Báo cáo khoa học: RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum pdf

RNAi-mediated knockdown of juvenile hormone acid
O-methyltransferase gene causes precocious
metamorphosis in the red flour beetle
Tribolium castaneum
Chieka Minakuchi*, Toshiki Namiki, Michiyo Yoshiyama and Tetsuro Shinoda
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
Insect juvenile hormone (JH) is a multifunctional hor-
mone that controls a variety of physiological events,
e.g. growth and development, reproduction, diapause
and caste determination in social insects [1]. The most
prominent role of JH is the control of insect metamor-
phosis, which has been studied extensively in many
species [2]. In holometabolous insects, for example, lar-
vae do not initiate larval–pupal metamorphosis until
JH in the hemolymph declines at the end of the larval
stage. If JH in the hemolymph is precociously elimi-
nated by surgical removal of the corpora allata (CA),
the specialized endocrine organs that secrete JH into
the hemolymph, precocious metamorphic change
occurs. In contrast, application of a JH mimic (JHM)
at the onset of larval–pupal metamorphosis prevents
metamorphosis and causes an extra larval moult in
Keywords
juvenile hormone; juvenile hormone acid
O-methyltransferase; metamorphosis; RNA
interference; Tribolium castaneum
Correspondence
T. Shinoda, National Institute of
Agrobiological Sciences, 1–2 Ohwashi,
Tsukuba, Ibaraki 305-8634, Japan
Fax: +81 29 838 6075

Tel: +81 29 838 6075
E-mail:
*Present address
Graduate School of Bioagricultural Sciences,
Nagoya University, Japan
(Received 4 February 2008, revised 24
March 2008, accepted 1 April 2008)
doi:10.1111/j.1742-4658.2008.06428.x
Juvenile hormone controls the timing of insect metamorphosis. As a final
step of juvenile hormone biosynthesis, juvenile hormone acid O-methyl-
transferase (JHAMT) transfers the methyl group from S-adenosyl-l-methi-
onine to the carboxyl group of farnesoic acid and juvenile hormone acid.
The developmental expression profiles of JHAMT mRNA in the silkworm
Bombyx mori and the fruitfly Drosophila melanogaster suggest that the sup-
pression of JHAMT transcription is critical for the induction of larval–
pupal metamorphosis, but genetic evidence for JHAMT function in vivo is
missing. In this study, we identified three methyltransferase genes in the
red flour beetle Tribolium castaneum (TcMT1, TcMT2 and TcMT3) that
are homologous to JHAMT of Bombyx and Drosophila. Of these three
methyltransferase genes, TcMT3 mRNA was present continuously from
the embryonic stage to the final larval instar, became undetectable before
pupation, and increased again in the adult stage. TcMT3 mRNA was local-
ized in the larval corpora allata. Recombinant TcMT3 protein methylated
farnesoic acid and juvenile hormone III acid, but TcMT1 and TcMT2 pro-
teins did not. Furthermore, RNA interference-mediated knockdown of
TcMT3 in the larval stage resulted in precocious larval–pupal metamorpho-
sis, whereas knockdown of either TcMT1 or TcMT2 showed no visible
effects on metamorphosis. Importantly, precocious metamorphosis caused
by TcMT3 RNA interference was rescued by an application of a juvenile
hormone mimic, methoprene. Together, these results demonstrate that

TcMT3 encodes a functional JHAMT gene that is essential for juvenile
hormone biosynthesis and for the maintenance of larval status.
Abbreviations
Bm, Bombyx mori; CA, corpora allata; DIG, digoxigenin; Dm, Drosophila melanogaster; EGFP, enhanced green fluorescent protein;
FA, farnesoic acid; JH, juvenile hormone; JHA III, juvenile hormone III acid; JHAMT, juvenile hormone acid O-methyltransferase; JHM,
juvenile hormone mimic; LA, lauric acid; MF, methyl farnesoate; PA, palmitic acid; RNAi, RNA interference; SAM, S-adenosyl-
L-methionine;
Tc, Tribolium castaneum.
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2919
some insect species [2]. Therefore, JH has a ‘status
quo’ action to prevent metamorphosis.
JH is a unique farnesoid with a methyl ester moiety
at the C1 position and an epoxide group at the C10–
11 position [3,4]. Natural compounds with these chem-
ical features have been found only in insects, with one
exception of JH III isolated from a Malaysian plant
Cyperus iria [5]. The biosynthetic pathway of JH
in CA is conventionally divided into two parts:
early steps and late steps. The early steps, starting
from acetyl-CoA or propionyl-CoA and leading to
(homo)farnesyl diphosphate, constitute the standard
mevalonate pathway and are conserved in various
organisms, including vertebrates [6,7]. In contrast, the
late steps, starting from (homo)farnesyl diphosphate
and leading to JH, are unique to JH biosynthesis [7].
As the final step of JH biosynthesis, farnesoic acid
(FA) is converted into active JH by methylation of a
carboxyl group and epoxidation at the C10–11 posi-
tion [8].
The identification of the genes encoding enzymes in

the late steps has been hampered because of a lack of
vertebrate and plant homologues. Recently, we identi-
fied and characterized the JH acid O-methyltransferase
(JHAMT) gene that encodes one of the late step
enzymes, first from the silkworm Bombyx mori [9], and
then from the fruitfly Drosophila melanogaster [10].
In vitro enzyme assays showed that recombinant
JHAMT proteins of B. mori (BmJHAMT) and D. mel-
anogaster (DmJHAMT, CG17330) methylated car-
boxyl groups in JH acid and FA in the presence of
S-adenosyl-l-methionine (SAM) [9,10]. JHAMT mRNA
is detected primarily in CA of both B. mori and
D. melanogaster, and its temporal expression profile
correlates well with a change in the JH titre in the he-
molymph, suggesting that the suppression of JHAMT
transcription at the end of the larval stage is critical
for the initiation of metamorphosis into a pupa [9,10].
However, direct evidence for the significance of
JHAMT in inducing larval–pupal metamorphosis
remains to be shown.
To reveal the function of JHAMT in vivo, overex-
pression and RNA interference (RNAi)-mediated
knockdown of JHAMT were performed in D. melanog-
aster [10]. Overexpression of DmJHAMT caused a
pharate adult lethal phenotype, as well as defects in
the rotation of adult male genitalia [10], both of which
are typically observed after treating wild-type insects
with an excess of JHM at the end of the larval stage
[11–14]. In contrast, RNAi-mediated knockdown of
DmJHAMT showed no visible effect on growth and

