An ecdysteroid-inducible insulin-like growth factor-like
peptide regulates adult development of the silkmoth
Bombyx mori
Naoki Okamoto
1
, Naoki Yamanaka
2,
*, Honoo Satake
3
, Hironao Saegusa
1,
, Hiroshi Kataoka
2
and
Akira Mizoguchi
1
1 Division of Biological Science, Graduate School of Science, Nagoya University, Japan
2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba,
Japan
3 Suntory Institute for Bioorganic Research, Osaka, Japan
Keywords
Bombyx mori; ecdysteroid; fat body;
insulin-like growth factors (IGFs);
insulin-like peptides
Correspondence
A. Mizoguchi, Division of Biological Science,
Graduate School of Science, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya
464-8602, Japan
Fax: +81 52 789 2511
Tel: +81 52 789 5039

Expression analysis reveals that this IGF-like peptide is predominantly
produced by the fat body, a functional equivalent of the vertebrate liver
and adipocytes, and is massively released during pupa–adult development.
Studies using in vitro tissue culture systems show that secretion of the
peptide is stimulated by ecdysteroid and that the secreted peptide promotes
the growth of adult-specific tissues. These observations suggest that this
peptide is a Bombyx counterpart of vertebrate IGFs and that functionally
IGF-like peptides may be more ubiquitous in the animal kingdom than
previously thought. Our results also suggest that the known effects of
ecdysteroid on insect adult development may be in part mediated by
IGF-like peptides.
Abbreviations
20E, 20-hydroxyecdysone; 8K-BLP, 8 kDa bombyxin-like peptide; BIGFLP, Bombyx mori insulin-like growth factor-like peptide; BrdU,
bromodeoxyuridine; DAPI, 4¢,6-diamidino-2-phenylindole; DILP, Drosophila insulin-like peptide; IGF, insulin-like growth factor; ILP, insulin-like
peptide; qRT-PCR, real-time quantitative RT-PCR.
FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS 1221
Members of the insulin-like peptide (ILP) family are
present in a wide variety of metazoans. In vertebrates,
insulin and insulin-like growth factors (IGFs) regulate
metabolism, growth, and development. Although these
peptides have similar amino acid sequences, they have
distinct domain organizations and physiological func-
tions: insulin is a heterodimeric peptide consisting of
an A-chain and a B-chain, whereas IGFs are single-
chain peptides with domains B, C, A and D, and the
major function of insulin is to control carbohydrate
metabolism, whereas that of IGFs is to promote tissue
growth [1,2]. They also differ in the mode of secretory
regulation; insulin secretion is modulated by blood
sugar concentration [3], whereas IGFs are secreted in a

bombyxin. Figure 1 shows the developmental fluctua-
tion of this 8 kDa bombyxin-like peptide (8K-BLP) in
Bombyx hemolymph assessed by western blotting with
the M7H2 antibody. Remarkably high levels of 8K-BLP
were detected during pupa–adult development, particu-
larly in females, whereas no visible bands were detected
in the larval or adult stages. This article describes the
purification and characterization of this peptide, and
shows that 8K-BLP is more like IGFs than like insulin
in many respects, and that 8K-BLP and bombyxin func-
tion in different developmental contexts.
Results
Purification of 8K-BLP
8K-BLP was purified from hemolymph of female
day 5 developing adults (see Experimental procedures
for definition) as follows: 20 mL of hemolymph was
subjected to heat treatment, M7H2 antibody affinity
chromatography (Fig. 2A), ion exchange HPLC
(Fig. 2B), and RP-HPLC (Fig. 2C). Antibody affinity
chromatography was so effective that 8K-BLP was
already the major component of the eluate (Fig. 2A).
Fractions from ion exchange HPLC were assayed for
bombyxin immunoreactivity by ELISA, which gave
two peaks of immunoreactivity: a large peak before
30 min, and a small peak shortly before 60 min. The
large peak contained 8K-BLP as assessed by western
blotting (Fig. S1A), whereas the smaller peak probably
contained bombyxin, because synthetic bombyxin-II
was eluted at the same retention time (Fig. S1B). The
8K-BLP-containing fractions were combined and sub-

