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599

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600

601

20

602

Figure legends

603

Figure 1. Phenotypes of Tribolium castaneum pupae that underwent topical application of JH or

604

JHM. Newly molted pupae within 12 h after molting were treated with solvent, pyriproxyfen, or

605

JH III. (A–C) Ventral view of pupae observed from pupa day 1 (P1) to pupa day 5 (P5) that had

606

received the solvent methanol (A), 40 pmol pyriproxyfen (B), and 0.80 nmol of JH III (C). (D–K)

607

Ventral view of pupae observed at P3 that had been treated with solvent (D), pyriproxyfen at 40

608

pmol (E), 0.12 nmol (F), 0.40 nmol (G), and 1.2 nmol (H), 0.40 nmol of fenoxycarb (I), 0.40 nmol

609

of (S)-hydroprene (J), and 40 pmol of (S)-(+)-methoprene (K). The numerator is the number of

610

phenotypes that were observed among the samples, and the denominator is the total number of test

611

insects used in the experiment.

612

613

Figure 2. Expression profiles of selected genes in the whole body of Tribolium castaneum pupae

614

after JHM treatment. Newly molted pupae within 12 h after molting were treated with 40 pmol of

615

pyriproxyfen or an equal volume of solvent. RNA extraction from the whole body followed by

616

quantitative RT-PCR were conducted at different pupal ages from pupa day 1 (P1) to pupa day 5

617

(P5). The transcript levels of Kr-h1 (A), br (B), E93 (C), E75 (D), HR3 (E), Ftz-f1 (F), Th (G),

618

DDC (H), Lac2 (I), CPR27 (J), and Y-y (K) were examined. Asterisks over the bars indicate

619

statistically significant differences from the solvent-treated controls (*P<0.05, **P<0.01,

620

***P<0.001, and ****P<0.0001; Student’s t-test). Means and standard errors of the mean (SEM)

621

are shown (N=6). P-values are available in Table S2.

622

623

Figure 3. Expression profiles of selected genes in the legs of Tribolium castaneum pupae after

624

JHM treatment. Newly molted pupae within 12 h after molting were treated with 40 pmol of

625

pyriproxyfen or an equal volume of solvent. RNA extraction from pooled legs followed by

626

quantitative RT-PCR were conducted from pupa day 3 (P3) to pupa day 5 (P5). The transcript

627

levels of Kr-h1 (A), br (B), E93 (C), E75 (D), Th (E), DDC (F), Lac2 (G), and CPR27 (H) were

628

examined. Asterisks over the bars indicate statistically significant differences from the solvent-

629

treated controls (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001; Student’s t-test). Means

630

and standard errors of the mean (SEM) are shown (N=6). P-values are available in Table S3.

21

631

632

Figure 4. Ventral view of the Tribolium castaneum pupae and an adult that were treated with

633

dsRNA of malE (A) and E75 (B). The prepupae were injected with approximately 100 nl of 5

634

µg/µl dsRNA. P stands for pupa; A stands for adult; 0 to 5, day age; scale bars, 500 µm.

635

636

Figure 5. Expression profiles of selected genes in the whole body of Tribolium castaneum pupae

637

and adults after E75 knockdown. Prepupae were injected with dsRNA of malE or E75. RNA was

638

extracted from the whole body, and quantitative RT-PCR was conducted from day 0 pupa (P0) to

639

day 0 adult or day 5 pupa (A0/P5). The transcript levels of Kr-h1 (A), br (B), E93 (C), E75 (D),

640

HR3 (E), Ftz-f1 (F), Th (G), DDC (H), Lac2 (I), CPR27 (J), and Y-y (K) were examined. Inset of

641

panel J shows the levels at A0/P5 by enlarging the Y-axis. Asterisks over the bars indicate

642

statistically significant differences from the dsmalE-treated controls (*P<0.05, **P<0.01,

643

***P<0.001, and ****P<0.0001; Student’s t-test). Means and standard errors of the mean (SEM)

644

are shown. Six samples each were used for P0 to P4, but only three samples were used for A0/P5.

645

P-values are available in Table S4.

