in the activation of JH degradation genes in L. 18
ACCEPTED MANUSCRIPT 399
4.5. LdCncC and LdKeap1 plays other physiological roles Except the L. decemlineata larval PGs, both LdCncC and LdKeap1 genes were
easily detectable in other tested larval tissues such as guts, and adult ovaries and testis.
Similarly, DmCncC and DmKeap1 mRNAs were abundantly expressed in the D.
melanogaster larval alimentary canal, Malpighian tubules, salivary glands and brain,
as well as adult female and male flies (Sykiotis and Bohmann, 2008). Since digestive
tract represents the first line of defense to environmental stressors, and the Malpighian
tubules are major sites of detoxification, the tissue expression profiles in both L.
decemlineata and D. melanogaster larvae are reminiscent of a crucial role of the
Keap1/Nrf2 module as a multiorgan protector in mammalians (Itoh et al., 1977;
Kensler et al., 2007; Kobayashi and Yamamoto, 2006; Leiser and Miller, 2010;
Venugopal and Jaiswal, 1996).
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Moreover, DmCncC/DmKeap1 pathway was necessary and sufficient for
xenobiotic-induced transcription of a wide range of detoxification genes in
insecticide-resistant D. melanogaster strains (Deng and Kerppola, 2013; Karim et al.,
2015; Misra et al., 2011; Misra et al., 2013; Sykiotis and Bohmann, 2008). In Aphis
gossypii, in vivo RNAi of AgCncC dramatically suppressed the expression of
AgCYP6DA2, and increased the sensitivity to gossypol (Peng et al., 2016). In
Tribolium castaneum, TcCncC and V-maf musculoaponeurotic fibrosarcoma oncogene
homolog regulated the expression of deltamethrin metabolism gene TcCYP6BQ (Kalsi
and Palli, 2015). Similarly, LdCncC and LdMaf are involved in the regulation of four
ACCEPTED MANUSCRIPT cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25 and CYP9Z29) that are
required for defense against both natural and synthetic chemicals (Kalsi and Palli,
2017). In the present paper, we found that the expression of 11 (out of 14) LdGSTd
and LdGSTe members was significantly suppressed in the LdCncC RNAi hypomorphs,
whereas the expression of 4 transcripts was significantly activated and 5 transcripts
was dramatically repressed in the LdKeap1 RNAi larvae.
In D. melanogaster, CncC and Keap1 mediate transcriptional responses to
xenobiotic genes and developmental signals using distinct mechanisms. DmKeap1
regulates xenobiotic response genes through inhibiting nuclear DmCncC levels. It can
interact with DmCncC and trigger its ubiquitination and proteasomal degradation in
the cytoplasm. Interference of this interaction in response to stimuli leads to
stabilization and nuclear accumulation of DmCncC (Itoh et al., 1999; Kobayashi et al.,
2004). In contrast, DmKeap1 regulates some developmental genes through facilitating
DmCncC binding to chromatin (Deng, 2014). In the work present here, we found that
knockdown of LdCncC resulted in the suppression of both xenobiotic agents and
several Halloween genes. In contrast, silencing of LdKeap1 led to the suppression of
several Halloween genes and a subset of xenobiotic genes, but resulted in the
upregulation of another subset of xenobiotic genes. Moreover, a subset of CYP genes
were regulated by CncC/Maf in both T. castaneum (Kalsi and Palli, 2015) and L.
decemlineata (Kalsi and Palli, 2017). Therefore, the two distinct mechanisms of CncC
signaling to mediate transcriptional responses are conserved in L. decemlineata,
although the specific gene subsets regulated are different between the beetle and D.
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ACCEPTED MANUSCRIPT 443
melanogaster (Deng, 2014; Itoh et al., 1999; Kobayashi et al., 2004) . In addition, our results showed that the expression levels of LdCncC, LdKeap1 and
the three LdGSTd genes were higher right after the molt. It gives the impression that
the active LdCncC/LdKeap1 signaling triggers the expression of detoxification genes,
such as CYPs and GSTs, during the early and mid instar stages. The resultant enzymes
may degrade xenobiotics from food to protect the larvae from poisoning when they
are actively feeding.
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This research was supported by the National Natural Science Foundation of China
(31272047 and 31360442), and the Fundamental Research Funds for the Central
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Fig. 1. Temporal expression patterns of LdCncC (A) and LdKeap1 (B) genes in L.
decemlineata. The cDNA templates are derived from the day 3 eggs, the whole bodies
of the first, second and third larval instars at an interval of one day, and from the
fourth larval instars at an interval of twelve hours (D0/H0 indicated newly ecdysed
larvae). 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 individuals at
specific developing stages to that in the day 1 third-instar larvae (A) or the eggs (B).
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.
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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.
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Fig. 4. Effects of knockdown of LdCncC (A) and LdKeap1 (B) genes in L.
decemlineata fourth-instar larvae. The newly-ecdysed fourth-instar larvae have
been allowed to ingest PBS (CK)-, dsegfp- and dsCncC-immersed leaves, or PBS
(CK)-, dsegfp- and dsKeap1-dipped leaves for 3 days, and normal foliage for an
additional 2 days. The relative transcripts (A, D) are measured on the 3 days after the
initiation of experiment. 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). The emergence rates in the same days after dsRNA exposure are
compared (B, E). Knockdown either gene causes development delay. The larvae and
pupae are weighed on the 5 and 10 days after the initiation of experiment (C, F).
Different letters above the bars indicate significant difference at P value < 0.05. The
larval and pupal sizes of dsCncC-, and dsKeap1-fed beetles are shown (G, H).
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Fig. 5. Knockdown of LdCncC and LdKeap1 genes in L. decemlineata
fourth-instar larvae affecting ecdysteroidogenesis. The newly-ecdysed fourth-instar
larvae have been confined in dishes containing PBS (CK)-, dsegfp- and
dsCncC-immersed leaves, or PBS (CK)-, dsegfp- and dsKeap1-dipped leaves for 3
days. The relative transcripts of five Halloween genes (Ldspo, Ldphm, Lddib, Ldsad
and Ldshd) (A, B) and the 20-hydroxyecdysone (20E) titer (C) are determined. 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.
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Fig. 6. Knockdown of LdCncC (A) and LdKeap1 (B) genes in L. decemlineata
fourth-instar larvae have ingested dsCncC-, or dsKeap1-dipped leaves for 3 days. The
PBS (CK)- and dsegfp-immersed leaves are used as controls. The relative transcripts
of five 20-hydroxyecdysone response genes (LdEcR-A, LdUSP, LdE75, LdHR3 and
LdFTZ-F1) are quantified. 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.
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Fig. 7. Rescuing effect of 20-hydroxyecdysone (20E) on the phenotypes of the
LdCncC and LdKeap1 knockdown larvae in L. decemlineata. The newly-ecdysed
fourth-instar larvae have ingested foliage immersed with dsCncC or dsCncC+20E
(A-C), or dsKeap1 or dsKeap1+20E (D-F) for 3 days. The dsegfp-immersed leaves
are used as control. The relative transcripts of LdCncC and LdKeap1 (A, D) are tested.
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 dsCncC or dsKeap1-ingested individuals to
dsegfp-fed ones. The emergence rates in the same days after dsRNA exposure are
compared (B, E). The fresh larval weights (C, F) are measured.
above the bars indicate significant difference at P value < 0.05. Ingestion of 20E
completely restores the development delay and overweight phenotypes in the LdCncC
and LdKeap1 knockdown larvae.
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.
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
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