Precocious Metamorphosis in the Juvenile Hormone–
Deficient Mutant of the Silkworm, Bombyx mori
Takaaki Daimon1,2, Toshinori Kozaki1, Ryusuke Niwa3, Isao Kobayashi1, Kenjiro Furuta1¤, Toshiki
Namiki1, Keiro Uchino1, Yutaka Banno4, Susumu Katsuma2, Toshiki Tamura1, Kazuei Mita1, Hideki
Sezutsu1, Masayoshi Nakayama5, Kyo Itoyama6, Toru Shimada2, Tetsuro Shinoda1*
1National Institute of Agrobiological Sciences, Tsukuba, Japan, 2Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Tokyo, Japan, 3Initiative for the Promotion of Young Scientists’ Independent Research, Graduate School of Life and Environmental
Sciences, University of Tsukuba, Tsukuba, Japan, 4Institute of Genetic Resources, Faculty of Agriculture, Kyushu University Graduate School, Fukuoka, Japan, 5Institute of
Floricultural Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan, 6School of Agriculture, Meiji University, Kawasaki, Japan
Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs).
JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper
size for metamorphosis. In the silkworm, Bombyx mori, several ‘‘moltinism’’ mutations have been identified that exhibit
variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the
identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious
metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval
hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional
cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated
that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs.
Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific
manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in
transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the
mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not
occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori.
Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of
the hormonal control of development and metamorphosis.
Citation: Daimon T, Kozaki T, Niwa R, Kobayashi I, Furuta K, et al. (2012) Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm,
Bombyx mori. PLoS Genet 8(3): e1002486. doi:10.1371/journal.pgen.1002486
Editor: David L. Stern, Janelia Farm Research Campus, Howard Hughes Medical Institute, United States of America
Received October 10, 2011; Accepted December 1, 2011; Published March 8, 2012
Copyright: ? 2012 Daimon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Scientific Research (Nos. 20688003, 22128004, and 23688008) and by Special Coordination Funds for
Promoting Science and Technology from MEXT, the Program for Promotion of Basic Research Activities for Innovative Biosciences (PRO-BRAIN), MAFF-NIAS
(Agrigenome Research Program), JST (Professional Program for Agricultural Bioinformatics), and National Bioresource Project, Japan. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan
The number of larval instars in insects varies greatly across
insect taxa, and can even vary at the intraspecific level [1,2,3]. In
general, phylogenetically higher insects tend to have fewer larval
instars (three to eight) compared to species from basal lineages,
such as Ephemeroptera, Odonata and Plecoptera (more than ten)
[1,2,3]. In many species, the number of larval instars is affected by
genetic and environmental factors, such as temperature, nutri-
tional conditions, photoperiod, humidity, injuries, and sex [1,2].
The variation in the number of larval instars in the insect lifecycle
is generally considered to be an adaptive response to diverse
environmental conditions in order to ensure the attainment of a
threshold-size for metamorphosis [1,2,3,4].
The silkworm Bombyx mori, a classic model organism for
endocrinology, has been reared by humans for thousands of
years, and more than 1,000 strains are currently maintained
[5,6,7]. Among these, several ‘‘moltinism’’ strains have been
identified that exhibit variations in the number of larval instars
[6,7]. Silkworms typically have five larval instars, but the
moltinism strains vary between three and seven [6,7]. For
example, precocious larval-pupal metamorphosis is observed in
the mod (dimolting, chromosome 11–27.4 cM), rt (recessive trimolting,
7–9.0) and M3(Moltinism, 6–24.1) strains, while extra larval
molting is observed in the M5(Moltinism, 6–24.1) strain [6,7]. To
date, however, none of these loci has been characterized at the
molecular level. Given the availability of whole genome data and
post-genomic tools in B. mori [8,9,10], these strains offer a valuable
resource for elucidating the molecular mechanism that underlies
plasticity in the number of larval instars.
Here we report the identification and characterization of the
gene responsible for the mod mutation that causes precocious
PLoS Genetics | www.plosgenetics.org 1March 2012 | Volume 8 | Issue 3 | e1002486
larval-pupal metamorphosis in the third or fourth instar . Most
mod larvae form larval-pupal intermediates, but some individuals
can become miniature moths with normal fertility. Thus, the mod
mutant strain can be maintained as homozygous stocks [6,11,12].
We demonstrate that the mod locus encodes CYP15C1, a
cytochrome P450 involved in the biosynthesis of juvenile
hormones (JHs), whose ‘‘status quo’’ action allows the progression
of multiple larval-larval molting until the larva attains the required
size for metamorphosis [13,14,15]. CYP15C1 is specifically
expressed in the corpus allatum (CA), an endocrine organ that
produces and secretes JHs. Enzymological analysis revealed that
CYP15C1 converts farnesoic acid (FA) to JH acid (JHA) in a
highly stereospecific manner. We further demonstrated that
CYP15C1 plays an indispensable role in JH biosynthesis, and its
molecular defect results in the loss of JHs in the hemolymph,
thereby causing precocious metamorphosis in the mod strain.
Remarkably, precocious larval-pupal transitions in mod larvae
always occur after the larval third instar, but not in the first or
second instar. Our data provide further evidence supporting the
hypothesis that authentic (epoxidized) JHs are essential for the
classic ‘‘status quo’’ molting in late larval stages (third and fourth
instar), but not in early larval stages (first and second instar) of B.