development [10]. However, whether the RNAi-medi-
ated knockdown of DmJHAMT is effective enough to
completely eliminate JH in the hemolymph needs to be
examined. Functional analysis using RNAi techniques
has not confirmed the significance of JHAMT in JH
biosynthesis.
In this study, the red flour beetle Tribolium castane-
um was chosen to analyse the in vivo function of
JHAMT. In this species, RNAi-mediated knockdown
of a gene of interest by injecting dsRNA into larvae is
effective and easy to perform [15]. Although previous
biochemical studies have disclosed the enzymatic prop-
erties of the JHAMT enzyme in intact CA of a related
beetle, Tenebrio molitor [16], the JHAMT gene has not
yet been identified in Coleoptera, including T. molitor.
We report here the identification and functional char-
acterization of three JHAMT-like methyltransferase
genes (TcMT1, TcMT2 and TcMT3) from T. castane-
um. Only TcMT3 of the three methyltransferase genes
was shown by developmental and spatial expression
profiles, and the enzymatic properties of the recombi-
nant proteins, to encode a functional JHAMT gene.
Furthermore, RNAi-mediated knockdown of JHAMT
(TcMT3), but not TcMT1 or TcMT2, caused preco-
cious larval–pupal metamorphosis, demonstrating that
the JHAMT gene is essential for JH biosynthesis and
maintenance of the larval status.
Results
Identification of three methyltransferase genes
in T. castaneum

Three putative JHAMT-like methyltransferase genes
were found in a genomic sequence contig (Con-
tig4620_Contig8031) by tblastn searches of the beetle
genome database with the sequences of BmJHAMT
and DmJHAMT. Hereafter, these methyltransferase
genes are called TcMT1, TcMT2 and TcMT3. The
cDNAs containing full ORFs of TcMT1, TcMT2 and
TcMT3 were amplified by RT-PCR using primers
designed from the genomic sequences, and then
sequenced. Comparison of the genomic sequence with
the cDNA sequence revealed that TcMT2, TcMT1 and
TcMT3 were located in this order (from the 5¢-end to
the 3¢-end) with the same orientation in a  15 kb
region (Fig. 1A). The deduced amino acid sequences of
TcMT1, TcMT2 and TcMT3 were homologous to
each other (amino acid identities, 42–50%), as well as
to JHAMT of B. mori or D. melanogaster (Fig. 1B).
The amino acid identities of TcMT1, TcMT2 and
TcMT3 compared to BmJHAMT were 31%, 32% and
36%, respectively. The putative SAM-binding motif
(motif I) is well conserved in all five methyltransferases.
Each of the three TcMT genes consisted of three
Function of JHAMT gene in Tribolium C. Minakuchi et al.
2920 FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS
exons, as far as we examined (Fig. 1A), and two
introns at positions 1 and 3 located in identical posi-
tions for the three TcMTs (Fig. 1B). The intron at
position 3 was also conserved in DmJHAMT and
BmJHAMT (Fig. 1B). Although DmJHAMT lacked
an intron at position 1, BmJHAMT had an intron at

position 1 and an extra intron at position 2 (Fig. 1B).
The similarity in exon–intron structures of the three
TcMT genes to that of the JHAMT genes in D. mela-
nogaster and B. mori further confirmed that these are
homologues of JHAMT.
Developmental expression profiles of TcMT1,
TcMT2 and TcMT3
To examine the developmental expression profiles of
TcMT1, TcMT2 and TcMT3 transcripts, quantitative
RT-PCR analysis was performed (Fig. 2). The amount
of TcMT1 transcript was relatively low during the
embryonic and larval stages, but high in the last two
days of adult development (Fig. 2A,B). The TcMT2
transcript was also weakly expressed during the embry-
onic and most of the larval stages, but showed a distinct
peak at the beginning of the prepupal stage when the
larval ocelli begin to retract and the insects become slug-
gish (Fig. 2C,D). The amount of TcMT3 transcript was
high in the embryonic stage, decreased gradually in the
second larval instar, decreased to a low level at the end
of the sixth instar (day 2), and increased just before
ecdysis to the seventh instar (Fig. 2E,F). The transcript
level of TcMT3 gradually decreased during the final lar-
val instar, but was still detectable in the prepupal stage
(Fig. 2F). TcMT3 was undetectable in the pupal stage
and during subsequent adult development, but increased
again in adults by day 7 (Fig. 2F). This increase was
observed in both males and females (data not shown).
Spatial expression profiles of TcMT1, TcMT2
and TcMT3

The tissue specificity of the TcMT1, TcMT2 and
TcMT3 transcripts was examined by quantitative
RT-PCR and in situ hybridization. Quantitative RT-
PCR showed that TcMT1 and TcMT2 transcripts were
A
B
Fig. 1. Structure of the three methyltransferase genes in Tribolium castaneum. (A) Organization of the methyltransferase genes in T. casta-
neum. Exons are shown as boxes. (B) Alignment of TcMT1 (GenBank accession number: AB360761), TcMT2 (AB360762), TcMT3
(TcJHAMT, AB360763), Bombyx mori JH acid O-methyltransferase (BmJHAMT, BAC98835) and Drosophila melanogaster JH acid O-methyl-
transferase (DmJHAMT, BAC98836) sequences. Amino acids common in three or four methyltransferases are indicated by grey shadowed
letters, and those common in all methyltransferases are indicated by white letters with a black background. The putative SAM-binding motif
(motif I) is boxed. The positions of the introns are indicated by red lines, and the numbers above indicate the positions of the introns as
described in the text.
C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2921
more abundant in the posterior part of the sixth larval
instar (Fig. 3A,B) and at the beginning of the prepupal
stage in the seventh larval instar (Fig. 3D,E). In the
sixth instar larvae, TcMT3 was specifically expressed
in the anterior part, which presumably includes CA,
where JH is synthesized (Fig. 3C). In contrast, the
TcMT3 transcript was detected in both anterior and
posterior parts of the seventh larval instar (Fig. 3F).
The localization of the TcMT3 transcript was fur-
ther examined in the anterior part of sixth instar larvae
by in situ hybridization (Fig. 4). With the antisense
RNA probe, mRNA localization was found in a pair
of small globular organs (Fig. 4C), but there was no
obvious hybridization in these tissues with the sense
RNA probe (Fig. 4B). These organs showing TcMT3