646

647

Figure 6. Expression profiles of selected genes in the legs of Tribolium castaneum pupae after E75

648

knockdown. Prepupae were injected with dsRNA of malE or E75. RNA was extracted from the

649

legs, and quantitative RT-PCR was conducted from P2 to P4. The transcript levels of Kr-h1 (A),

650

br (B), E93(C), E75 (D), Th (E), DDC (F), Lac2 (G), and CPR27 (H) were examined. Asterisks

651

over the bars indicate statistically significant differences from dsmalE-treated controls (*P<0.05,

652

**P<0.01, ***P<0.001, and ****P<0.0001; Student’s t-test). Means and standard errors of the

653

mean (SEM) are shown. Six samples each were used for P3 and P4, but only three samples were

654

used for P2. P-values are available in Table S5.

655

656

Figure 7. A model for the regulatory pathway of adult cuticular formation in Tribolium castaneum.

657

(A) In normal pupae with undetectable JH, E75 induces adult cuticular formation in a proper

658

timing; the expression of CPR27, Y-y, and Lac2 is upregulated, and Th is suppressed. (B) In JHM-

22

659

or dsE75-treated pupae, E75 was downregulated compared with normal pupae, which disturbed

660

adult development of the cuticle; CPR27, Y-y, and Lac2 were downregulated, and Th was induced

661

precociously. E75 is involved in regulating the transcription of CPR27 and Th, but does not affect

662

the transcription of Y-y or Lac2. See the Discussion section for details.

663

664

Supplementary Materials

665

Figure S1. The structure of E75 isoforms of Tribolium castaneum.

666

Double-headed arrows represent the target region of quantitative RT-PCR primers, and the double

667

underlines show the region of E75 dsRNA. Arrowheads represent the position of the start codon

668

in each isoform, and asterisks show the stop codon. Numbers in bars are length in bp.

669

670

Figure S2. Expression profiles of selected genes in whole body of Tribolium castaneum pupae and

671

newly emerged adults.

672

RNA extraction from the whole body followed by quantitative RT-PCR were conducted at the

673

different ages from pupa day 0 (P0) to adult day 0 (A0). Expression profiles of Kr-h1 (A), br (B),

674

E93 (C), E75 common region (D), HR3 (E), Ftz-f1 (F), Th (G), DDC (H), Lac2 (I), CPR27 (J), Y-

675

y (K), E75 isoforms (L–P) were examined. Means and standard errors of the mean (SEM) are

676

shown (N=4).

677

678

Figure S3. Expression profiles of E75 isoforms using whole body pupae of Tribolium castaneum

679

after JHM treatment (A) and after E75 knockdown (B).

680

(A) Expression profiles in the whole body of Tribolium castaneum pupae after JHM treatment.

681

The same set of cDNAs as in Figure 2 was used. Newly molted pupae within 12 h after molting

682

were treated with 40 pmol of pyriproxyfen or an equal volume of solvent. Asterisks over the bars

683

indicate statistically significant differences from the solvent-treated controls (*P<0.05, **P<0.01,

684

and ***P<0.001; Student’s t-test). Means and standard errors of the mean (SEM) are shown (N=6).

685

P-values are available in Table S2.

23

686

(B) Expression profiles in the whole body of Tribolium castaneum pupae after E75 knockdown.

687

The same set of cDNAs as in Figure 5 was used. Prepupae were injected with dsRNA of malE or

688

E75. RNA was extracted from the whole body, and quantitative RT-PCR was conducted. Asterisks

689

over the bars indicate statistically significant differences from the dsmalE-treated controls

690

(*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001; Student’s t-test). Means and standard errors

691

of the mean (SEM) are shown. Six samples each were used for P0 to P4, but only three samples

692

were used for A0/P5. P-values are available in Table S4.

693

694

Table S1 Primers used in this study.

695

696

Table S2 The P-values of the transcript levels of selected genes in the whole body of Tribolium

697

castaneum pupae after treatment of 40 pmol of pyriproxyfen or solvent. The P-values indicating

698

there is not statistically significant differences (P>0.05) are shadowed. The transcript levels are

699

shown as graphs in Figure 2 and Figure S3.