The mod strain is a JH–deficient mutant
Larvae of standard B. mori strains undergo molting four times
and thus have five larval instars; these larvae are conventionally
termed ‘‘tetramolter’’ in silkworm genetics. The spontaneous
mutant mod was identified in a standard strain  and mod larvae
undergo precocious metamorphosis in the third (dimolter) or
fourth instar (trimolter). First, we obtained a detailed develop-
mental profile of larvae from two batches of the mod strain. All mod
larvae underwent precocious metamorphosis in the fourth instar
and no individuals reached the fifth instar (Figure 1A and 1B). We
plotted the timing of the onset of spinning in the mod larvae
(Figure 1C and 1D). Consistent with previous reports [11,12], we
found that spinning occurred at two clearly distinguishable
timings: (1) from 56 to 80 h and (2) from 112 to 144 h after the
third molt: these larvae were termed early- and late-maturing
trimolters, respectively. This segregation in the timing of the onset
of spinning was not observed in the standard strain p50T
(Figure 1C) or other moltinism strains , and thus is a unique
characteristic of the mod strain. Importantly, development in
almost all early-maturing trimolters was arrested and they
remained as larval-pupal intermediates (93.4%, 85/91 larvae);
only 3 of the 91 larvae (3.3%) successfully survived to adulthood
(Figure 1B). In contrast, the late-maturing trimolters did not show
such severe developmental impairment and 88.5% (77/87)
became miniature adults (Figure 1B). In the larval-pupal
intermediates, we usually observed prothetelic phenotypes such
as a mixed pupal cuticle on the exoskeleton of animals having
overall a larval appearance (Figure 1A), suggesting that hormonal
switching of molting and metamorphosis may be aberrant in the
mod strain. Notably, despite their small body size, reproduction in
mod moths seemed normal, and their eggs hatched without
In the silkworm, premature metamorphosis can be induced by
the loss of or low levels of JH signaling, which can occur due to the
surgical removal of the CA  or to overexpression of the JH-
degrading enzyme . We therefore hypothesized that preco-
cious metamorphosis in the mod strain was caused by the
prevention of JH biosynthesis or signaling. To examine this
hypothesis, we first determined whether the mod phenotype could
be rescued by treatment with methoprene, a JH analogue. We
topically applied several doses of methoprene to newly-molted
third or fourth instar mod larvae and found that a fourth larval
molting was induced by the treatment (Figure 1E). Fifth instar
larvae that had undergone fourth larval ecdysis grew normally,
began to spin after ,6 days, and eventually metamorphosed to
pupae and adults that were normal and fertile. This result suggests
that JH reception and subsequent JH signaling is normal in the
mod strain. Therefore, we next compared the JH titers in the
hemolymph of third instar larvae of mod and p50T strains at 24 h
after molting to the third instar. JHs were extracted from the
hemolymph and their methoxyhydrin derivatives were analyzed
by liquid chromatography-mass spectrometry (LC-MS). We
detected JH I and JH II in the hemolymph of p50T, whereas
the JH titer in the hemolymph of the mod strain was below the
detectable level (Figure 1F). These results indicate that the mod
strain is a JH-deficient mutant in which complete (or almost
complete) loss of JH caused precocious metamorphosis.
Positional cloning of the mod locus
To identify the gene responsible for the mod locus, we performed
positional cloning using backcross 1 progeny (BC1) obtained from
crossing females of the mod strain (t011 strain, see http://www.
shigen.nig.ac.jp/silkwormbase/index.jsp) with F1 heterozygote
males of mod and p50T strains (see Figure S1). We mapped the
mod locus within ,400 kb region on the scaffold Bm_scaf16
(chromosome 11)  using 792 BC1individuals. Twenty-five genes
were predicted to be present within this region. Among them, we
focused on BGIBMGA011708, a gene encodes a cytochrome P450
monooxygenase. Based on sequence homology and phylogenetic
analysis (Figure 2B), the gene was designated as CYP15C1. We
found that CYP15C1 shares high homology with the CYP15A1 of
the cockroach Diploptera punctata, which is involved in JH
biosynthesis in CA of the cockroach . Given that the mod
phenotype is a result of the loss of the JH titer (Figure 1F), we
speculated that the mod phenotype is due to the loss of function of
CYP15C1. To examine this possibility, we first determined the
The number of larval instars in insects varies greatly across
insect taxa and can even vary at the intraspecific level.
However, little is known about how the number of larval
instars is fixed in each species or modified by the
environment. The silkworm, Bombyx mori, provides a
unique bioresource for investigating this question, as
there are several ‘‘moltinism’’ strains that exhibit variations
in the number of larval molts. The present study describes
the first positional cloning of a moltinism gene. We
performed genetic and biochemical analyses on the
dimolting (mod) mutant, which shows precocious meta-
morphosis with fewer larval–larval molts. We found that
mod is a juvenile hormone (JH)–deficient mutant that is
unable to synthesize JH, a hormone that prevents
precocious metamorphosis and allows the larvae to
undergo multiple rounds of larval–larval molts. This JH–
deficient mutation is the first described to date in any
insect species and, therefore, the mod strain will serve as a
useful model for elucidating the molecular mechanism of
JH action. Remarkably, precocious larval–pupal transition
in mod larvae does not occur in the first or second instar,
suggesting that morphostatic action of JH is not necessary
for young larvae of B. mori.
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Figure 1. Characterization of the mod mutant. (A) Precocious metamorphosis observed in mod larvae. (left panel) Lateral and dorsal views and
(middle panel) a magnified view of a larval-pupal intermediate. In intermediate animals, the new head capsule of the next instar (fifth) is formed
(arrowhead). Beneath the old cuticles (asterisk), a new exoskeleton with larval eye spot markings (arrows) and brown-colored pupal cuticles are
formed. (Right panel) Late-maturing trimolters form small cocoons and are able to develop into small but normal adults with normal fertility. (B) The
developmental profiles of two batches of mod larvae (t011 strain). All of the larvae underwent precocious metamorphosis in the fourth instar, and no
dimolters or tetramolters were observed. Larvae could be classified into two groups (early- and late-maturing trimolters) on the basis of the timing of
onset of spinning. The numbers in parentheses indicate the sex of the moths (male/female). (C) Timing of the onset of spinning in mod (red, n=178)
and p50T (black, n=28) strains after final larval molting. As highlighted by the grey ellipses, spinning was induced at two distinct timings in the mod
strain, unlike the p50T strain. (D) Comparison of timings of the onset of spinning among early- and late-maturing trimolters of the mod strain and
normal strain larvae that had been allatectomized (CAX) at the beginning of the fourth instar. Data on CAX larvae are from ; these larvae were
reared at relatively low temperatures (23.0–25.5uC), which delays the timing of the onset of spinning to some extent. (E) Methoprene treatment of
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nucleotide sequence of the full-length CYP15C1 cDNA from p50T
and mod strains. We identified a 68-bp deletion in the mod allele
that introduces a premature stop codon in the coding region of
CYP15C1 (Figure 2C–2E). This deletion seemed to produce a
functionally null mutation in CYP15C1, since a heme-binding
motif, which is essential for enzymatic activities in P450s , was
eliminated in the mod allele (Figure 2D). This result indicates that
CYP15C1 is a strong candidate for the mod locus. Therefore, we
further characterized CYP15C1 and its gene product.