expression are the putative CA of T. castaneum. After
removing the remaining head capsule with forceps, we
located the putative CA on the ventral side of the
brain (Fig. 4D). The size of the putative CA was
approximately 15 lm in diameter.
Enzymatic properties of recombinant TcMT1,
TcMT2 and TcMT3 proteins
The enzymatic activities of recombinant TcMT1,
TcMT2 and TcMT3 proteins were examined against
two potential substrates, FA and JH III acid
(JHA III). Recombinant TcMT1 and TcMT2 protein
did not show detectable activity to methylate these
substrates. In contrast, recombinant TcMT3 protein
catalysed the methylation of FA and JHA III to give
methyl farnesoate (MF) and JH III, respectively
(Table 1). The TcMT3 protein showed weak methyl-
transferase activity with normal saturated fatty acids,
such as lauric acid (LA) or palmitic acid (PA), much
lower than against FA and JHA III (Table 1).
JHA and JH have a chiral centre in the epoxide moi-
ety at the C10–11 position. The stereospecificity of
TcMT3 against a mixture of (10R)- and (10S)-enantio-
mers of JHA III was investigated by analysing the prod-
uct with enantioselective HPLC. Under the conditions
Fig. 2. Developmental expression profiles of TcMT1, TcMT2 and TcMT3 transcripts in Tribolium castaneum. Transcript levels of TcMT1
(A, B), TcMT2 (C, D) and TcMT3 (E, F) were analysed by quantitative RT-PCR, and the signal intensity was normalized to the intensity
of TcRp49. In the embryonic stage and the first, second and third larval instars, RNA was isolated from a mass of eggs or larvae. From the
sixth larval instar until the adult stage, RNA was isolated from individuals (three larvae in the sixth and seventh larval instars, three males
and three females for pupae and adults for each time point). The means and standard deviations of expression are shown. The highest val-
ues during development (day 5 pupa for TcMT1, 84–96 h in the seventh instar for TcMT2 and the embryonic stage for TcMT3) were desig-

nated 100% for each gene.
Function of JHAMT gene in Tribolium C. Minakuchi et al.
2922 FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS
used in this study, (10R)- and (10S)-enantiomers of
racemic JH III can be completely separated (Fig. 5A).
The ratio of (10R)-JH III to (10S)-JH III in the product
obtained with TcMT3 was 87 : 13 (Fig. 5B), indicating
that TcMT3 catalyses the methylation of (10R)-JHA III
more favourably than (10S)-JHA III.
Effects of RNAi-mediated knockdown of TcMTs
on larval–pupal metamorphosis
To examine the role of methyltransferase genes in the
larval stage in vivo, RNAi-mediated knockdown of
TcMT1, TcMT2 and TcMT3 was performed by inject-
ing dsRNA at the beginning of the third instar.
dsRNA for enhanced green fluorescent protein (EGFP)
was injected as a control. First, the transcript levels
3–7 days after injection of dsRNA were quantified to
confirm the efficiency of RNAi-mediated knockdown.
As shown in Fig. 6A, injection of TcMT1 dsRNA sup-
pressed the transcript level of TcMT1 itself compared
with EGFP dsRNA-injected controls. In addition, the
transcript level of TcMT2 was suppressed by injection
of TcMT2 dsRNA (Fig. 6B), and the transcript level
of TcMT3 was suppressed 3 days (Fig. 6C) and 6 days
(Fig. 6D) after injection of TcMT3 dsRNA.
In the controls that received EGFP dsRNA at either
1.5–2.0 or 5.0 lgÆlL
)1
, no significant effect on growth

or metamorphosis was observed, and all of these lar-
vae pupated at the end of the seventh or eighth larval
instar and eclosed normally (Table 2). All the larvae
that received TcMT1 or TcMT2 dsRNA also pupated
and eclosed normally without undergoing precocious
metamorphosis (Table 2). In contrast, TcMT3 RNAi
caused precocious pupation, and most of the larvae
pupated at the end of the sixth instar (Table 2;
Fig. 7A). These pupae and adults appeared normal in
their external morphology, but were much smaller than
normal animals (Fig. 7). Three larvae that had been
injected with TcMT3 dsRNA showed prepupal charac-
teristics, such as larval ocellar retraction, at the end of
the fifth larval instar, but only one larva of these three
larvae succeeded in pupation followed by eclosion,
whereas the other two arrested either as prepupa or
pupa (Table 2). No significant difference in the effect
of RNAi as a result of the dose of dsRNA was
observed in this study.
As stated above, the TcMT1 transcript was
expressed strongly in the last 2 days of adult develop-
ment, whereas the TcMT2 transcript was expressed
strongly at the beginning of the prepupal stage
(Fig. 2B,D). To examine the role of TcMT1 and
TcMT2 when expression levels are normally high,
A B
C