700

701

Table S3 The P-values of the transcript levels of selected genes in the legs of Tribolium

702

castaneum pupae after treatment of 40 pmol of pyriproxyfen or solvent. The P-values indicating

703

there is not statistically significant differences (P>0.05) are shadowed. The transcript levels are

704

shown as graphs in Figure 3.

705

706

Table S4 The P-values of the transcript levels of selected genes in the whole body of Tribolium

707

castaneum pupae after injecting dsRNA of malE and E75. The P-values indicating there is not

708

statistically significant differences (P>0.05) are shadowed. The transcript levels are shown as

709

graphs in Figure 5 and Figure S3.

710

711

Table S5 The P-values of the transcript levels of selected genes in the legs of Tribolium

712

castaneum pupae after injecting dsRNA of malE and E75. The P-values indicating there is not

24

713

statistically significant differences (P>0.05) are shadowed. The transcript levels are shown as

714

graphs in Figure 6.

715

716

25

Figure 1

Click here to access/download;Figure;Fig 1_R3_2.tiff

Figure 2

Figure 2

Solvent

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Pyriproxyfen

0.15

Kr-h1

0.40

***

0.40

**

0.30

****

E93

0.30

***

0.10

0.20

***

***

0.05

***

****

0.20

****

**

0.10

0.10

**

P1

1.50

P2

P3

P4

P1

P5

E75

0.60

1.25

0.50

1.00

0.40

0.75

0.30

***

0.25

****

***

P2

P3

P4

P5

0.30

Th

0.25

1.00

0.20

0.75

0.15

0.50

0.10

0.25

0.05

0.00

0.00

P1

P5

0.40

P2

P3

P4

P5

150

***

P1

P2

P3

****

P4

P5

P2

P3

P4

P5

Ftz-f1

0.00

P2

P3

P4

P1

P5

DDC

0.80

Lac2

0.60

0.40

**

0.20

***

***

**

0.00

P2

P3

P4

P5

Y-y

0.05

**

P5

****

100

P4

0.10

**

0.10

50

P3

0.20

P1

0.15

CPR27

P2

0.30

P1

1.25

200

P4

0.00

P1

P3

HR3

0.10

0.00

1.50

P2

0.20

0.50

**

0.00

0.00

0.00

P1

Relative mRNA levels (/rpL32)

br

***

0.00

P1

P2

P3

P4

***

P5

P1

P2

P3

P4

P5

Figure 3

Figure 3

Solvent

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Pyriproxyfen

0.10

Kr-h1

2.0

br

2.5

**

0.08

1.5

***

2.0

1.5

****

0.06

1.0

1.0

0.04

0.5

0.02

**

0.0

0.0

P3

P4

P3

P5

E75

P4

0.5

**

0.00

E93

P3

P5

Th

2.5

P4

P5

DDC

2.0

1.5

**

1.0

**

P4

P5

P3

Lac2

800

P4

P5

CPR27

600

1.0

400

0.5

**

200

0.0

P3

P4

P5

****

P3

**

P4

P5

0.0

P3

1.5

0.5

****

P3

P4

P5

Figure 4

Figure 4

dsmalE

P0

P1

P2

P3

P4

P1

P2

P3

P4

P5

dsE75

P0

Figure 5

Figure 5

dsmalE

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

dsE75

0.05

E93

0.40

0.30

**

0.30

0.03

0.20

0.20

0.02

0.10

0.01

0.00

2.00

P1

P2

P3

P4 A0/P5

P0

0.50

E75

P1

P2

P3

P0

P4 A0/P5

0.40

HR3

0.40

1.50

0.10

0.00

0.00

P0

P1

Ftz-f1

P2

P3

P4 A0/P5

****

0.30

0.30

1.00

0.20

****

****

0.00

P1

P2

P3

P4 A0/P5

P0

0.25

Th

****

2.00

P1

1.50

**

P2

P3

****

**

0.00

P4 A0/P5

P0

1.00

DDC

0.20

**

**

0.10

0.10

0.00

0.20

**

0.50

2.50

****

P1

P2

P3

P4 A0/P5

P2

P3

P4 A0/P5

Lac2

0.80

0.60

0.15

***

1.00

0.10

0.50

0.05

P0

100

P1

0.40

0.20

***

0.00

Relative mRNA levels (/rpL32)