Temporal and spatial expression of CYP15C1
The strict regulation of JH biosynthesis in CA is critical for the
successful development and reproduction of insects [14,15,20]. We
next examined the spatial expression pattern of CYP15C1 mRNA.
We examined 12 tissues at four different developmental stages and
found that CYP15C1 mRNA was highly specific to the corpus
cardiacum (CC)-CA complex (Figure 3A). A whole mount in situ
hybridization experiment in the brain (Br)-CC-CA complex
(Figure 3B and Figure S2) showed that the signal for CYP15C1
was strictly limited to CA, where JH is synthesized, and could not
be detected in the brain or CC. These results showed a close
spatial correlation between CYP15C1 expression and JH biosyn-
Next, we carried out a detailed analysis of the temporal
expression pattern of CYP15C1 in the CC-CA complex and
compared it to that of the gene for JHA methyltransferase
(JHAMT), a key enzyme that acts in the final step of the JH
biosynthetic pathway in CA . CYP15C1 mRNA was
constitutively expressed in CA from the first instar larval to adult
stages (Figure 3D), even when JH is not synthesized (Figure 3C)
; no apparent differences in levels of CYP15C1 mRNA were
observed between males and females during pupal and adult stages
(Figure 3D). In contrast, the temporal expression pattern of
JHAMT correlates well with the JH synthetic activity of CA
(Figure 3D and Figure S2). JHAMT transcript completely
disappeared by day 4 of the fifth instar when CA ceased
production of JH (see Figure 3C). It reappeared from the mid-
pupal stage and increased to a very high level in the female CA.
This was consistent with the temporal profile of JH biosynthesis
activity in CA as this occurs only in females during the pupal and
adult stages . Taken together, our results strongly indicate that
CYP15C1 is involved in JH biosynthesis in CA, but does not
appear to act as a rate-limiting factor for JH biosynthesis.
Enzymatic properties of CYP15C1
The cockroach CYP15A1, the ortholog of B. mori CYP15C1,
catalyzes the epoxidation of (2E,6E)-methyl farnesoate (MF) to JH
III . Although biochemical studies predicted the presence of
FA epoxidase in the CA of the lepidopteran insect Manduca sexta
[22,23], the corresponding gene has not been identified to date.
Therefore we examined the enzymatic activity of B. mori
CYP15C1 against two plausible substrates, FA and MF. First,
we employed a transient expression system using Drosophila S2
cells. When S2 cells expressing CYP15C1 were incubated with
medium containing FA, a major HPLC peak was generated that
had the same retention time (15.1 min) as standard JH III acid
(JHA III) (Figure 4A, middle). This peak did not appear when S2
cells expressing GFP were used (Figure 4A, bottom). The ESI-MS
spectrum of this peak gave an [M-H]2at m/z 251, consistent with
the C15H24O3formula of JHA III, confirming that CYP15C1
catalyzes the conversion of FA to JHA III. The enzymatic
properties of CYP15C1 was further examined in a stable Sf9 cell
line (Sf9/BmCYP15C1) that constitutively expresses CYP15C1.
When the Sf9/BmCYP15C1 cells were cultured in medium
containing FA, significant levels of JHA III were detected; in
contrast, JHA III production was difficult to detect when original
Sf9 cells were used (Table S2, Exp.1). When Sf9/BmCYP15C1
cells were cultured in medium containing MF, JH III generation
was detected at low levels. However, a similar level of JH III
production was also detected in the original Sf9 cells when they
were cultured in the same medium (Table S2, Exp.1). These
results suggest that JH III production observed in Sf9/
BmCYP15C1 was might be due to the presence of endogenous
P450 epoxidases in Sf9 cells, which have been reported previously
to have lower substrate specificity and stereospecificity [18,24].
The addition of the JH esterase inhibitor 3-octylthio-1,1,1-
trifluropropan-2-one (OTFP) did not increase production of JHA
III (Table S2, Exp. 2), indicating that the degradation of JH III by
intrinsic JH esterases in the cells was negligible. Therefore, we
were able to estimate the conversion ratio of FA and MF to JH III
by CYP15C1. This showed that CYP15C1 exhibited at least 18-
fold higher activity for FA than MF (Table S2, Exp. 1), a result
that is consistent with previous biochemical studies on lepidopter-
an FA epoxidase in CA.
To further examine the stereospecificity of CYP15C1, the JHA
III generated by Sf9/CYP15C1 was chemically methylated and
analyzed by a Chiral-HPLC. The methylated product had a major
(R)-JH III and a minor (S)-JH III peak (R:S=97:3) (Figure 4B).
These results show that B. mori CYP15C1 encodes a functional
P450 epoxidase that preferentially converts FA to JHA III rather
than MF to JH III, and does so in a highly (R)-enantioselective
manner (Figure 4C).
