Tài liệu Báo cáo khoa học: An ecdysteroid-inducible insulin-like growth factor-like peptide regulates adult development of the silkmoth Bombyx mori docx

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

E-mail:
Present address
*Department of Genetics, Cell Biology
& Development, University of Minnesota,
Mineapolis, MN, USA
Department of Pharmacology and Neurobi-
ology, Graduate School of Medicine, Tokyo
Medical and Dental University, Japan
Database
The sequence reported in this article has
been deposited in the GenBank database
( />under accession numbers AB360450–
AB360454
(Received 10 October 2008, revised 11
December 2008, accepted 16 December
2008)
doi:10.1111/j.1742-4658.2008.06859.x
Insulin-like growth factors (IGFs) play essential roles in fetal and postnatal
growth and development of mammals. They are secreted by a wide variety
of tissues, with the liver being the major source of circulating IGFs, and
regulate cell growth, differentiation and survival. IGFs share some biologi-
cal activities with insulin but are secreted in distinct physiological and
developmental contexts, having specific functions. Although recent analyses
of invertebrate genomes have revealed the presence of multiple insulin fam-
ily peptide genes in each genome, little is known about functional diversifi-
cation of the gene products. Here we show that a novel insulin family
peptide of the silkmoth Bombyx mori, which was purified and sequenced
from the hemolymph, is more like IGFs than like insulin, in contrast to
bombyxins, which are previously identified insulin-like peptides in B. mori.
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
developmentally regulated manner [4].

ILPs are also found in insects [5,6]. Among these,
bombyxins, a family of peptides produced by the brain
of the silkmoth Bombyx mori, were the first to be dem-
onstrated to have structural homology to vertebrate
insulins [7,8]. Bombyxin-II, one of the purified bomb-
yxins, is a heterodimeric molecule in which two peptide
chains are held together by disulfide bonds in exactly
the same manner as in insulin [8]. Subsequently, many
putative ILPs from a number of nonvertebrate animals
have been deduced by cDNA and gene cloning [5,6].
The genomes of insects contain multiple genes for
ILPs; there are more than 30 in B. mori [9], seven in
the fruit fly Drosophila melanogaster [10], eight in the
yellow fever mosquito Aedes aegypti [11], and four in
the red flour beetle Tribolium castaneum [12]. Mole-
cular genetic studies in Drosophila have shown that
Drosophila ILPs (DILPs) regulate diverse functions,
including growth, metabolism, fecundity, and lifespan
[10,13–17]. Expression analysis of DILP genes revealed
that each gene is differentially expressed in a tissue-
specific and stage-specific manner during development
[10], suggesting that these peptides might have distinct
functions; however, little is known about the func-
tional diversification of invertebrate ILPs within a
given organism.
Our recent studies on Bombyx hemolymph using a
mouse monoclonal antibody against bombyxin-II
(M7H2) have revealed the presence of a novel 8 kDa
immunoreactive substance in addition to 6.5 kDa
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-
jected to RP-HPLC, which concentrated the ELISA-

positive material into a peak so sharp that we judged
that sufficient purification had been achieved.
Determination of the structure of 8K-BLP
MALDI-TOF MS analysis of the purified material
from the final HPLC yielded a monoisotopic mass of
7409.2 ([M + H]
+
) (Fig. 3A). Amino acid sequence
analysis of the purified peptide determined the
Fig. 1. Fluctuation of 8K-BLP in Bombyx hemolymph during pupa–
adult development. Hemolymph taken at each stage was subjected
to M7H2 antibody affinity chromatography, resolved by Tri-
cine ⁄ SDS ⁄ PAGE, and analyzed by western blotting with M7H2 anti-
body. Fifty microliter (females) or 100 lL (males) of hemolymph
equivalents of the sample were loaded in each lane. Bbx, synthetic
bombyxin-II (15 ng). Bombyxin, present at a far lower level than
8K-BLP in these samples, formed no visible band. Pn, n days after
pupal ecdysis, taking the day of pupation as P0; A0, the day of
adult eclosion.
Silkmoth IGF-like peptide N. Okamoto et al.
1222 FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS
37 N-terminal amino acids, with five unidentified inter-
vening residues (Fig. 3B). Using this sequence, a blast
search of the Bombyx expressed sequence tag database
on kaikoblast ( />revealed five cDNA clones that encoded an identical
peptide sequence composed of a putative 20 amino
acid signal peptide and a 68 amino acid peptide
belonging to the insulin family (Fig. 3C). If the signal
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
eclosion (Fig. 4B). The 8K-BLP peptide became detect-
able in the fat body after pupal ecdysis (Fig. 4C), in