DE F
Fig. 3. Spatial expression pattern of TcMT1, TcMT2 and TcMT3

transcripts in Tribolium castaneum. RNA was isolated from four lar-
vae in the sixth instar (A–C) and from four larvae 84 h after ecdysis
to the final (seventh) instar (D–F), which were cut in half between
thoracic segments T2 and T3, and the transcript levels of TcMT1
(A, D), TcMT2 (B, E) and TcMT3 (C, F) in the anterior and posterior
parts were examined by quantitative RT-PCR. Signal intensities rela-
tive to the highest values in the developmental expression profiles
(see Fig. 2) are shown. A, anterior part; P, posterior part.
A BCD
Fig. 4. In situ hybridization of TcMT3 transcript in Tribolium castaneum. (A) Dorsal view of head and thoracic segments of a normal sixth
instar larva. The area that was used for in situ hybridization and subsequent imaging (B–D) is boxed. (B–D) In situ hybridization of TcMT3.
Heads of sixth instar larvae that were dissected with some part of the head capsule still attached were fixed, hybridized with sense (B) or
antisense (C, D) RNA probes for the TcMT3 transcript, and detected. Pictures were taken before (B, C) and after (D) removing the head cap-
sules with forceps. mRNA localization in the putative corpora allata is indicated by arrows, and non-specific staining in the cuticle is indicated
by asterisks. A, anterior; BR, brain; D, dorsal; P, posterior; SG, sub-oesophageal ganglion; V, ventral.
C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2923
TcMT1 dsRNA was injected in the prepupal stage and
TcMT2 dsRNA was injected at the beginning of the
final larval instar. In both cases, quantitative RT-PCR
confirmed that RNAi-mediated knockdown suppressed
the transcript levels (Fig. 6E,F). However, all TcMT1
dsRNA-injected insects (n = 13) eclosed to form nor-
mal adults, and all TcMT2 dsRNA-injected insects
(n = 4) pupated and eclosed normally.
Effects of JHM treatment on precocious
metamorphosis induced by TcMT3 RNAi
To confirm that the observed precocious metamorpho-
sis was a result of JH deficiency caused by TcMT3
knockdown, the JHM methoprene was topically

applied to larvae that had been injected with TcMT3
dsRNA at the beginning of the fourth larval instar. As
shown in Table 3, 84% of the larvae that received
TcMT3 dsRNA and were then treated with the solvent
precociously pupated at the end of the sixth instar. In
contrast, 62% of the larvae (n = 21) that received
TcMT3 dsRNA moulted into the seventh instar after
treatment with JHM either at the fourth or fifth instar.
If JHM was applied at the beginning of the sixth instar
to the larvae that had received TcMT3 dsRNA, the
majority (94%, n = 16) moulted into the seventh
instar (Table 3). Thus, JHM application at the begin-
ning of the sixth instar was more effective in rescuing
TcMT3 RNAi-mediated precocious pupation than was
JHM application in the fourth or fifth instar.
Table 1. Enzymatic activity of recombinant TcMT3 protein to FA,
JHA III and saturated fatty acids. The average and standard devia-
tion were calculated from independent enzyme assays (n = 3).
Substrate
Activity
[molÆ(mol enzyme)
)1
Æmin
)1
]
FA 0.59 ± 0.04
JHA III 0.56 ± 0.03
Lauric acid 0.016 ± 0.002
Palmitic acid 0.002 ± 0.001
0

500
A
B
mAU
100
0
mAU
05101520
05101520
10S
10R
Retention time (min)
10S
10R
Fig. 5. Enantioselective HPLC profiles of racemic JH III (A) and the
metabolites from racemic JHA III produced by recombinant TcMT3
protein (B). Arrows indicate (10S)-JH III and (10R)-JH III.
AB
CD
EF
Fig. 6. Efficiency of RNAi-mediated knockdown in Tribolium casta-
neum. (A) The level of TcMT1 transcript 6 days after injection with
dsRNA of EGFP or TcMT1 in the third larval instar (n = 6). (B) The
level of TcMT2 transcript 7 days after injection with dsRNA of
EGFP or TcMT2 in the third larval instar (n = 4). (C, D) The level of
TcMT3 transcript 3 days (C) and 6 days (D) after injection with
dsRNA of EGFP or TcMT3 on day 0 of the fourth larval instar
(day 0_4th; n = 4). (E) The level of TcMT1 transcript in pharate
adults after injection with dsRNA of EGFP or TcMT1 in the prepupal
stage (n = 3). (F) The level of TcMT2 transcript 3 days after injec-

tion with dsRNA of EGFP or TcMT2 on day 0 of the seventh larval
instar (day 0_7th; n = 3). Means and standard deviations are
shown, and the intensity in EGFP dsRNA-injected insects was set
at 100% in each graph.
Function of JHAMT gene in Tribolium C. Minakuchi et al.
2924 FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS
After injecting TcMT3 dsRNA and treating the lar-
vae with JHM at the beginning of the sixth instar, 13
insects (n = 16) either arrested at eclosion or eclosed
with the exuviae stuck on the elytra, whereas three
eclosed successfully into adults with pupal-like uro-
gomphi (data not shown). These phenomena may be
the result of the effect of residual methoprene, as simi-
lar defects were also observed in wild-type larvae trea-
ted with JHM.
Discussion
In this study, we performed expressional and func-
tional analyses of three methyltransferase genes
(TcMT1, TcMT2 and TcMT3) identified from T. cas-
taneum. Only TcMT3 was expressed strongly in the
larval putative CA, the primary organ for JH biosyn-
thesis. Recombinant TcMT3 protein methylated FA
and JHA III, bur recombinant TcMT1 and TcMT2
proteins did not. Furthermore, RNAi-mediated knock-
down of TcMT3 in the larval stage resulted in preco-
cious metamorphosis into a pupa, presumably because
of precocious shutdown of JH biosynthesis. These
results demonstrate that TcMT3 encodes a functional
JHAMT that is essential for JH biosynthesis. Hereaf-
ter, TcMT3 is called TcJHAMT.