Transgenic rescueexperiments using theGAL4/UASsystem
To obtain direct evidence that CYP15C1 is responsible for the
mod mutation, we performed transgenic rescue experiments using
the GAL4/UAS system . We generated transgenic silkworm
lines carrying the UAS-CYP15C1 transgene with the eye-specific
3xP3-EGFP marker . The UAS-CYP15C1 transgene was driven
using a silkworm enhancer trap line ET14 in which GAL4 was
strongly expressed in CA (Figure 5A), although weak expression
was also detected in peripheral tissues including fat bodies and the
midgut [9,27]. As these lines were generated using the standard
Shiro-C (w-1; +mod) strain, we changed the genetic background to
w-1/w-1; mod/mod by crossing to the mod strain. The resultant w-1;
mod; ET14/+ females were then crossed with w-1; mod; UAS-
CYP15C1/+ males to determine whether the mod phenotype could
be rescued by CYP15C1 overexpression. We used two independent
UAS-CYP15C1 lines with ET-14 (Figure 5B). In both UAS-
CYP15C1 lines, CYP15C1 overexpression efficiently prevented
precocious metamorphosis and 97.1% of the larvae (34/35 in
total) underwent the fourth larval molt to become fifth instar
larvae (Figure 5B and 5C). Only one larva (1/35) became a late-
maturing trimolter, but neither dimolters nor early-maturing
mod larvae. Selected doses of methoprene (0.01–10 mg/larva) were topically applied to newly molted third and fourth instar larvae (8–12 h after
molting). As highlighted in blue, precocious pupation could be blocked by methoprene treatment. (F) Measurement of the JH titer in the hemolymph
of third instar larvae of p50T and mod strains at 24 h after molting. Hemolymph was collected from ,400 larvae using a microsyringe and the pooled
sample was analyzed. JH in the hemolymph was converted to its corresponding methoxyhydrin derivatives and analyzed by GC-MS. JHs were not
detected (ND) in the hemolymph of mod larvae.
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Figure 2. Positional cloning of the mod locus. (A) Physical map showing the outcome of the linkage analysis using 792 BC1individuals. The mod
locus was narrowed to the genomic region flanked by the PCR markers M2 and Q, as indicated by the orange arrows. Putative genes predicted by the
Gene model program [8,9] are shown below the map, and CYP15C1 (BGIBMGA011708) is shown in red. For more details refer to Figure S1. (B) A
phylogenetic tree showing the relationship of CYP15C1 and other related P450 genes. The rootless tree was constructed based on the entire amino
acid sequence by the neighbor-joining method using the ClustalX program . Sequences were retrieved from public databases, and the species
names are abbreviated as follows: Aedes, A. aegypti; Anopheles, A. gambiae; Apis, A. mellifera; Bombyx, B. mori; Diploptera, D. punctata; Drosophila, D.
melanogaster; and Tribolium, T. castaneum. The scale bar indicates the number of amino acid substitutions per site. Note that CYP15 was not found in
D. melanogaster. (C) The genomic structure of CYP15C1 in the wild-type (p50T) strain. White box, grey box, and a black bar indicate untranslated,
coding, and intronic regions, respectively. (D) Transcripts of CYP15C1 from p50T and mod strains. A 68-bp deletion was found in CYP15C1 of the mod
strain, and this deletion introduced a premature stop codon as indicated in red. Heme-binding motifs of P450s  are indicated in orange. (E)
Genomic PCR showing the presence of the 68-bp deletion in CYP15C1 from the mod strain. PCR primers (Table S1) that flank the deletion are shown
by arrows in (C).
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Figure 3. Temporal and spatial expression of CYP15C1. (A) qRT-PCR analysis of the spatial expression of CYP15C1 in the silkworm strain
Kinshu6Showa. ‘‘CYP15C1/rp49’’ on the vertical axis indicates the level of CYP15C1 mRNA normalized to that of internal rp49 mRNA. RNAs were
collected from larvae on day 1 of the fourth instar (4th D1), fourth instar larvae showing head capsule slippage (4th HCS), larvae on day 2 of the fifth
instar (5th D2), and larvae on day 1 after the onset of spinning (Spin+1). CC-CA, corpus cardiacum-corpus allatum complex; PG, prothoracic gland; Br,
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trimolters appeared. This result was in contrast to what was
observed in control larvae or larvae carrying either the GAL4 or
UAS construct alone: approximately half of the larvae became
dimolters and the remainder became trimolters, while no larvae
became tetramolters. We also measured the JH titer in the
hemolymph (Figure 5D). As expected, the JH titers in control,
ET14, and UAS larvae were below the detectable limit. In contrast,
we were able to detect JH I and JH II in the hemolymph of mod
larvae carrying both ET14 and UAS-CYP15C1 constructs. Taken
together, these results provide direct evidence that CYP15C1 is
responsible for the mod mutation and is essential for JH
In this study, we identified and characterized the gene
responsible for the mod locus that causes precocious larval-pupal
metamorphosis in B. mori. The data we present here have two
important implications. First, we provide direct genetic evidence
for the significance of P450 epoxidase in the late step of the JH
biosynthetic pathway, whose expression is essential for normal
growth and metamorphosis. Second, we show that the mod strain is
a JH-deficient mutant strain carrying a null allele of CYP15C1, in
which developmental abnormalities are mostly limited to larval-
pupal transitions and are not observed before the second larval
Biochemical and physiological function of CYP15C1
JH III is the most common JH in many insect orders, although
its ethyl-branched homologs (JH I and II) are the major JHs in the
order Lepidoptera [22,28]. Biochemical studies have shown that in
the late steps of JH biosynthesis in many insect species, including
cockroaches and locusts, FA is first methylated to MF and then
epoxidized to JH III in CA . However, the final two steps of
JH biosynthesis are reversed in Lepidoptera: ethyl-branched
homologs of FA (homo-FAs) are first epoxidized and the resultant
brain; FB, fat body; MG, midgut; Ep, epidermis; Ms, muscle; Mp, Malpighian tubule; SiG, silk gland; SaG, salivary gland; Ts, testis; and Ov, ovary. (B) In
situ mRNA hybridization of CYP15C1 and JHAMT. Br-CC-CA complexes on day 2 of the fourth instar and day 4 of the fifth instar were used for analysis.
Signals of both genes were limited to CA as indicated by arrows, but JHAMT was not detected on day 4 of the fifth instar. The purple coloration in the
brain is primarily due to ommochrome pigments and does not reflect gene expression. The result of control experiments using sense probes are
shown in Figure S2. (C) Developmental changes in the rate of JH biosynthesis by B. mori CA in vitro. The data are based on Kinjoh et al. (2007). Black,
red, and blue lines indicate CA from unsexed larvae, female and male animals, respectively. The activity in CA on day 1 of the fourth instar was set as
100. (D) Temporal expression patterns of JHAMT (upper) and CYP15C1 (lower) in the Br-CC-CA (first and second instar larvae) or CC-CA (third to fifth
instar larvae, pupae, and adults) complex. Developmental stages are defined as h/days after certain developmental events [i.e., molting, head capsule
slippage (HCS), spinning, or emergence] or by a spiracle index (si) . Animals were unsexed during larval stages, while sexed during pupal and adult
stages (female in red and male in blue). The expression profile of JHAMT after the second larval instar is based on published data (20). Expression
levels measured on day 2 of the 4th larval instar are arbitrarily set at 100 (for actual transcript numbers per rp49) and are shown in a log scale.