agreement with the fluctuation pattern of 8K-BLP
levels in hemolymph (Fig. 1). Production of 8K-BLP by
the fat body was also confirmed by immunohistochem-
istry using a novel monoclonal antibody D7H3, specific
for the C-domain of 8K-BLP (Fig. 4D). Although the
staining intensity of the cells increased during adult
development, the number of fat body cells decreased
dramatically from mid-adult development onwards, due
to their disruption. These results indicate that the fat
body is the main source of the hemolymph 8K-BLP.
Induction of 8K-BLP gene expression and
secretion by ecdysteroid
The onset of 8K-BLP production shortly before or
after pupal ecdysis suggested the involvement of
A
B
C
Fig. 2. Purification of 8K-BLP. (A) Enrichment of 8K-BLP by M7H2
antibody affinity chromatography. Day 5 female developing adult
hemolymph was heat-treated and subjected to affinity chromatogra-
phy. Aliquots of the input and the eluate of the chromatography
were resolved by Tricine ⁄ SDS ⁄ PAGE and then analyzed by Coo-
massie Brilliant Blue staining (left) and western blotting (right).
Lane 1: input (2 lL of hemolymph equivalents). Lane 2: eluate
(200 lL of hemolymph equivalents). Lane 3: input (0.5 lL of hemo-
lymph equivalents). Lane 4: eluate (50 lL of hemolymph equiva-
lents). Arrows indicate 8K-BLP. (B, C) The last two steps of
purification, ion exchange HPLC (B) and RP-HPLC (C), are shown.
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
in males (Fig. S2) and because preliminary experiments
showed that the effect of 8K-BLP on bromodeo-

xyuridine (BrdU) incorporation into genital disks was
similar between concentrations of 20 and 200 nm.
Genital disks of either sex dissected from pharate
pupae shortly (< 12 h) before pupation and cultured
in the presence of 8K-BLP for 5 days became larger
than controls (Fig. 6A,B). The protein content in the
8K-BLP-treated disks increased significantly, and was
A
E
B
C
D
Fig. 3. Determination of the structure of 8K-BLP. (A) MALDI-TOF MS analysis of the purified peptide. (B) The N-terminal sequence of
8K-BLP as determined by sequence analysis. X, unidentified residues. (C) The sequence of a putative 8K-BLP precursor polypeptide revealed
by an expressed sequence tag database search. The highlighted sequence represents the signal peptide. The underlined portion is identical
to the sequence in (B). (D) Sequence alignment of 8K-BLP with homologous peptides. Highlighted amino acid residues are completely con-
served among these peptides. Inverted filled triangles indicate cysteine residues. Dibasic, potential cleavage sites are boxed. (E) A phyloge-
netic tree showing the relationship between 8K-BLP (boxed) and bombyxin subfamilies. The phylogenetic tree was generated on the basis
of the entire amino acid sequences of 8K-BLP and probombyxins by using the
CLUSTALW program ( />The numbers on the branches denote bootstrap values per 1000 replications. Only the values > 500 (50%) are shown. The scale bar
indicates an evolutionary distance of 0.1 amino acid substitutions per position.
Silkmoth IGF-like peptide N. Okamoto et al.
1224 FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS
30% (female) or 63% (male) higher than in control
disks at the end of the culture (Fig. 6C,D). The effect
of 8K-BLP on cell proliferation was also investigated
by measuring BrdU incorporation and cell number.
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
is secreted as a single-chain peptide, instead of as a
heterodimer. Second, 8K-BLP was predominantly pro-
duced by the fat body, a functional equivalent of the

liver and adipose tissue of vertebrates, and the liver is
the major source of circulating IGF, although most
AC
D
B
Fig. 4. Production of 8K-BLP by fat body.
(A) Relative levels of 8K-BLP gene expres-
sion in various tissues taken from day 6 and
day 9 fifth instar larvae (V6, V9) and day 2
developing adults (P2), as assessed by qRT-
PCR. FB, fat body; OV, ovary; TE, testis;
BR, brain; MG, midgut; MT, Malpighian
tubule; MU, muscle; WD, wing disk; AG,
abdominal ganglia. (B) Developmental
changes in 8K-BLP gene expression level in
the female fat body examined by qRT-PCR.
Vn, n days after final larval ecdysis; Pn,
n days after pupal ecdysis. (C) Changes in
8K-BLP abundance in the fat body (0.25 g)
around the time of pupation, analyzed by
western blotting using M7H2 antibody. (D)
Immunohistochemical detection of 8K-BLP
in the P5 female fat body with an antibody
against 8K-BLP (red). Nuclei were stained
with DAPI (blue). Scale bar: 30 lm.
B
A
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
L
M
Fig. 6. Growth-promoting effects of 8K-BLP on imaginal anlagen. (A, B) Female (A) and male (B) genital disks from pharete pupae were cul-

