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

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 the

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: shinoda@affrc.go.jp

*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 moriand 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 TcMT3in 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.

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

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 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 methylO-methyl-transferases 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.

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.

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 beginninject-ing 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

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 larlar-vae 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.

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.

TcMT1dsRNA 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))1Æmin)1]

0

500

A

B

100

0

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.

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.

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 simi-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.

Lethal phase

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.

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)1dsRNAs 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

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-HAMTare 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 30C 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

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 (http://www.bioinformatics.ksu.edu/BeetleBase/)

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 posteante-rior 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 lL of template cDNAs or standard plasmids PCR conditions were 95C for one 10 s cycle, followed by 40–50 cycles at

95C 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 situhybridization 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⁄ Pi, and most of the head capsules were carefully removed with forceps Tissues were fixed in 4% parafor-maldehyde at 4C 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