peptide is eliminated and disulfide bridges are formed
as in other insulin family peptides, the theoretical
monoisotopic mass value is 7409.3 ([M + H]
+
), which
matches the mass value of 8K-BLP determined by
MALDI-TOF MS analysis. 8K-BLP consists of three
peptide domains, B, C and A (Fig. 3D). Although two
dibasic sites are present in the C-domain (boxed),
determination of the sequence beyond the first dibasic
site and the good agreement between the predicted and
measured mass values of 8K-BLP led us to conclude
that 8K-BLP is a single-chain peptide. This structural
feature indicates that this peptide is more similar to
IGFs than to insulin. Phylogenetic analysis revealed
that 8K-BLP does not belong to any known subfamily
of bombyxins (Fig. 3E). The phylogenetic relationship
between 8K-BLP and IGFs could not be determined,
because amino acid sequences are highly diverged
between mammalian and insect ILPs.
Production of 8K-BLP by fat body
To identify the tissue that produces 8K-BLP, gene
expression of 8K-BLP in various tissues was analyzed
by real-time quantitative RT-PCR (qRT-PCR). The
8K-BLP gene was predominantly expressed in the fat
body after pupal ecdysis (Fig. 4A). Gene expression
was very low during the larval stage but increased
slightly at the pharate pupal stage, and this was fol-
lowed by a steep increase after pupal ecydsis. There-
after, the expression further increased towards adult

The solid line denotes the UV absorbance, the broken line denotes
the NaCl or acetonitrile gradient, and the solid squares at the bot-
tom indicate ELISA-positive fractions. The immunoreactive fractions
before 30 min in (B) were combined and subjected to the next
chromatography step.
N. Okamoto et al. Silkmoth IGF-like peptide
FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS 1223
ecdysteroid in 8K-BLP secretion. To test this, we
investigated the in vitro effect of 20-hydroxyecdysone
(20E) on the fat body of pharate pupae shortly
(< 12 h) before pupation. Both 8K-BLP gene expres-
sion and secretion were significantly increased by the
addition of 20E to the fat body culture (Fig. 5A,B).
Growth-promoting effects of 8K-BLP on imaginal
anlagen
Bombyx pupae initiate adult development shortly after
pupal ecdysis, when larval tissues degenerate while
adult tissues, including the reproductive system and
flight muscles, undergo growth and differentiation.
Considering its structural similarity to IGFs and the
timing of secretion, we hypothesized that 8K-BLP reg-
ulates the growth of adult-specific tissues. To examine
this possibility, we first tried knockdown of the 8K-
BLP gene by injection of dsRNA; however, this did
not reduce 8K-BLP mRNA levels. Therefore, we inves-
tigated its potential growth-promoting effects in vitro
by addition of the purified 8K-BLP to the culture at a
concentration of 20 nm. This concentration was chosen
because its titer in the hemolymph on the day of pupa-
tion was approximately 400 nm in females and 150 nm

When genital disks were cultured for 24 h with or
without 8K-BLP and then pulse-labeled for 2 h with
BrdU, many more cells were labeled in the 8K-BLP-
treated disks, especially at their posterior end (female)
or both ends (male) (Fig. 6E,F). These intensely
labeled regions of the disks develop into the mucous
glands (female) or accessory glands and ejaculatory
duct (male) in adults [18]. The effect of 8K-BLP was
dose-dependent, with a concentration as low as 2 nm
being effective (Fig. 6I). Stimulation of BrdU labeling
by 8K-BLP was also observed in other adult-specific
tissues, such as sperm ducts (Fig. 6G,K) and flight
muscle anlagen (Fig. 6H,L). In contrast, no or little
stimulation of BrdU labeling was detected in larval tis-
sues, including the fat body, midgut and epidermis
(data not shown), all of which remain for a short per-
iod after pupation but are reconstructed or replaced
by adult tissues at later stages [19]. Cell number was
determined for female genital disks. After 5 days of
cultivation, the number of the disk cells was approxi-
mately 30% larger than in the control (Fig. 6M). Con-
sidering the BrdU incorporation in specific areas of the
disks (Fig. 6E), the rate of cell proliferation must be
much higher in those areas. Overall, these results
strongly suggest that 8K-BLP functions as a growth
factor to regulate adult development in B. mori.
Discussion
In the present study, we identified a novel insect pep-
tide 8K-BLP that shows greater similarity to vertebrate
IGFs than to insulin in many respects. First, 8K-BLP

Fig. 5. Induction of 8K-BLP gene expression and secretion by
ecdysteroid. Fat bodies from pharate pupae < 12 h before pupation
were cultured for 1 day or 2 days (B, right) in the presence (+) or
absence ()) of 20E (2 l
M). (A) 8K-BLP gene expression was
assessed by qRT-PCR. Values are the means and standard errors
of the mean (n = 5). Student’s t-test; *P < 0.05. (B) 8K-BLP
released into culture medium was extracted using a Sep-Pak C8
cartridge and analyzed by western blotting with antibody against
8K-BLP.
N. Okamoto et al. Silkmoth IGF-like peptide
FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS 1225
other tissues also secrete this peptide at a lower level
[20–22]. The predominant secretion of 8K-BLP and
IGFs by analogous tissues may imply related physio-
logical functions. Third, the titer of 8K-BLP in hemo-
lymph was remarkably high, with the titers during
early adult development being  800 nm in females
and  200 nm in males (Fig. S2), which are much
more similar to those of IGFs in human adults
A B
E
F
G
H
C
D
I
J
K