TcJHAMT is expressed in a tissue-specific and
stage-specific manner
In both B. mori and D. melanogaster, JHAMT mRNA
was detected in large amounts in the larval CA [9,10].
In B. mori, JHAMT mRNA was detected in the third
and fourth larval instars, but decreased rapidly at the
beginning of the final (fifth) larval instar [9]. The
JHAMT transcript of D. melanogaster was abundant
in the larval stage, but was not detected in the pupal
stage or during most of adult development [10]. These
observations indicate that JHAMT is the key enzyme
Table 2. Phenotypes of Tribolium larvae injected with dsRNAs on day 0 of the third instar. Numbers of animals, and the instar when they
pupated, are indicated. The insects that underwent precocious metamorphosis are shown in bold.
dsRNA injection Pupation
Lethal phasedsRNA Concentration (lgÆlL
)1
) n 5th 6th 7th 8th
EGFP 1.5–2.0 22 – – 18 4
5.0 13 – – 12 1
TcMT1 1.5 6 – – 3 3
TcMT2 1.7 10 – – 4 6
TcMT3 1.5–2.0 15 – 12 2 – Pupal arrest (5th), 1
5.0 19 1142 – Prepupal arrest (5th), 1; pupal arrest (7th), 1
A
B
Fig. 7. Effects of TcMT3 RNAi-mediated knockdown in the larval
stage in Tribolium castaneum. The larval instar from which each
larva pupated is indicated in parentheses in each photograph.
(A) Ventral view of pupae that were injected with dsRNA of EGFP
or TcMT3 on day 0 of the third larval instar (day 0_3rd). Scale bar,

500 lm. (B) Dorsal and ventral views of adults injected with dsRNA
of EGFP or TcMT3 in the larval stage. Scale bar, 500 lm.
C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2925
determining the timing of larval–pupal metamorphosis
by controlling the rate of JH biosynthesis. In this
study, we analysed the spatial and temporal expression
patterns of a JHAMT orthologue in T. castaneum. The
TcJHAMT transcript was expressed in the embryonic
and larval stages, and decreased at the end of the final
larval instar (Fig. 2E,F). In addition, the TcJHAMT
transcript was detected specifically in the larval CA
(Fig. 4). Although the developmental profile of JH
titre has not yet been examined in T. castaneum, the
temporal expression profile of TcJHAMT may corre-
late with JH biosynthetic activity in CA as observed in
B. mori [17].
In B. mori, the BmJHAMT transcript is expressed
specifically in CA until the beginning of the final larval
instar [9]. In contrast, the BmJHAMT transcript is
undetectable in CA at the beginning of the spinning
stage, but is detected at low levels in the testis and
ovary [9]. In D. melanogaster, the DmJHAMT tran-
script is expressed very strongly in the larval CA, and
a small amount of DmJHAMT is also detected in the
testis of wandering third instar larvae [10]. In this
study, we found that the TcJHAMT transcript was
expressed exclusively in the putative CA of the sixth
instar (Figs 3C and 4), but the TcJHAMT transcript
was detected in both the anterior and posterior parts

of the body at the beginning of the prepupal stage
(Fig. 3F). These results suggest that TcJHAMT is
expressed in tissues other than CA in the prepupal
stage.
Quantitative RT-PCR analysis showed that the TcJ-
HAMT transcript exists in the prepupal stage.
Recently, Parthasarathy et al. [18] have reported that
the JH level in T. castaneum decreases just before
entrance into the quiescent (prepupal) stage, but
increases again during the prepupal stage. In the
Cecropia silkworm and the tobacco hornworm M. sex-
ta, JH reappears in the wandering stage just before
pupation, and removal of CA from the final larval
instar causes precocious adult differentiation of certain
imaginal structures [19,20]. Whether JH in the prepu-
pal stage of T. castaneum plays a role in preventing
precocious adult development needs to be examined.
TcJHAMT methylates FA and JHA III
We have shown that recombinant TcJHAMT protein
methylates (10R)-JHA III more favourably than
(10S)-JHA III. JHAMT of D. melanogaster has also
been reported to catalyse (10R)-JHA III preferentially
over the (10S)-enantiomer [10]. To date, the absolute
configuration of the chiral epoxide of natural JH III
has been reported to be 10R in the lepidopteran
M. sexta [21], coleopteran Tenebrio molitor [22] and
orthopterans Schistocerca vaga and Locusta migratoria
[23,24]. Although the chemical structure and stereo-
chemistry of JH in T. castaneum has not yet been elu-
cidated, it is probably the same as in other insect

species.
In JH biosynthesis, FA is converted into active JH
by methylation of the carboxyl group and epoxidation
at the C10–11 position. Biochemical studies using CA
homogenates from lepidopteran species suggest that
FA is epoxidized into JH acid first, and then JH acid
is methylated to JH [6,25]. In contrast, in other insect
orders, such as Orthoptera and Dictyoptera, biochemi-
cal studies indicate that FA is methylated to MF, and
then epoxidation occurs [6,26]. This observation is fur-
ther supported by a recent study that showed that
recombinant CYP15 protein of the cockroach Diplop-
tera punctata epoxidizes MF but does not epoxidize
FA [8]. In both D. melanogaster [10] and T. castaneum
(Table 1), recombinant JHAMT protein methylates
FA and JHA III at similar rates. Therefore, either
order of reactions is possible for the late steps in JH
biosynthesis in these species.
Table 3. Phenotypes of Tribolium larvae injected with 5.0 lgÆlL
)1
dsRNAs on day 0 of the fourth instar, and treated with a JH mimic. Num-
bers and percentages of animals, and the instar when they pupated, are indicated. The insects that underwent precocious metamorphosis
are shown in bold. Each larva was topically treated with 25 ng of methoprene (JHM) or the same volume of solvent as the control.
dsRNA
Hormonal treatment Number of pupae (%)
Compound Instar n 5th 6th 7th 8th
EGFP (No treatment) – 43 – – 30 (70) 13 (30)
Solvent 4th–5th 23 – – 21 (91) 2 (9)
JHM 4th–5th 23 – – 18 (78) 5 (22)
TcMT3 Solvent 4th–5th 19 – 16 (84) 2 (11) 1 (5)