Asterisks indicate that data were not available.
Figure 4. Enzymatic properties of B. mori CYP15C1. (A) Enzymatic activity against FA. Medium containing FA was incubated with Drosophila S2
cells transiently expressing CYP15C1 (middle) or GFP (bottom), and analyzed by HPLC. Standard JHA III (top). Arrows indicate peaks of JHA III. (B)
Stereospecificity. JHA III generated from FA by Sf9 cells stably expressing CYP15C1 (Sf9/CYP15C1) was chemically methylated and analyzed by a
Chiral-HPLC. R and S indicate peaks of (R)- and (S)-JH III enantiomers, respectively. The R:S ratio of standard racemic JH III (top) was 50:50, while that of
CYP15C1-produced JH III (bottom) was 97:3. (C) The late JH biosynthetic step in B. mori, in which major JHs in the hemolymph are JH I and II .
Ethyl-branched farnesyl diphosphates (homo-FPPs) are converted to homo-FAs, epoxidized to JHAs by the cytochrome P450 epoxidase CYP15C1 (this
study), and then methylated by the JHA methyltransferase (JHAMT) . JH I: R1=R2=C2H5, JH II: R1=C2H5, R2=CH3.
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JHAs (i.e., JHA I and II) are then methylated to the authentic JHs
(i.e., JH I and II) . This study showed that B. mori CYP15C1
epoxidizes FA to JHA III in a highly stereospecific manner.
CYP15C1 might also epoxidize MF to JH III, but in a far less
efficient manner (Table S2). Given that B. mori JHAMT can
methylate both FA and JHAs with similar efficiencies , our
data clearly demonstrate the major JH biosynthetic pathway in B.
mori: homo-FAs are first epoxidized to JHAs by CYP15C1, and
then methylated to JHs by JHAMT (Figure 4C and Figure 6A).
Interestingly, D. punctata CYP15A1 does not convert FA to JHA III
. Thus, the difference in specificity of CYP15 to the substrates
FA and MF may determine the order of the final steps of JH
biosynthesis in insects.
The expression of most early JH biosynthetic enzyme genes and
JHAMT in B. mori is limited to the CA and shows dynamic
developmental fluctuations [20,21,29]. In particular, the temporal
expression profile of JHAMT correlates well with JH biosynthetic
activity in B. mori [20,21,30,31] and in the Eri silkworm Samia
cynthia ricini , indicating that JHAMT is a key regulatory gene
whose transcriptional control is critical for the regulation of JH
biosynthesis in Lepidoptera. Here, we found that expression of
CYP15C1 was also limited in CA but in a different pattern to other
JH biosynthesis genes in that it was constitutively expressed from
larval to adult stages. This result suggests that the transcriptional
regulation of CYP15C1 is less important than JHAMT for the
temporal regulation of JH production in B. mori. CA of the
Figure 5. Transgenic rescue of mod. (A) Visualization of GAL4 expression in CA of the enhancer trap line ET14 carrying the UAS-GFP construct. GFP
expression (green) is limited to CA (arrowhead). Red fluorescence in the optic nerve is due to DsRed2 expression driven by the 3xP3 promoter . Br,
brain; SOG, suboesophageal ganglion; and CA, corpus allatum. (B) Developmental profiles of binary GAL4/UAS transgenic lines. Male moths with a w-
1; mod background and carrying UAS-CYP15C1 were crossed with w-1; mod female moths carrying ET14, and their progenies were analyzed.
Tetramolters appeared in GAL4/UAS transgenic lines, but not in nonbinary lines. (C) Images of pupae and moths of GAL4/UAS transgenic lines. Larvae
carrying both ET14 and UAS-CYP151 constructs entered the fifth larval instar and eventually formed larger adults. Control animals did not carry
transgenic vectors. (D) Measurement of the JH titer in the hemolymph of GAL4/UAS transgenic lines on the w-1; mod background. Hemolymph was
collected from fourth instar larvae at 24 h after molting and analyzed. JH was detected only in GAL4/UAS lines, but not in nonbinary lines. ND, not
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silkworm ceases JH biosynthesis by day 3 of the last (fifth) instar
; however, it is speculated that CA synthesizes and secretes
JHAs during the following prepupal period. Our data indicate that
this endocrine switch can be explained by constitutive CYP15C1
expression and the shut-off of JHAMT expression in CA
(Figure 6A). During the larval-pupal transition, homo-FAs are
constantly converted to JHA I and II by CYP15C1, and the
resultant JHAs are secreted from the gland without further
conversion because of the absence of JHAMT.
CYP15 P450 family members are found in both hemimetabo-
lous and holometabolous insects . In a similar manner as
CYP15C1 expression in B. mori, CA-specific CYP15 expression has
also been observed in two cockroach species, D. punctata and
Blattella germanica [18,34], in the locust Schistocerca gregaria , and
in the mosquito Aedes aegypti , suggesting a conserved function
in JH biosynthesis. However, the enzymatic properties of CYP15
products, with the exception of those of D. punctata  and B. mori
(this study), have not been studied and the physiological role of
CYP15s in the development of other insects remains unknown. By
characterizing the CYP15C1-null mutant silkworm, we have
demonstrated here that CYP15C1 plays an essential role in JH
biosynthesis and for the maintenance of the proper timing of
Accumulating data have suggested that CYP15 genes are
evolutionarily diversified in terms of their gene regulation and
nature. For example, unlike B. mori CYP15C1, A. aegypti CYP15
shows developmentally and dynamically regulated changes of
expression, which appear to correlate well with the JH synthetic
activity in the CA . In addition, CYP15 is not present in the
genome of D. melanogaster, but a P450 gene (Cyp6g2) is expressed in
CA in a highly tissue-specific manner . More extensive
research on the transcriptional controls and enzymatic properties
of JH epoxidases across a broader range of insect taxa will shed
light on the roles of these enzymes.