mals [4]. Finally, like that of IGFs, the secretion of
8K-BLP is developmentally regulated and independent
of nutrient intake; the 8K-BLP peptide is mainly
secreted during pupa–adult development, when insects
never feed. In contrast, bombyxin secretion is stimu-
lated by hyperglucosemia associated with feeding [28].
As bombyxin regulates tissue growth [29] as well as
carbohydrate metabolism [30] in larvae, this hormone
is thought to serve as a link between nutrition and
growth [29], as insulin does in humans. Thus, bom-
byxin and 8K-BLP appear to have different physiolo-
gical roles. In light of the similarities between 8K-BLP
and IGFs, we propose that this peptide be named
Bommo-IGFLP (BIGFLP), for Bombyx mori IGF-like
peptide.
It is difficult to detect homologous peptides of BIG-
FLP in the genomes of other insects on the basis of
sequence homology, because amino acid sequences of
insect ILPs are highly diverged between insect orders,
except for some critical residues necessary for appro-
priate processing and tertiary structure. However, on
the basis of sequence features, some ILPs are predicted
to be more similar to IGFs than to insulin. For exam-
ple, one of the ILPs in A. aegypti and one of those in
T. castaneum have a truncated C-peptide and a C-ter-
minal extension, features consistent with IGFs [11,12].
One of seven Drosophila ILPs, DILP6, also has a short
C-peptide [10,11]. Furthermore, it may be possible to
find BIGFLP homologs by examining gene expression
patterns. A characteristic feature of the BIGFLP gene

although they are still very immature. The imaginal
disks or rudiments of the adult appendages gradually
grow and develop beneath the epidermis throughout
the larval stages, and rapidly grow and evaginate at
the time of pupal molt to give rise to adult-like struc-
tures [19]. Thus, the BIGFLP surge after pupation can-
not be involved in these processes. However, as a
small amount of BIGFLP, which is detectable by a
fluoroimmunaoassay but not by western blotting, is
already present in the hemolymph shortly before pupa-
tion (Fig. S2), it is possible that this peptide also serves
as a growth factor for the developing appendages.
Therefore, we tested the effect of the peptide using the
wing disks from day 6 fifth instar larvae. BrdU incor-
poration into the disks was strongly promoted by BIG-
FLP (Fig. S3), suggesting that this peptide may also
affect the development of the wing disks at the prepu-
pal stages. It is interesting to note that bombyxin-II
also stimulated the growth of the wing disks in the
butterfly Precis coenia and the hawkmoth Mandu-
ca sexta [29,32]. Both peptides may have potentially
the same activity but function in different developmen-
tal contexts, with bombyxin being used mainly in the
larval stage and BIGFLP during pupa–adult develop-
ment. The above-mentioned study on bombyxin action
on the Manduca wing disk also suggested that bomb-
yxin by itself has little or no effect on disk growth but
enhances the growth-promoting effect of 20E [32]. In
the present study, BIGFLP stimulated BrdU incorpo-
ration into the wing disk in the absence of 20E. How-

described [38]. Pupae initiated adult development 1 day
after ecdysis. Adults emerged 10 days (males) or 11 days
(females) after pupation. The insects within a day after
pupal ecdysis were termed pupae, and those at later stages
were termed developing adults, with day n developing
adults representing the insects n days after pupal ecdysis.
Antibodies and hormones
An antibody against bombyxin-II (M7H2) was previously
produced in our laboratory [39]. A mouse monoclonal anti-
body against 8K-BLP (D7H3) was produced essentially as
described previously [40], using a synthetic peptide (GED-
WSWLSASGRKDGAVTEN) corresponding to the
C-domain of 8K-BLP as an immunogen. Upon immuniza-
tion, this peptide was conjugated to BSA through carbodii-
mide coupling. A mouse monoclonal antibody against
BrdU (G3G4) was obtained from Developmental Studies
Hybridoma Bank (Iowa City, IA, USA). Anti-mouse IgGs
labeled with horseradish peroxidase and Cy3 were
purchased from Jackson ImmunoResearch (West Grove,
PA, USA) and Amersham Biosciences (Little Chalfont,
UK), respectively. Bombyxin-II was chemically synthesized
[41]. 20E was purchased from Sigma (St Louis, MO, USA).
Affinity chromatography and western blotting
M7H2 antibody was bound to a Hi-Trap NHS-activated col-
umn (1 mL, Amersham) according to the manufacturer’s
instruction. Hemolymph and fat body homogenate were pre-
treated before being applied to the column. Hemolymph was
collected as previously described [39], diluted with the same
volume of 50 mm NaCl ⁄ Tris (pH 8.0), and heated at 70 °C
for 5 min. After centrifugation at 1200 g for 15 min, the