tured in the absence (control) or presence of 8K-BLP (20 n
M) for 5 days. Scale bars: 200 lm. (C, D) Protein contents in the female (C) and
male (D) disks before and after cultivation for 5 days. Values are the means and standard errors of the mean (n = 6). (E–H) Confocal images
of tissues with BrdU immunoreactivity (red). Female (E) and male (F) genital disks and sperm ducts (G) from pharate pupae < 12 h before
pupation and flight muscle anlagen (H) from day 1 developing adults were cultured in the absence (control) or presence of 8K-BLP (20 n
M)
for 24 h, and this was followed by BrdU labeling for 2 h. Scale bars: 100 lm. (I) Dose-dependent effects of 8K-BLP on BrdU incorporation
into the posterior end of female genital disks. (J–L) Graphic representations of the effects of 8K-BLP on BrdU incorporation into male genital
disks (J), sperm ducts (K) and flight muscles (L). Values are the means and standard errors of the mean (n = 4–6). Student’s t-test;
*P < 0.05, **P < 0.01 against control. (M) Cell numbers in the female genital disks before and after cultivation for 5 days. Values are the
means and standard errors of the mean (n = 6).
Silkmoth IGF-like peptide N. Okamoto et al.
1226 FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS
(20–80 nm) [4] than to those of bombyxin and insulin,
which are of the order of 100 pm [23,24]. The very
high titer of 8K-BLP may be due to a remarkably high
level of gene expression in the fat body and to the
large volume of this tissue. Fourth, 8K-BLP showed a
growth-promoting effect on some adult-specific tissues.
IGFs are essential growth factors for normal growth
of mammals [22], playing especially critical roles in
fetal and pubertal development [25–27], when tissues
grow rapidly. Because, in Bombyx, the adult structures
develop rapidly in a short period after pupation, the
surge of 8K-BLP release in this period is reminiscent
of the massive release of IGF-I and IGF-II during
pubertal and fetal development, respectively, in mam-
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
is its very high level of expression in the fat body dur-
ing pupa–adult development. Therefore, insulin family
peptides in other insects showing such an expression

pattern are good candidates for being the functional
equivalent of BIGFLP. Although bombyxin genes and
the majority of known insect ILP genes are mainly
expressed in medial neurosecretory cells of the brain
[9,10,13,14], some ILP genes are expressed outside the
brain [10,11]. Among them is the DILP6 gene. Inter-
estingly, our preliminary experiments showed that dilp6
was predominantly expressed in the fat body during
pupa–adult development at remarkably high levels as
compared with other DILP genes (N. Okamoto,
N. Yamanaka, H. Kataoka & A. Mizoguchi, unpub-
lished results).
It is worth noting that BIGFLP is secreted mainly
after pupal ecdysis, and the secretion is stimulated by
the ecdysteroid 20E. Ecdysteroids are well known to
be essential for adult development of insects; without
them, pupae cease development and undergo diapause
[31]. However, little is known about the mechanisms
by which ecdysteroids regulate adult development. The
induction of BIGFLP secretion by 20E, together with
the observed growth-promoting effects of BIGFLP on
some adult-specific tissues, may suggest that the roles
of ecdysteroids in adult development are in part medi-
ated by BIGFLP. In B. mori, as in many other holo-
metabolous insects, the basal external structure of
adult appendages such as wings, legs and antennae
have already been built at the time of pupation,
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-
ever, the observed synergistic effect of bombyxin
and 20E in other insects suggests the possibility that
N. Okamoto et al. Silkmoth IGF-like peptide

FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS 1227
BIGFLP also exert a greater growth-promoting effect
in the presence of 20E. Further studies are required to
determine the functional relationship between BIGFLP
and ecdysteroid.
Although we have clearly demonstrated the growth-
promoting effects of BIGFLP using an in vitro tissue
culture system, in vivo studies are also important to
establish the roles of BIGFLP in Bombyx develop-
ment. However, such studies seem to be difficult in
B. mori, for the following reasons: (a) it is impossible
to remove the BIGFLP-producing cells, because the
fat body is a large, diffuse tissue; (b) inhibition of
BIGFLP activity by antibodies is difficult, because of
its very high titer in hemolymph; and (c) in B. mori,as
in other members of the Lepidoptera, genetic
approaches using gene knockout or knockdown tech-
nology have not yet been established [33,34], although
a few successful cases have been reported [35–37].
Comparative studies using other insects in which
genetic approaches are applicable may advance our
understanding of how IGF-like peptides regulate adult
development in insects.
Experimental procedures
Animals
A racial hybrid of B. mori, Kinshu · Showa (Ueda Sanshu,
Ueda, Japan), was used. Larvae were reared as previously
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
supernatant was filtered through a 0.2-lm filter. Fat body
was homogenized in NaCl ⁄ Tris with a glass–glass homo-
genizer, and the homogenate was processed in the same way.