Precocious pupation in mod larvae
Our results consistently indicate that the mod strain is a JH-
deficient mutant that is unable to synthesize JHs in CA. One
unique characteristic of the precocious pupation in the mod strain is
the variation in the timing of the onset of spinning (Figure 1). The
Figure 6. A model for JH biosynthetic pathway in the CA of wt and mod silkworms. (A) In the B. mori CA, constitutive CYP15C1 expression
allows the consistent conversion of homo-FAs to JHAs (predominantly JHA I and II in Lepidoptera). When JHAMT is expressed in CA, JHAs are further
converted to JHs, and released from CA, thereby preventing precocious metamorphosis. When JHAMT expression is shut off (e.g., in the prepupal
stage), JHAs are likely to be released from CA. (B) In CA of the mod strain, homo-FAs are not converted to JHAs because of the loss of CYP15C1, but
instead, homo-FAs are converted to ethyl-branched homologs of MF (homo-MFs, i.e., unepoxidized JH I and II) by JHAMT. The loss of CYP15C1 does
not allow the conversion of homo-MFs to the authentic JHs. Therefore, neither JHs is synthesized in nor released from CA of the mod strain, thereby
causing precocious metamorphosis. The synthesized homo-MFs might be released from CA of the mod strain, similar to that of higher dipteran
insects . JH I: R1=R2=C2H5, JH II: R1=C2H5, R2=CH3.
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feeding period in early-maturing trimolters was unusually short
(50 h after molting) compared with that observed in surgical
allatectomy of newly molted fourth instar larvae. In the latter
larvae, the feeding period was comparable in length to that of the
late-maturing trimolters [e.g. ,130 h ] and no timing
segregation was observed . In addition, most of the early-
maturing trimolters displayed a larval-pupal intermediate pheno-
type and eventually died, unlike allatectomized larvae, most of
which successfully developed into small but normal pupae .
One explanation for this phenomenon is that the early-maturing
trimolters were destined to undergo larval molting to the fifth
instar on day 2, while the late-maturing trimolters were destined
for pupation after a prolonged fourth instar, similar to allatecto-
mized larvae  (Figure 1D). Molting in early-maturing
trimolters on day 2 usually resulted in the formation of larval-
pupal intermediates. One possible explanation for this mixed
phenotype is that metamorphosis in the mod strain is induced in the
presence of homo-MFs (unepoxidized JH I and II), presumed
products instead of epoxidized JH I and II in CA of the mod strain
(see Figure 6B). MF is known as a crustacean JH and has recently
been reported to have JH activity in D. melanogaster [38,39].
Therefore, MF and its homologs might have JH-like activity but
not able to fully substitute for authentic (epoxidized) JHs in the
physiology of the silkworm. Alternatively, other P450 epoxidases
in B. mori that have low substrate specificity and stereospecificity,
like CYP9E1  and CYP6A1  in other insects, might
substitute for the absence of CYP15C1 in peripheral tissues of mod
larvae, and such locally-synthesized JHs may prevent precocious
metamorphosis in the first and second instar larvae carrying the
mod mutation. Further studies are needed to elucidate the
mechanism for this unique characteristic of the mod strain.
We found that the precocious phenotype was more severe in the
w-1; mod strain compared to that in t011, a genetic stock of the mod
strain. We rarely observed dimolter larvae in the t011 stock
(Figure 1B). However, in the original manuscript in 1956, it was
reported that 28–92% of mod larvae became dimolters . This
difference might have developed as a consequence of unintended
artificial selection during stock maintenance that favored broods
producing trimolters in higher proportions, as it is difficult to
obtain sufficient number of eggs using dimolter moths [11,12].
Thus, we speculate that the present t011 stock may be genetically
fixed to produce mostly trimolters, and that this attribute can be
varied by outcrossing to other strains.
In the silkworm, premature metamorphosis can be induced by
surgical removal of JH-producing CA (allatectomy) , by
application of an imidazole-based insect growth regulator KK-42
 or an anti-juvenile hormone agent KF-13S [41,42], or by
continuous overexpression of the JH-degrading enzyme, JH
esterase . In any case, however, premature pupation is not
induced in larvae younger than the third instar. In agreement with
these studies, we did not observe precocious pupation in first or
second instar mod larvae, nor did we observe apparent develop-
mental abnormalities during these early instars. Therefore, our
data support the hypothesis that there are two physiological phases
in the life of silkworm larvae : the JH-independent phase (first
and second instar) in which JH does not have a morphogenetic
function; and, the JH-dependent phase (third instar and thereafter)
in which the morphostatic action of JH is required to prolong the
larval stage until the attainment of the appropriate body size for
metamorphosis. Given that most generally the minimum number
of the larval instar in insects is three [1,2], our data further imply
that insect larvae need to experience at least one [e.g., L2 pupae in
D. melanogaster ] or two (e.g., B. mori) larval-larval molts and/or
require a certain length of time of postembryonic development in
order to acquire competence for metamorphosis.
The silkworm is a classic model organism that has been used for
pioneering studies in genetics, physiology, and biochemistry .
The availability of whole genome data , post-genomic tools
, and unique mutant resources , together with the classic
‘‘status quo’’ responses to JHs in this insect [14,15,17], makes the
silkworm well-suited for study of hormonal control of growth and
development. Indeed, these advantages have greatly contributed to
the identification of essential components in the biosynthesis of
ecdysteroids, the insect molting hormones . Moreover, recent
success in targeted gene disruption using a zinc-finger nuclease
 increases the utility of this model organism. We are hopeful
that our present study will encourage further studies on other
‘‘moltinism’’ strains in the silkworm, and consequently pave the
way for a greater understanding of physiological control,
developmental plasticity, and evolutionary history of the number
of larval molting in insects, which may reflect adaptive strategies of
insects to diverse environmental conditions. It is also noteworthy
that the late step of the JH biosynthetic pathway is insect-specific
and is therefore a potential target for biorational insecticides .
