peptide regulates adult development of the silkmothBombyx mori Naoki Okamoto1, Naoki Yamanaka2,*, Honoo Satake3, Hironao Saegusa1,, Hiroshi Kataoka2and Akira Mizoguchi1 1 Division of Bio
Trang 1peptide regulates adult development of the silkmoth
Bombyx mori
Naoki Okamoto1, Naoki Yamanaka2,*, Honoo Satake3, Hironao Saegusa1,, Hiroshi Kataoka2and Akira Mizoguchi1
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: mizoguch@bio.nagoya-u.jp
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
(http://www.ncbi.nlm.nih.gov/Genbank)
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 physiologibiologi-cal 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.
Trang 2Members 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-tissue-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
Bombyxhemolymph 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.
Trang 337 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 (http://kaikoblast.dna.affrc.go.jp/) 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 hemohemo-lymph
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.
Trang 4ecdysteroid 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
Bombyxpupae 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
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 phylogephyloge-netic tree was generated on the basis
of the entire amino acid sequences of 8K-BLP and probombyxins by using the CLUSTALW program (http://clustalw.ddbj.nig.ac.jp/top-j.html) 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.
Trang 530% (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
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.
Trang 6other 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
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).
Trang 7(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
Trang 8BIGFLP 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
Trang 9of 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 4C 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
A492 nmvalue 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
RpL3were 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 ± 0.5C 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
Trang 10denatured 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⁄ Pi and
individually incubated with agitation in NaCl⁄ Picontaining
0.5% trypsin, 0.2% EDTA and 2 lgÆmL)1DAPI for 4 h at
37C 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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