The pretreated samples were applied to the column equili-
brated with NaCl ⁄ Tris, and the adsorbed materials were
eluted with 100 mm glycine–HCl buffer (pH 2.8). For desalt-
ing, the eluate was mixed with 1 ⁄ 1000 volume of trifluoro-
acetic acid and applied to a Sep-Pak Vac C8 cartridge (3 mL,
200 mg; Waters, Boston, MA, USA). After washing of the
cartridge, the adsorbed materials were eluted with 40% ace-
tonitrile in 0.1% trifluoroacetic acid and then lyophilized,
unless otherwise stated. Western blotting was performed as
previously described [39], using samples thus prepared. The
immunoreactive band was detected using an ECL system
(Amersham) and a Polaroid camera (Amersham).
Purification of 8K-BLP
8K-BLP was purified from hemolymph of female developing
adults through three steps of purification: M7H2 antibody
affinity chromatography, ion exchange HPLC, and RP-
HPLC. Pooled hemolymph (20 mL) from day 5 female
developing adults was heat-treated and filtered as above.
The filtrate was divided into 10 parts, each of which was
subjected to affinity chromatography, followed by desalting
using the Sep-Pack cartridge. Each eluate from the cartridge
was condensed by vacuum centrifugation for 30 min, and
the condensates in 10 tubes were combined. To this solu-
tion, Trizma base was added to adjust the pH to 8.0, and
NaCl to give a final concentration of 50 mm. Thus prepared
solution was applied to a Bio-Scale Q2 column (52 · 7 mm;
Bio-Rad Laboratories, Hercules, CA, USA) equilibrated
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,
MA, USA) and incubated overnight at 4 °C. After blocking
with 5% skimmed milk in NaCl ⁄ Tris for 2 h, the wells were
sequentially incubated with M7H2 antibody (1 lgÆmL

)1
) and
1 : 3000 diluted horseradish peroxidase-labeled second anti-
body for 2 and 1 h, respectively, and this was followed by
development with o-phenylenediamine. The wells showing an
A
492 nm
value of 0.2 or higher were regarded as positive.
MALDI-TOF MS analysis
Mass spectra were obtained with a Voyager-DE STR mass
spectrometer (Applied Biosystems, Foster City, CA, USA).
A saturated solution of a-cyano-4-hydroxycinnamic acid in
methanol ⁄ water (1 : 1 by volume) was used as the matrix
solution. All spectra were measured in the reflector mode.
Amino acid sequence analysis
Sequence analysis was performed using an Applied Biosys-
tems (ABI) Procise protein sequencer Model 492 equipped
with a 140C Microgradient System (ABI) and a Series 200
UV–visible detector (Perkin-Elmer, Wellesley, MA, USA).
qRT-PCR
Total RNAs were prepared from various tissues and con-
verted to cDNA as previously described [42]. qRT-PCR was
performed on a Smart Cycler System (Cepheid, Sunnyvale,
CA, USA) as previously described [43]. The 8K-BLP-specific
primers used are as follows: sense primer, 5¢-TTGTGATC
CTCCTCGTTCTACTGACGG-3¢; and antisense primer,
5¢-AGTAGGAAAGCAGAACCTCTAGGGTGC-3¢. Serial
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
trehalose and supple-
menting with 1% BSA and 100 unitsÆmL
)1

penicillin and
100 lgÆmL
)1
streptomycin) and then transferred to the
wells of a 24-well culture plate containing 450 lL of the
same medium. The tissues were precultured overnight
before exposure to 8K-BLP or 20E. 8K-BLP and 20E were
added to the culture to give final concentrations of 20 nm
and 2 lm, respectively, by distributing 50 lL of their stock
solution, except for the dose–response experiment, where
the stock solution was diluted before addition to the cul-
tures. Control cultures received 50 lL of the modified
Grace’s medium. All the cultures were maintained at
25 ± 0.5 °C under 40% oxygen partial pressure. The cul-
ture medium was not renewed throughout experiments.
Images of the cultured genital disks were obtained using a
Leica DFC480 digital camera on a Leica MZ16FA micro-
scope (Leica, Heerbrugg, Switzerland).
Protein assay
The protein content of the genital disk was determined using
a protein assay kit (Bio-Rad). The disks were individually
homogenized in NaCl ⁄ Tris with a glass–glass homogenizer,
frozen and thawed three times, and centrifuged at 1200 g for
15 min to remove precipitates. The resultant supernatants
were used for protein assay, with BSA as the standard.
BrdU labeling
After 24 h of tissue cultivation with 8K-BLP, BrdU stock
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
(JSPS).
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Supporting information
The following supplementary material is available:
Doc. S1. Supplementary method and reference.
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.
This supplementary material can be found in the
online version of this article.
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sponding author for the article.
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1232 FEBS Journal 276 (2009) 1221–1232 ª 2009 The Authors Journal compilation ª 2009 FEBS