JHM 4th–5th 21 – 8 (38) 13 (62) –
Solvent 6th 15 – 14 (93) 1 (7) –
JHM 6th 16 – 1 (6) 15 (94) –
Function of JHAMT gene in Tribolium C. Minakuchi et al.
2926 FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS
Functions of TcMT1 and TcMT2 genes
In this study, we have demonstrated that TcMT3
encodes a functional TcJHAMT gene. Although there
are two more putative methyltransferase genes
(TcMT1 and TcMT2) in the Tribolium genome, we
conclude that they do not catalyse the methylation
reaction in JH biosynthesis, because recombinant
TcMT1 and TcMT2 proteins do not methylate FA or
JHA III, and RNAi-mediated knockdown of TcMT1
or TcMT2 in larvae does not cause precocious larval–
pupal metamorphosis. As TcMT1, TcMT2 and TcJ-
HAMT are located in the same vicinity in the genome,
and the positions of the introns are very similar in the
three genes, they may have been derived through gene
duplication events. In contrast with the CA-specific
expression of the TcJHAMT transcript, the TcMT1
and TcMT2 transcripts are abundant in the posterior
part of the sixth instar larvae (Fig. 3A–C). Interest-
ingly, the temporal expression profiles of these three
methyltransferase genes are quite different (Fig. 2),
suggesting that the transcription of these genes may be
regulated by hormones or other unknown factors in
different ways.
At this point, the functions of TcMT1 and TcMT2
are unknown because the substrates for TcMT1 and

TcMT2 have not been identified. TcMT1 and TcMT2
have putative SAM-binding motifs, and therefore it is
likely that they methylate compounds with carboxyl
groups, such as aliphatic or aromatic carboxylic acids.
Further studies, such as in situ hybridization and
enzyme assays using a variety of candidate substrates,
are needed to elucidate the functions of TcMT1 and
TcMT2.
Significant role of TcJHAMT in the regulation
of JH biosynthesis and maintenance of the larval
status
In this study, we have shown that RNAi-mediated
knockdown of JHAMT in the larval stage causes pre-
cocious pupation. Importantly, this phenotype was res-
cued by the application of exogenous JHM, indicating
that precocious metamorphosis is caused by precocious
shutdown of JH biosynthesis. Therefore, we conclude
that the JHAMT gene is essential for JH biosynthesis,
and continuous expression in the larval stage is neces-
sary for the maintenance of the larval status. Although
the TcJHAMT transcript was suppressed significantly
3 days after dsRNA injection, i.e. day 0 of the fifth
larval instar (day 0_5th; Fig. 6C), precocious metamor-
phosis did not occur until the end of the sixth larval
instar in most cases. We assume that this time lag is
caused by a long half-life for the TcJHAMT protein.
Alternatively, it may take time for JH to be completely
eliminated from the hemolymph because enzymes such
as JH esterase and JH epoxide hydrolase are necessary
for the degradation of JH in the hemolymph and

tissues [27].
In some insect species, such as B. mori, it has been
reported that precocious larval–pupal metamorphosis
is caused by surgical removal of CA [28] or the appli-
cation of chemicals with anti-JH action, such as the
imidazole derivative KK-42 [29]. Recently, it has been
reported that overexpression of the JH esterase gene
in transgenic B. mori also results in precocious larval–
pupal metamorphosis, probably as a result of preco-
cious degradation of JH in the hemolymph [30]. As
demonstrated in this study, RNAi-mediated knock-
down of JH biosynthetic enzymes is a novel method
to induce precocious metamorphosis. Although preco-
cious metamorphosis can also be induced by the
injection of dsRNA of the Methoprene-tolerant (Met)
gene of Tribolium, probably a mediator of JH signals
[31], most larvae arrest as prepupae, probably because
Met function is necessary for normal pupation. In
contrast, JHAMT RNAi results in miniature pupae
and adults that appear normal in their external mor-
phology.
RNAi-mediated knockdown by the injection of
dsRNA into larvae or nymphs has also been reported
to be effective in other insect species, such as lacewings
[32], cockroaches [33–35] and milkweed bugs [36]. As
demonstrated in this study, the RNAi technique is par-
ticularly useful to suppress JH biosynthesis in small
insects for which it is extremely difficult to eliminate
JH by traditional surgical methods. We anticipate that
the RNAi technique will contribute to the elucidation

of the physiological functions of JH and the molecular
mode of JH action.
Materials and methods
Beetles
The wild-type strain of T. castaneum used in this study was
provided by the National Food Research Institute, Tsu-
kuba, Ibaraki, Japan. T. castaneum was raised in whole
wheat flour at 30 °C. To collect eggs, adult beetles were
kept in wheat flour for 1–3 days, and beetles and eggs were
separated using sieves. To stage the larvae, they were indi-
vidually raised in 24-well microtitre plates, and exuviae
were checked every day. T. castaneum larvae do not
develop synchronously: in our hands, they pupated either
at the seventh or eighth larval instar. To distinguish the
instar in which they pupate, the head capsule widths of
C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2927
early sixth and seventh instar larvae were measured using a
microscope [Leica Microsystems MZ16FA ⁄ DFC500 system
(Leica Microsystems, Heerbrugg, Switzerland)]. Larvae with
head capsule widths of 566 ± 20 lm (mean ± SD;
n = 30) in the sixth instar and 671 ± 22 lm(n = 37) in
the seventh instar pupated at the end of the seventh larval
instar. Larvae with head capsule widths of 529 ± 19 lm
(n = 7) in the sixth instar and 633 ± 22 lm(n = 7) in the
seventh instar pupated at the end of the eighth larval instar.
Approximately 83% of larvae (n = 81) pupated at the end
of the seventh larval instar, and 17% pupated at the end of
the eighth larval instar. To investigate the developmental
profile using quantitative RT-PCR, sixth instar larvae with

