ELECTRONIC SUPPLEMENTARY MATERIAL
(a)
Silkworm strain
A multivoltine, non-diapausing silkworm strain, Nistari, was used in this study. Silkworm larvae were
reared on fresh mulberry leaves at 25°C under standard conditions as described previously.
(b)
cDNA synthesis and quantitative real-time PCR
Total RNA was isolated from larval tissues using TRIzol reagent (Invitrogen) following the
manufacturer’s instructions. A total of 1 μg RNA was used for cDNA synthesis using the ReverAid
First Strand cDNA Synthesis Kit (Vazyme Biotech Co., ltd.). Relative mRNA levels of BmEO were
determined by qRT-PCR using SYBR Green Real-time PCR Master Mix (TOYOBO). PCR conditions
used are as follows: initial incubation of 95°C for 1 min, followed by 40 cycles of 95°C for 10 s and
55°C for 20 s. A pair of primers (EORTF: 5-CCACCGTACCTGAGTTTGCT-3 and EORTR:
5-GCAGGACAGCAGCAATTGTA-3) designed based on the sequence obtained from the GenBank
(JF433972) were used in qRT-PCR. Primers that amplified a 136-bp fragment of B. mori ribosomal
protein 49 (Bmrp49) were used as the internal control. Three independent biological replicates were
used for each treatment. Other qRT-PCR primers used are included in the electronic supplementary
material, table S2.
(c)
To
Plasmid construction
establish
a
binary
transgenic
CRISPR/Cas9
system,
an
activator
plasmid
pBac[3xp3-DsRed2-IE1-Cas9] (IE1-Cas9), which constitutively expresses Cas9 under the control of a
baculovirus immediate-early gene promoter IE1, and an effector plasmid pBac[3xp3-EGFP-U6-BmEO
sgRNA] (U6-BmEOsgRNA) which expresses sgRNA targeting BmEO under the control of the silkworm
small nuclear RNA promoter U6 were constructed through a series of cloning steps. The primers used
in cloning are included in the electronic supplementary material, table S2.
For construction of the Gal4/UAS transgenic system, a ubiquitous activator line A3-Gal4 and a
MSG-specific line Ser-Gal4 described in our recent publications were used. The open reading frame
(ORF) sequences of BmEO were sub-cloned into the initial plasmid pBac[UAS-3xp3-EGFP] using
ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co., ltd.) and used to generate the effector
line UAS-BmEO.
(d)
Silkworm germline transformation
Preblastoderm embryos were injected with transgenic plasmids mixed with the helper plasmid and
incubated in a humidified chamber at 25°C for 10–12 days until hatching. The G0 moths were sib-mated
or backcrossed with wild-type (WT) moths and the presence of the selection marker gene in G1 progeny
was scored during late embryonic stage using a fluorescence microscope (Nikon AZ100). Heterozygous
progeny between IE1-Cas9 and U6-BmEOsgRNA lines or between Gal4 (Ser-Gal4 or A3-Gal4) and
UAS-BmEO lines were scored for double fluorescence and used in subsequent experiments.
(e)
RNA-seq analysis
A total of 20 μg RNA samples extracted from the MSG dissected from Ser>BmEO and UAS-BmEO
was used to construct RNA-seq libraries using Illumina kit. Total mRNA was enriched using Sera-mag
Magnetic Oligo (dT) Beads (Illumina). The RNA was fragmented (200 nt) and used for cDNA synthesis
using random hexamer (N6) primers. The cDNA was then subjected to end-repair with 3-adenylation
and adapters addition (Illumina). After 15 rounds PCR amplification with PCR primer PE 1.0 and PCR
primer PE 2.0, the libraries were sequenced using the Illumina HiSeq 2000 platform (Shanghai OE
BIOTECH CO., LTD). Sequenced raw data were qualified, filtered and then mapped to the reference
silkworm genome database (http://silkworm.genomics.org.cn/) by tophat/bowtie2. Unigene abundances
were measured by Fragment per kilobase of exon per million fragments mapped (FPKM). Differentially
expressed genes (DEGs) were functionally annotated by GO and KEGG annotation.
(f)
Ecdysteroid titer determination
Ecdysteroids were extracted from the tissues of transgenic or control silkworms that are in the
wandering stage, when 20E titer reaches its maximum levels in the WT animals. Tissues were dissected
on ice, homogenized and extracted with methanol as described by Warren et al. [23]. The extracts were
evaporated, re-dissolved in EIA buffer (Cayman Chemical), and subjected to enzyme immunoassays
using the EIA kit (Cayman). 20E purchased from Sigma was used as the standard. 20E is specifically
recognized by the anti-20E antiserum and quantified by its competitor acetylcholinesterase-conjugated
20E. The acetylcholinesterase activity was measured using Ellman’s buffer.