Materials and Methods
Insects and cell lines
Silkworms were reared on an artificial diet or mulberry leaves at
25–27uC under standard conditions as described previously .
The silkworm strain t011 (mod/mod) was obtained from Kyushu
University . The Spodoptera frugiperda Sf9 and Drosophila
melanogaster S2 cells were maintained as described previously
. To determine the developmental profile of mod, larvae from
two batches of t011 were individually reared in plastic dishes, and
their developmental stages were recorded at ,8-h intervals.
The JH analog, methoprene (a kind gift from S. Sakurai) was
applied to newly molted third or fourth instar larvae (,8–12 h
after molting). Methoprene was diluted with acetone and the
selected doses (0.01–10 mg/larva) were topically applied to the
dorsum using a 10-ml Hamilton microsyringe. The same volume of
acetone was applied as a control.
Positional cloning of the mod locus
Positional cloning of the mod locus was performed as described
previously . Codominant PCR markers and p50T-specific
PCR markers were generated for each position of the scaffold
Bm_scaf16 (chromosome 11) , and used for genetic analysis
(Figure 2A and Figure S1). Homozygotes of the mod locus were
collected from the BC1population [t0116(p506t011)] based on
the phenotype of precocious pupation.
Cloning of CYP15C1
Total RNAs were collected from CA of day 0 fifth instar larvae
of p50T and Kinshu6Showa strains and used for 59- and 39-rapid
amplification of cDNA ends (RACE) using the GeneRacer Kit
(Invitrogen). PCR was performed using the primers listed in Table
S1. The PCR products were subcloned and sequenced as
described previously . The obtained cDNA sequence was
deposited in the GeneBank (accession number: AB124839).
Quantitative RT–PCR (qRT–PCR) analysis
qRT-PCR was performed essentially as described previously
. The primers used for the quantification of the CYP15C1
transcript are listed in Table S1.
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In situ hybridization
In situ hybridization was performed essentially as described
previously . A CYP15C1 cDNA fragment (,1.1 kb) was
amplified by PCR listed in Table S1 and subcloned into a pDrive
plasmid vector (QIAGEN).
(2E,6E)-farnesoic acid (FA) and (2E,6E)-methyl farnesoate (MF)
were purchased from Echelon Research Laboratories (Salt Lake
City) and racemic JH III from Sigma. JH III acid was prepared
from the racemic JH III as described previously . (R)-JH III
was a kind gift from W.G. Goodman.
Enzyme assays of CYP15C1 in S2 cells
CYP15C1 overexpression in S2 cells was achieved using a
GAL4/UAS system . To generate a vector for expressing
CYP15C1 under the control of the UAS promoter (UAS-CYP15C1-
HA), a cDNA fragment coding the entire CYP15C1 ORF was
ligated into the pUAST vector. UAS-GFP.RN3  was used as a
negative control. UAS-CYP15C1-HA or UAS-GFP.RN3 was trans-
fected with the Actin5C-GAL4 construct (a gift from Yasushi
Hiromi, National Institute of Genetics, Japan). Forty-eight hours
after transfection of S2 cells in a 60-mm dish, the old medium was
replaced with 2 ml of fresh medium. S2 cells were detached from
the bottom of the dish by pipetting, and 1 ml of the cell suspension
was transferred to a siliconized glass test tube. FA or MF (100 mM
at final concentration) was then added to the tube. After
incubation at 25uC for 16 h, 500 ml of medium was collected
and mixed with 500 ml of acetonitrile. Samples were centrifuged
for 10 min at 15,000 rpm, followed by incubation at 25uC for
10 min. After filtration using a 0.2 mm filter, 10–20 ml of each
sample was subjected to HPLC analysis as described below.
Establishment of Sf9 cells stably expressing CYP15C1 and
A cDNA with the full ORF of CYP15C1 cDNA was subcloned
into the pIZT/V5-His vector (Invitrogen). The plasmid was
transfected into Sf9 cells with Cellfectin reagent (Invitrogen), then
cells transiently expressing CYP15C1 were selected successively
with Zeocin according to the manufacture’s instruction and a cell
line (Sf9/CYP15C1) stably expressing CYP15C1 was established.
Sf9/CYP15C1 cells were placed in a glass tube (12675 mm)
coated with PEG20,000 and cultured in SF900-II SFM medium
containing FA or MF (10 mg/ml) for either 2 or 6 h at 26uC. In
some experiments, the specific JH esterase-specific inhibitor OTFP
(6 mM) was added to the medium to prevent possible degradation
of the generated JH III by intrinsic JH esterase present in the cells.
After incubation, an equal volume of CH3CN was added to the
medium, vortexed vigorously and centrifuged for 4,800 rpm for
10 min to remove cell debris. The supernatant was directly
subjected to an HPLC analysis as described below for JH III acid
or JH III, which were expected to be generated from FA and MF,
HPLC and ESI-MS analyses of JH III and JH III acid
JH III was analyzed by reversed-phase HPLC as described
previously . JH III acid was analyzed by reversed-phase HPLC
(column, Shiseido ODS UG80, 150 mm63.0 mm ID; solvent,
CH3CN-20 mM CH3COONH4, pH 5.5, 35:65, flow rate,
0.5 ml/min; detection, UV 219 nm). ESI-MS spectrum of JH
III acid was obtained by TSQ system (Thermo Quest Finnigan,
Analysis of the stereospecificity of JH III acid generated
The stereospecificity of the epoxide group of JH III acid formed
by CYP15C1 was analyzed as follows under semi-dark conditions.
Sf9/CYP15 cells were cultured in medium containing 10 mg/ml
FA for 48 hrs. An equal volume of CH3CN was added to the
medium (2 ml), vortexed vigorously and centrifuged at 4,800 rpm
for 10 min. One ml of 1 M CH3COONH, (pH 5.5) was added to
the supernatant and extracted with 5 ml of CH2CH2; this step was
performed 5 times. The extract was dehydrated with anhydrous
Na2SO4and evaporated to dryness in vacuo at 40uC, then the
residue was dissolved in 200 ml of CH2Cl2, 50 ml of MeOH and
100 ml of TMS-diazomethane were then added and the solution
was incubated at room temperature for 30 min. The reaction was
dried with an N2gas stream, the residue dissolved in 100 ml of
hexane, and subjected to a normal-phase HPLC (column, Shiseido
SG80, 25064.6 mm ID; solvent, hexane-EtOH, 99:1; flow rate,
0.5 ml/min; detection, UV 211 nm). The peak corresponding to
JH III (r.t.=9.8 min) was collected. The stereospecificity of the
epoxide group of the JH III was analyzed by a chiral-HPLC
(column, Chiralapack IA, 25064.6 mm ID, DAICEL; solvent,
hexane-EtOH, 99:1; flow rate, 0.5 ml/min; detection UV
219 nm) as described previously .