head capsules wider than 570 lm were considered as penul-
timate instar larvae, and seventh instar larvae with head
capsules wider than 690 lm were considered as final instar
larvae, and were used for RNA isolation.
cDNA cloning of methyltransferase genes
tblastn searches were performed using the beetle genome
database ( />with the sequences of B. mori and D. melanogaster JHAMT
proteins, and a contig (Contig4620_Contig8031) containing
three putative methyltransferase genes (TcMT1, TcMT2
and TcMT3) was identified. RT-PCR was performed to
amplify the ORF of TcMT1 (828 bp) by Advantage 2
DNA Polymerase (Clontech Laboratories, Mountain View,
CA, USA) with TcMT1_start and TcMT1_stop primers.
Similarly, the TcMT2 ORF (846 bp) was amplified with
TcMT2_start and TcMT2_stop primers, TcMT3 ORF
(834 bp) with TcMT3_start and TcMT3_stop primers, and
TcRp49 ORF (402 bp) with TcRp49_start and TcRp49_
stop primers. It should be noted that the recognition site of
the NdeI restriction enzyme was added to the 5¢-end of
TcMT1_start, TcMT2_start and TcMT3_start primers. The
PCR products were subcloned into a pGEM-T vector
(Promega Corporation, Madison, WI, USA). The DNA
sequence data of TcMT1, TcMT2 and TcMT3 (TcJHAMT )
were deposited in GenBank (accession numbers: AB360761
for TcMT1, AB360762 for TcMT2 and AB360763 for
TcJHAMT). The sequences of the primers are listed in
supplementary Table S1.
Quantitative RT-PCR analysis
The TcMT1, TcMT2 and TcMT3 transcripts were quanti-
fied using a real-time thermal cycler (LightCycler 2.0, Roche

Diagnostics, Basle, Switzerland). Total RNA was isolated
from the whole body of T. castaneum using an RNeasy Plus
Mini Kit (Qiagen, Valencia, CA, USA). To analyse the
developmental expression profile, several insects were com-
bined for RNA isolation of the embryonic stage and the
first, second and third larval instars, whereas RNA was iso-
lated from individuals for the sixth and seventh larval in-
stars, pupal and adult stages. To examine the tissue
specificity of these genes in the sixth and seventh instars (at
84 h after ecdysis for the seventh instar), four larvae were
cut in half between thoracic segments T2 and T3, and ante-
rior and posterior parts were collected separately for RNA
isolation. cDNAs were synthesized with an oligo(dT)
18
pri-
mer and M-MLV reverse transcriptase (Clontech Laborato-
ries). Quantitative RT-PCR was carried out in a 20 lL
reaction volume containing SYBR Premix Ex Taq (Takara
Bio, Shiga, Japan), 0.2 lm of each primer and 2–3 lLof
template cDNAs or standard plasmids. PCR conditions
were 95 °C for one 10 s cycle, followed by 40–50 cycles at
95 °C for 5 s and 60 °C for 20 s. The primers used for quan-
tification are listed in supplementary Table S1. After PCR,
the absence of unwanted byproducts was confirmed by melt-
ing curve analysis. For standards, serial dilutions of a plas-
mid containing the ORF of each gene were used. TcRp49
was used as a reference gene. Transcript levels of TcMT1,
TcMT2 and TcMT3 were normalized with TcRp49 in the
same samples. For each gene, the highest intensity in the
developmental expression profile (Fig. 2) was set as 100%.

In situ hybridization
In situ hybridization was carried out according to a method
reported for Drosophila brains [37]. The full coding region
of TcMT3 was subcloned into a pGEM-T vector, and a lin-
earized plasmid was used as the template for RNA synthe-
sis. Digoxigenin (DIG)-labelled sense and antisense RNA
probes were prepared using a DIG RNA Labelling Kit and
SP6 or T7 RNA polymerase (Roche Applied Science,
Mannheim, Germany), according to the manufacturer’s
instructions. Heads of sixth instar larvae were dissected in
NaCl ⁄ P
i
, and most of the head capsules were carefully
removed with forceps. Tissues were fixed in 4% parafor-
maldehyde at 4 °C for 40 min, and treated with 5 lgÆmL
)1
Proteinase K for 75 s. Re-fixation, hybridization and detec-
tion with pre-adsorbed, alkaline phosphate-conjugated
anti-DIG FAB fragments and nitroblue tetrazolium ⁄ 5-
bromo-4-chloroindol-2-yl phosphate (Roche Applied Sci-
ence) were performed as described previously [37,38]. After
hybridization and detection, the remaining head capsule,
fat body and muscles were carefully removed with forceps,
so that the brain and CA could be seen well.
Preparation of recombinant proteins
and enzyme assays
Full-length ORFs of TcMT1, TcMT2 and TcMT3 cloned
into the pGEM-T vector described above were excised with
NdeI and NotI restriction enzymes and subcloned into
pET28a(+) expression plasmid vector (Novagen, Madison,

WI, USA) that was linearized with the same restriction
enzymes. The resulting constructs, TcMT1⁄ pET28a(+),
TcMT2 ⁄ pET28a(+) and TcMT3 ⁄ pET28a(+), were used
Function of JHAMT gene in Tribolium C. Minakuchi et al.
2928 FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS
individually to transform the Escherichia coli BL21(DE3)
strain to express N-terminal 6· His-tagged recombinant
TcMT1, TcMT2 and TcMT3 proteins, respectively. Expres-
sion and purification procedures were essentially the same
as described previously [9]. After adding glycerol (final con-
centration, 25%), the purified protein aliquots were frozen
in liquid nitrogen and stored at )80 °C until further analy-
sis. Under these conditions, no obvious decrease in enzy-
matic activity was observed for at least 1 year.
Enzymatic analyses were performed essentially as
described previously [9,10]. Briefly, purified recombinant
methyltransferases were incubated individually in 500 lLof
50 mm Tris ⁄ Cl buffer (pH 7.5) containing SAM (500 lm)
and one of the following acids as a substrate: FA
(50 lm), racemic JHA III (50 lm), LA (100 lm)orPA
(100 lm). After 5–60 min of incubation at 25 °C, the reac-
tions were stopped by the addition of 500 lLofCH
3
CN
and vortexing. Incubation times were adjusted so as not to
consume more than 15% of the initial substrates. To ana-
lyse the generation of methylated FA (MF) and methylated
JHA III (JH III), after removing the precipitate from the
samples by centrifugation, the supernatants were analysed
directly by RP-HPLC [10]. To analyse the methyl esters