with 20 mm Tris ⁄ HCl buffer (pH 7.8) containing 10% ace-
tonitrile and 50 mm NaCl. Anion exchange chromatography
on this column was performed with isocratic elution for
45 min, followed by linear gradient elution (50–520 mm
NaCl) for 25 min at a flow rate of 0.8 mLÆmin
)1
using a
Biologic medium-pressure liquid chromatography system
(Bio-Rad). Fractions of the eluate were assayed for bomb-
yxin-like immunoreactivity by ELISA. The ELISA-positive
fractions containing 8K-BLP were combined, acidified
(pH 3.0) with HCl and trifluoroacetic acid (0.1%), and then
applied to a Hi-Pore RP-304 column (250 · 4.6 mm;
Bio-Rad), on which reversed-phase chromatography was
performed using the Biologic system with a linear gradient
Silkmoth IGF-like peptide N. Okamoto et al.
1228 FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS
of 10–60% acetonitrile in 0.1% trifluoroacetic acid for
100 min at a flow rate of 0.5 mLÆmin
)1
. The purified mate-
rial was quantified using a NanoDrop ND-1000 spectropho-
tometer (NanoDrop Technologies, Wilmington, DE, USA).
ELISA
Aliquots (50 lL) of the 2 mL fractions from ion exchange
HPLC were directly used as samples for ELISA. Aliquots
(50 lL) of the 2 mL fractions derived from reversed-phase
chromatography were lyophilized, dissolved in 50 lL NaCl ⁄
Tris, and used as samples for ELISA. These samples were
pipetted into wells of an ELISA plate (Coster, Cambridge,

dilutions of plasmids containing cDNAs of 8K-BLP and
RpL3 were used for standards, and the transcript levels of
8K-BLP were normalized with RpL3 levels in the same
samples [44].
Whole mount immunohistochemistry
Tissues were immunostained essentially as previously
described [45]. They were incubated sequentially with
D7H3 antibody (2 lgÆmL
)1
) and 1 : 500 diluted Cy3-conju-
gated second antibody, and this was followed by counter-
staining with 4¢,6-diamidino-2-phenylindole (DAPI) (Wako,
Osaka, Japan). The stained tissues were mounted in Vecta-
shield H-1200 (Vector Laboratories, Burlingame, CA,
USA) and observed using a Zeiss LSM510 confocal laser
scanning microscope (Carl Zeiss, Oberkochen, Germany).
The specificity of the signals was established by including
appropriate controls.
In vitro culture of tissues
Insects were dissected under sterile conditions as previously
described [38]. All tissues were dissected from pharate
pupae shortly (< 12 h) before pupal ecdysis, except for
flight muscle anlagens, which were dissected from insects
1 day after pupation, because they were not found at earlier
stages. Genital disks, sperm ducts and flight muscle anlagen
were identified and dissected as previously described [46–
48]. Other tissues were easily identified. Dissected tissues
were rinsed with modified Grace’s medium (prepared by
replacing glucose with 2 mgÆmL
)1

solution was added to the culture (final concentration,
100 lm). Two hours later, the tissues were fixed and immu-
nostained as previously described [49], except that they were
N. Okamoto et al. Silkmoth IGF-like peptide
FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS 1229
denatured with 2 m HCl instead of 0.2 m HCl. Confocal
images of the tissues were obtained as above. The numbers
of BrdU-labeled cells were counted on the acquired images,
and were compared between the corresponding areas of the
control and 8K-BLP-treated tissues.
Cell number counting
Cultured genital disks were washed once in NaCl ⁄ P
i
and
individually incubated with agitation in NaCl ⁄ P
i
containing
0.5% trypsin, 0.2% EDTA and 2 l g ÆmL
)1
DAPI for 4 h at
37 °C. The completely dispersed cells were counted using a
hemocytometer under a Nikon ECLIPSE E800 microscope
with UV illumination.
Acknowledgements
We thank L. I. Gilbert for useful discussion. This
work was supported by Grants-in-aid for Scientific
Research (20570056) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
N. Yamanaka was a recipient of a research fellowship
from the Japan Society for the Promotion of Science

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Fig. S1. Separation of 8K-BLP and bombyxin by ion
exchange HPLC.
Fig. S2. Developmental changes in hemolymph levels
of 8K-BLP determined by time-resolved fluoroimmu-
noassay.
Fig. S3. 8K-BLP-stimulated BrdU incorporation by
wing disks.
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