Purification of JHs from hemolymphs and preparation for
Ten microliters of deuterium-substituted JH III (d3-JH III) 
in toluene (67.1 pg/ml) was transferred to a clean glass tube to
which 0.5 ml of methanol was added. The hemolymph sample
(100 ml) was then added and mixed vigorously, and 1.5 ml of 2%
NaCl was added to the JH sample. JH was extracted by partition
with 0.5 ml hexane; this step was performed three times. The
combined solvent containing JH (1.5 ml) was evaporated under a
stream of nitrogen. One hundred microliters of methanol and 2 ml
trifluoroacetic acid were added to the crude JH extract and
mixture heated at 60uC for 30 min. After removal of the
methanol, methoxyhydrin derivatives of JH (JH-MHs) were
purified using a Pasture pipette packed with 1.0 g of aluminum
oxide (activity grade III, ICN Ecochrom) prewashed with hexane.
After loading the extract and washing with 2 ml of 30% ether in
hexane, JH-MHs were eluted with 2 ml of 50% ethyl acetate in
hexane and then dried under a stream of nitrogen. The residue
was dissolved in 25 ml of 80% acetonitrile containing 5 mM
Analytical condition for LC-MS
The HP1100MSD system (Agilent) was equipped with a
15063 mm C18 reversed phase column (UG80, Shiseido)
protected by a guard column with 70% acetonitrile containing
5 mM sodium acetate at a flow rate of 0.4 ml/min. For MS
analysis, electrospray ionization in the positive mode was used
under the conditions of drying gas temperature at 320uC with
10 l/min flow rate, ionization voltage of 70 V. Under these
conditions, selected ion masses for each JH-MH were monitored
as [M+Na]+, i.e., m/z 321, 324, 335, and 349 for JH III, d3-JH
III, JH II, and JH I, respectively.
Transgenic rescue experiments
Overexpression of CYP15C1 was performed in transgenic
silkworms using the GAL4/UAS system as described previously
[25,27,54]. A coding sequence of CYP15C1 was introduced into a
silkworm UAS vector carrying the marker gene 3xP3-EGFP. B.
mori transformants were established using standard protocols .
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To overexpress CYP15C1 on the mod/mod background, established
UAS lines and an enhancer trap line ET14  were crossed with
the t011 strain, and the resultant F1animals were sib mated to
obtain the F2 generation. In the F2 generation, we collected
animals showing premature pupation with white eyes (i.e., mod/
mod; w-1/w-1) and confirmed the presence of the fluorescent
marker gene using a fluorescent microscope (SZX12, Olympus).
The established w-1; mod lines carrying UAS-CYP15C1 or ET14
were crossed, and their offspring were examined to determine
whether precocious metamorphosis was blocked by CYP15C1
locus. (A) Mating scheme for mapping the mod locus. A single-pair
cross between a female p50T (wt) and a male t011 (mod/mod) 
produced the F1offspring. Then, the male informative cross (t011
female6F1male) produced the BC1progeny. We collected and
analyzed 792 BC1individuals with the mod phenotype (premature
pupation). (B) The result of fine mapping of the mod locus. We
generated 12 PCR markers for each position of the scaffold
Bm_scaf16  that showed polymorphisms between p50T and
t011 strains. We analyzed 792 BC1individuals and the results are
summarized in the Table. ‘‘m’’ indicates the t011/t011 homozy-
gous genotype, ‘‘p/m’’ indicates the p50T/t011 heterozygous
genotype, and ‘‘p’’ indicates the genotype carrying a p50T-specific
allele in each marker. The genomic region for the mod locus lies
between the Q and M2 markers, as indicated by red arrows. (C)
PCR markers used in this study.
Detailed procedure for positional cloning of the mod
CA complex. Whole-mount in situ hybridization of CYP15C1 and
Whole-mount in situ hybridization in the brain-CC-
JHAMT in the brain-CC-CA complex on day 2 of the fourth instar
and day 4 of the fifth instar. Magnified images of CAs indicated by
arrows are shown below each panel. Signals were not detected
when sense probes were used for analysis.
PCR primers used in this study.
or Sf9/CYP15C1 cells (,1.26106) were cultured with 200 ml of
medium containing 2 mg of FA or MF at 26uC for 2 h (Exp. 1) or
6 h (Exp. 2), and the production of JHA III or JH III in the
medium was quantified by HPLC. Mean 6 SD (N=3). ND, not
Substrate specificity of CYP15C1 to FA and MF. Sf9
We thank S. Sakurai (Kanazawa Univ.) for providing methoprene, W. G.
Goodman (Univ. of Wisconsin-Madison) for (R)-JH III, Y. Hiromi (Natl.
Inst. Genet.) for the Actin5C-GAL4 plasmid, M. Yoshiyama for technical
assistance, and M. Kawamoto for clerical assistance.
Conceived and designed the experiments: T Daimon, T Kozaki, R Niwa, S
Katsuma, H Sezutsu, T Shimada, T Shinoda. Performed the experiments:
T Daimon, T Kozaki, R Niwa, I Kobayashi, K Furuta, T Namiki, K
Uchino, M Nakayama, K Itoyama, T Shinoda. Analyzed the data: T
Daimon, T Kozaki, R Niwa, T Namiki, T Shinoda. Contributed reagents/
materials/analysis tools: I Kobayashi, K Uchino, Y Banno, T Tamura, K
Mita, H Sezutsu. Wrote the paper: T Daimon, T Kozaki, R Niwa, K
Furuta, T Shinoda.
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