produced from LA and PA, the supernatants were
extracted with hexane-containing 5 lgÆmL
)1
methyl tride-
canoate as an internal standard and analysed by GC-MS
[10]. To analyse the selectivity of recombinant TcMT3 pro-
tein between (10R)- and (10S)-enantiomers of JHA III,
TcMT3 protein was incubated with racemic JHA III for
10 min, and the configuration of the metabolites extracted
with hexane was analysed further by enantioselective HPLC
with a ChiralPak IA column (250 mm · 4.6 mm inside
diameter; Daicel, Osaka, Japan), as described previously
[39].
RNAi experiments
Template DNA fragments for the synthesis of dsRNA were
prepared by PCR as follows. A DNA fragment containing
the 828 bp TcMT1 ORF with a T7 promoter sequence at
the N-terminal end was amplified with TcMT1_start_T7
and TcMT1_stop primers to synthesize sense RNA, and a
fragment containing the TcMT1 ORF with a T7 promoter
sequence at the C-terminal end was amplified with
TcMT1_start and TcMT1_stop_T7 primers to synthesize
antisense RNA. RNA was synthesized using the T7 Ribo-
MAX Express RNAi System (Promega Corporation). The
in vitro transcription reaction was carried out at 37 °C for
1 h with T7 RNA polymerase, and the template DNA was
digested by DNase I. Both strands of TcMT1 RNA were
mixed and incubated at 70 °C for 10 min, and were gradu-
ally cooled to room temperature for annealing. dsRNA in
the mixture was then purified by ethanol precipitation and

dissolved in DNase ⁄ RNase-free water. To synthesize
dsRNA for TcMT2, TcMT3 and EGFP, DNA fragments
with T7 promoter sequences on both ends were amplified
by PCR as follows: a fragment containing a 368 bp gene-
specific region of TcMT2 (positions 1–368 in TcMT2 ORF)
with T7 promoter sequences on both ends was amplified
with TcMT2_start_T7 and TcMT2_3¢_T7 primers, and a
fragment containing a 359 bp TcMT3 sequence (positions
1–359 in TcMT3 ORF) with T7 promoter sequences on
both ends was amplified with TcMT3_start_T7 and
TcMT3_3¢_T7 primers. The template for EGFP dsRNA
(720 bp) was prepared with EGFP-F-T7 and EGFP-R-T7
primers and a plasmid containing the EGFP sequence
(pEGFP, Clontech Laboratories). In vitro transcription was
carried out at 37 °C for 1 h with T7 RNA polymerase, so
that both strands would be synthesized simultaneously. The
reaction mixture was then treated with DNase I, annealed
and purified by ethanol precipitation. The primer sequences
are listed in supplementary Table S1.
To knockdown genes in the larval stage, third or
fourth instar larvae within 24 h after ecdysis were anaes-
thetized with ether for 3 min, aligned on double-sided
tape, and dsRNA solution (approximately 20 nL for the
third instar and 30–40 nL for the fourth instar) was
injected into the abdomens at a concentration of 1.5–
5.0 lgÆlL
)1
using a capillary tube pulled by a Narishige
needle puller. To examine the efficiency of RNAi-medi-
ated knockdown, total RNA was isolated individually

from dsRNA-injected larvae several days later for quanti-
tative RT-PCR. To knockdown TcMT1 in adult develop-
ment, prepupae were injected with about 150 nL of
2.5 lgÆlL
)1
TcMT1 dsRNA, and three pharate adults
were homogenized individually for quantification of the
TcMT1 transcript levels 6 days later. To knockdown
TcMT2 in the final larval instar, newly moulted final
instar larvae were injected with approximately 150 nL of
1.7 lgÆlL
)1
TcMT2 dsRNA, and three larvae were
homogenized individually for quantification of TcMT2
levels 3 days later. EGFP dsRNA was injected as a con-
trol. After injection, insects were reared individually in
24-well microtitre plates at 30 °C.
Hormonal treatments
Methoprene (SDS Biotech, Tokyo, Japan) was dissolved in
methanol to 0.31 lgÆlL
)1
(1 mm), and this stock solution
was diluted to 0.19 lgÆlL
)1
with acetone. Larvae that were
injected with dsRNA on day 0 of the fourth larval instar
(day 0_4th) were anaesthetized with ether for 2.5–3 min,
aligned on double-sided tape, and approximately 130 nL
of 0.19 lgÆ lL
)1

methoprene solution (containing approxi-
mately 25 ng of methoprene) was topically applied on
the dorsum using a 10 lL Hamilton microsyringe, on
day 3_4th, day 0_5th or day 0_6th. The same volume of
solvent was applied as a control. After hormonal treat-
ment, larvae were individually reared in 24-well microtitre
plates.
C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2929
Acknowledgements
We thank Professor L. M. Riddiford (Janelia Farm,
Howard Hughes Medical Institute, Ashburn, VA,
USA), Professor D. Taylor (University of Tsukuba,
Japan) and Professor H. Wojtasek (University of
Opole, Poland) for critical comments on the manu-
script, Dr A. Miyanoshita and Dr M. Murata
(National Food Research Institute, Tsukuba, Ibaraki,
Japan) for providing T. castaneum, Dr Y. Tomoyasu
and Dr Y. Arakane (Kansas State University, Man-
hattan, KS, USA) for technical advice on RNAi
experiments, and Professor S. Sakurai (Kanazawa
University, Japan) for kindly providing methoprene.
C. M. was supported by a research fellowship from
the Japan Society for the Promotion of Science
(JSPS). This work was supported by the Program
for Promotion of Basic Research Activities for Inno-
vative Biosciences (PROBRAIN).
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Supplementary material
The following supplementary material is available
online:
Table S1. Oligonucleotide primers used in RT-PCR
for ORF amplification, quantitative RT-PCR and
amplification of template DNA for dsRNA synthesis.

This material is available as part of the online article
from
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C. Minakuchi et al. Function of JHAMT gene in Tribolium
FEBS Journal 275 (2008) 2919–2931 ª 2008 The Authors Journal compilation ª 2008 FEBS 2931