0.50

br

**

0.04

P0

Relative mRNA levels (/rpL32)

0.40

Kr-h1

**

0.00

0.00

P2

P3

P4 A0/P5

P0

P1

P2

P3

P4 A0/P5

0.15

CPR27

0.25

0.20

0.15

0.10

0.05

0.00

80

60

Y-y

0.10

A0/P5

40

0.05

20

**

P0

P1

P2

P3

***

P4 A0/P5

**

0.00

P0

P1

P2

P3

P4

A0/P5

P0

P1

Figure 6

Figure 6

dsmalE

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

dsE75

0.04

0.03

br

E93

0.04

0.02

**

***

0.02

0.01

0.00

0.00

P2

P3

P2

P4

E8

E75

P3

P4

P2

0.8

Th

P4

P3

P4

DDC

****

0.6

**

0.4

0.2

****

****

P3

P4

2.5

P3

***

P2

Relative mRNA levels (/rpL32)

0.06

Kr-h1

600

Lac2

0.0

P2

P3

P4

CPR27

2.0

400

1.5

1.0

200

0.5

0.0

P2

P3

P4

P2

P3

P4

P2

Figure 7

Figure 7

In normal pupae

Observed phenotypes

Undetectable JH

Adult development of the

cuticle in a proper timing

E75

Th

CPR27

Y-y, Lac2

JH mimic- or dsE75-treated pupae

Exogenous JH mimic

dsE75

Disturbed adult development of

the cuticle

E75

CPR27

Th

Y-y, Lac2

Table S1. Primers used in this study

Gene

Forward primer 5’ to 3’

Reverse primer 5’ to 3’

5'RACE PCR

TcE75A-RR1

CGGTAATTGTCCACTGCTATCCGGTGA

TcE75B-RR1

CATGTCAGGCGGCCACCATTGTTTAT

TcE75C-RR1

ACTTGAACACGATGGTGGCGGTCTG

TcE75D-RR1

TCACATGTGCACCAGCAGCACGATAG

TcE75E-RR1

CGGTGGCACAGGTGGTCTCTTTGAA

qRT-PCR

E75 common

GCTTCATCATGGTCGTCCTCTT

AGGAGGTGATTTCTGAGGAGCA

E75A

CGGAATCCACCACGGATAGTAACA

AAGCCCTTGCATCCTTCGCA

E75B

GGCTCAGTGCTCCGTCGAATTT

GTTTTATCCGAACTCTGGGGCG

E75C

TAACGGGGGTGACGACATGCAG

TCCTCCTGCGTCTTGGGCTT

E75D

GACAGGCTATCGTGCTGCTGGT

GCTTTCTCCCTTTTCGGAACGC

E75E

GACAGCGAGAACCTCCTGGG

AGTGGACGCCGTAGTGGAA

E93

CTCTCGAAAACTCGGTTCTAAACA

TTTGGGTTTGGGTGCTGCCGAATT

br

CCAGCGGTCTCTTTCGTCGTTT

CGTCCTCCACTAACTTCTCGGTGAA

Kr-h1

TGCCATTGAATAACACCACCAA

CCAAGGGGTCTTCGGTGTAATA

HR3

GAACGACACGGGAAGCTTAATG

AAGTACGTGTGTGTGCGTCTGA

Ftz-f1

CTAACATCGTCGCCCGACTC

GAGCCCCAGAGCTTGTTGTC

JHAMT

CATCTCGCCCTATCACCATTCG

CCGCTGAAACCGATTTTGACAA

Tyrosine hydroxylase

CCAGACGCTGAAAAGGCTCT

TGACATACTGCCCCTTGGTG

DDC

TGAGGCTGGCCTTATTCCAT

GCAGCCAGATGTTGTTCGAG

Laccase 2

CGTTTTCAGGTGAACGATACGA

GTTGGTATGGCCCTTTGGCATA

Yellow-y

GGAAACACAACCCAAAACCCGT

TGGGTTTGTGTCTTCAGGTCGT

CPR27

GCCCAAGGGGGAGAAGGTTA

CTCCCATTGGTGGTGGAAGTC

rpL32

CAGGCACCAGTCTGACCGTTATG

GCTTCGTTTTGGCATTGGAGC

dsRNA synthesis (T7 promoter sequence is underlined)