(g)
Paraffin embedding and LysoTracker staining
The MSG dissected from pre-pupal day 2 (PP2) animals (UAS-BmEO and Ser>BmEO lines) were fixed
in Qurnah’s fixative (ethanol:chloroform:acetic acid=6:3:1) and subjected to 100% ethanol treatment
three times followed by xylene treatment three times, and embedded in paraffin overnight. Five
micrometre thick cross sections were cut using the Leica RM2235 microtome. The sections were
processed with xylene, 95, 80 and 70% ethanol, distilled water and phosphate-buffered saline (PBS).
Rehydrated sections were stained with 50 nM LysoTracker Deep Red (Invitrogen) for 1 h at 37°C and
washed with PBS for three times. Samples were observed under a FV10-ASW Confocal microscope
(Olympus). For the fat body and midgut, dissected tissues were separated into small pieces, washed
with PBS three times and stained directly.
(h)
Transmission electron microscopy
MSG, fat body and midgut from PP2 animals were fixed in 2.5% glutaraldehyde at 4°C overnight. Then
the tissues were washed with PBS twice, post-fixed with 0.5% osmium tetroxide for 2 h and embedded
in Epon resin. Sections were cut and stained with Reynold’s lead citrate. Autophagic structures were
investigated under a H7650 transmission electron microscope (Hitachi).
(i)
Antibody production and western blotting
The BmEO ORF sequence was cloned into the pET-28a(+) (Novagen) plasmid to express and purify
recombinant protein. Rabbits were immunized with BmEO protein four times (1.5 mg each time). Then
antibody produced was purified with a Protein A column. The selected tissues were dissected and
proteins
were
extracted
in
PBS
(phosphate-buffered
saline)
containing
1%
PMSF
(phenylmethanesulfonyl fluoride). The proteins were quantified using the BCA kit (Thermo). Proteins
were separated using SDS/PAGE (10%) and transferred to a nitrocellulose membrane (GE Healthcare).
BmEO protein was detected with rabbit anti-BmEO primary antibody (1:1000 dilution, Youke Biotech).
Silkworm α-tubulin, detected by the mouse anti-rabbit α-tubulin primary antibody (1:5000 dilution;
Vazyme biotech co., ltd.), was used as the control. The horseradish peroxidase conjugated goat
anti-mouse or anti-Rabbit IgG (1:5000 dilution; Beyotime) was used as the secondary antibody. The
signal was detected by the ECL Plus Western Blotting Detection Kit (GE Healthcare).
(j)
Statistical analysis of data
A two-tailed Student’s t-test was used to perform statistical analysis. At least three independent
replicates were used for each treatment and the error bars show means±s.e.m.
References
1. Tan A, Tanaka H, Tamura T, Shiotsuki T. 2005 Precocious metamorphosis in transgenic silkworms
overexpressing juvenile hormone esterase. Proc. Natl Acad. Sci. USA 102, 11751–11756.
(doi:10.1073/pnas.0500954102)
2. Li Z, Zeng B, Ling L, Xu J, You L, Aslam AF, Tan A, Huang Y. 2015 Enhancement of larval RNAi
efficiency by over-expressing Argonaute2 in Bombyx mori. Int. J. Biol. Sci. 11, 176–185.
(doi:10.7150/ijbs.10235)
3. Tan A et al. 2013 Transgene-based, female-specific lethality system for genetic sexing of the
silkworm, Bombyx mori. Proc. Natl Acad. Sci. USA 110, 6766–6770. (doi:10.1073/pnas.1221700110)
4. Warren JT, Yerushalmi Y, Shimell MJ, O’Connor MB, Restifo LL, Gilbert LI. 2006 Discrete pulses
of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster:
correlations with changes in gene activity. Dev. Dyn. 235, 315–326. (doi:10.1002/dvdy.20626)
Table S1. Developmental profile of transgenic silkworms.
No. of
No. of larvae
1st
with
5th
No. of pupae
No. of
with
No. of
survived
retained
survived
MSG
Adult
instar
extended
larvae
instar
pupae
UAS-BmEO
47
0
44
0
41
A3>BmEO
43
0
6
0
0
Ser>BmEO
87
0
85
85
83
U6-BmEOsg
41
0
39
0
39
33
20
32
0
32
Genotype
RNA
IE1>BmEOs
gRNA