E75 common

TAATACGACTCACTATAGGGAAGTTCGAATTCGC TAATACGACTCACTATAGGGATGAGTTCGGTGTT

GTTCTCTG

CCTCAAGC

HR3

TAATACGACTCACTATAGGGGAGATAATCCCGTG TAATACGACTCACTATAGGGACGCTGTTGTACTG

CAAAGTATGTG

TCCACGTAATC

Table S2 The P-values of the transcript levels of selected genes in the whole body of

Tribolium castaneum pupae after treatment of 40 pmol of pyriproxyfen or solvent. The Pvalues indicating there is not statistically significant differences (P>0.05) are shadowed.

The transcript levels are shown as graphs in Figure 2 and Figure S3.

P1

P2

P3

P4

P5

Kr-h1

<0.0001

0.0002

0.0001

0.0003

0.0004

br

<0.0001

<0.0001

0.0002

0.0086

0.0708

E93

0.0012

0.0024

0.1973

0.5080

0.0030

E75

0.1986

0.0005

<0.0001

0.0003

0.4209

HR3

0.6923

0.0021

0.0001

<0.0001

0.0644

Ftz-f1

0.8876

0.0780

0.6222

0.6959

0.0635

Th

0.8120

0.1340

0.1796

0.0619

0.0611

DDC

0.4892

0.0163

0.0470

0.0477

0.0064

Lac2

0.1615

0.0573

0.0003

0.0003

0.0010

CPR27

0.8639

0.2683

0.0020

<0.0001

0.0112

Y-y

0.1145

0.3757

0.1479

0.0002

0.0002

E75A

0.2638

0.0433

0.1553

0.9408

0.7730

E75B

0.2637

0.0756

0.0063

0.0041

0.3878

E75C

0.0152

0.0952

0.2040

0.4043

0.9252

E75D

0.8496

0.2027

0.0032

0.2284

0.6483

E75E

0.7043

0.4942

0.0002

0.0001

0.0675

Table S3 The P-values of the transcript levels of selected genes in the legs of Tribolium

castaneum pupae after treatment of 40 pmol of pyriproxyfen or solvent. The P-values

indicating there is not statistically significant differences (P>0.05) are shadowed. The

transcript levels are shown as graphs in Figure 3.

P3

P4

P5

Kr-h1

0.0008

<0.0001

0.0074

br

0.0139

0.0051

0.0073

E93

0.0254

0.1876

0.0385

E75

<0.0001

0.0235

0.1966

Th

0.0018

0.0466

0.0053

DDC

0.5783

0.0631

0.0171

Lac2

0.2018

0.1678

0.0045

<0.0001

0.0020

0.0553

CPR27

Table S4 The P-values of the transcript levels of selected genes in the whole body of

Tribolium castaneum pupae after injecting dsRNA of malE and E75. The P-values

indicating there is not statistically significant differences (P>0.05) are shadowed. The

transcript levels are shown as graphs in Figure 5 and Figure S3.

P0

P1

P2

P3

P4

P5/A0

Kr-h1

0.0782

0.7537

0.1743

0.0743

0.4346

0.0099

br

0.4246

0.4821

0.2117

0.1651

0.3393

0.0206

E93

0.0972

0.0866

0.9114

0.0082

0.2709

0.1178

E75

0.3038

0.0478

0.0015

<0.0001

0.0153

0.2425

HR3

0.9330

0.1279

0.0229

<0.0001

0.0028

0.1983

Ftz-f1

0.1135

<0.0001

<0.0001

0.0016

0.3723

0.0920

Th

0.5663

0.0180

0.0037

<0.0001

0.0035

0.1476

DDC

0.9037

0.0004

<0.0001

0.0006

0.1701

0.0243

Lac2

0.1992

0.0059

0.3549

0.3805

0.8006

0.7881

CPR27

0.6511

0.2243

0.2801

0.0368

0.0006

0.0481

Y-y

0.4952

0.0053

0.3929

0.0805

0.0012

0.3574

E75A

0.3625

0.2610

0.0639

0.0290

0.6250

0.3252

E75B

0.0015

<0.0001

0.0201

<0.0001

0.0331

0.5682

E75C

0.0323

0.0010

0.5047

0.1892

0.8929

0.1733

E75D

0.0172

0.0866

0.0007

0.0008

0.9339

0.0022

E75E

0.7539

0.0129

0.2266

0.0003

0.0012

0.6341

Table S5 The P-values of the transcript levels of selected genes in the legs of Tribolium

castaneum pupae after injecting dsRNA of malE and E75. The P-values indicating there

is not statistically significant differences (P>0.05) are shadowed. The transcript levels

are shown as graphs in Figure 6.

P2

P3

P4

Kr-h1

0.1787

0.1671

0.6509

br

0.1555

0.0271

0.1443

E93

0.3204

0.0006

0.0013

E75

0.0495

<0.0001

<0.0001

Th

0.1157

<0.0001

0.0003

DDC

0.0032

0.2917

0.4224

Lac2

0.6396

0.1811

0.8154

CPR27

0.3104

0.0327

0.0351

Figure S1

Isoform-specific

region

241

Common region

88 147

100 bp

222

E75 dsRNA

1991

272

(E75D)

E75 common

280

541

A, B, C, E-specific

A, C, E-specific

Specific region to each isoform

Common region among isoforms

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Figure S2

0.004

Kr-h1

0.040

0.001

0.80

0.200

0.000

0.000

P0 P1 P2 P3 P4 P5 A0

E75

P0 P1 P2 P3 P4 P5 A0

2.000

Relative mRNA levels (/rpL32)

HR3

P0 P1 P2 P3 P4 P5 A0

0.200

0.60

1.500

0.150

0.40

1.000

0.100

0.20

0.500

0.050

0.00

2.500

0.000

0.000

Th

2.000

P0 P1 P2 P3 P4 P5 A0

0.400

0 P1

1 P2

2 P3

3 P4

4 P5

5 A0

P0

Lac2

1.000

0.500

0.100

0.500

0.000

0.000

0.000

P0 P1 P2 P3 P4 P5 A0

CPR27

P0 P1 P2 P3 P4 P5 A0

0.15

200

0.10

100

0.05

1.500

Ftz-f1

0.200

1.000

DDC

0.300

1.500

300

E93

0.020

P0 P1 P2 P3 P4 P5 A0

Relative mRNA levels (/rpL32)

0.600

0.400

0.060

0.002

br

0.080

0.003

0.000

0.100

Y-y

0.00

0 P1

1 P2

2 P33 P44 P55 A0

P0

P0 P1 P2 P3 P4 P5 A0

P0 P1 P2 P3 P4 P5 A0

Figure S2, continued

0.15

E75A

0.40

E75B

0.30

0.10

0.15

E75C

0.10

0.20

0.05

0.00

0.20

0.05

0.10

P0 P1 P2 P3 P4 P5 A0

E75D

0.15

0.00

0.15

0P0 P1

1 P2

2 P3

3 P44 P55 A00

E75E

0.10

0.10

0.05

0.05

0.00

0.00

P0 P1 P2 P3 P4 P5 A0

0 P1

1 P2

2 P33 P44 P55 A00

P0

0.00

P0 P1 P2 P3 P4 P5 A0

Relative mRNA levels (/rpL32)

Relative mRNA levels (/rpL32)

Figure S2

0.004

Kr-h1

0.040

0.001

0.80

0.200

0.000

0.000

P0 P1 P2 P3 P4 P5 A0

E75

P0 P1 P2 P3 P4 P5 A0

2.000

Relative mRNA levels (/rpL32)

HR3

P0 P1 P2 P3 P4 P5 A0

0.200

0.60

1.500

0.150

0.40

1.000

0.100

0.20

0.500

0.050

0.00

2.500

0.000

0.000

Th

2.000

P0 P ...