Juvenile hormone resistance gene Methoprenetolerant controls entry into metamorphosis
in the beetle Tribolium castaneum
Barbora Konopova* and Marek Jindra†‡
*Department of Molecular Biology, University of South Bohemia, and †Biology Center, Czech Academy of Sciences, 37005 Ceske Budejovice, Czech Republic
Edited by David L. Denlinger, Ohio State University, Columbus, OH, and approved May 1, 2007 (received for review April 23, 2007)
Besides being a spectacular developmental process, metamorphosis is key to insect success. Entry into metamorphosis is controlled
by juvenile hormone (JH). In larvae, JH prevents pupal and adult
morphogenesis, thus keeping the insect in its immature state. How
JH signals to preclude metamorphosis is poorly understood, and a
JH receptor remains unknown. One candidate for the JH receptor
role is the Methoprene-tolerant (Met) Per-Arnt-Sim (PAS) domain
protein [also called Resistance to JH, Rst (1)JH], whose loss confers
tolerance to JH and its mimic methoprene in the fruit fly Drosophila
melanogaster. However, Met deficiency does not affect the larval–
pupal transition, possibly because this process does not require JH
absence in Drosophila. By contrast, the red flour beetle Tribolium
castaneum is sensitive to developmental regulation by JH, thus
making an ideal system to examine the role of Met in the antimetamorphic JH action. Here we show that impaired function of
the Met ortholog TcMet renders Tribolium resistant to the effects
of ectopic JH and, in a striking contrast to Drosophila, causes
early-stage beetle larvae to undergo precocious metamorphosis.
This is evident as TcMet-deficient larvae pupate prematurely or
develop specific heterochronic phenotypes such as pupal-like cuticular structures, appendages, and compound eyes. Our results
demonstrate that TcMet functions in JH response and provide the
critical evidence that the putative JH receptor Met mediates the
antimetamorphic effect of JH.
insect metamorphosis 兩 postembryonic development 兩
endocrine regulation 兩 bHLH-PAS domain 兩 RNAi
M
etamorphosis is a marked change between larval and adult
forms that occurs in all winged insects (1, 2). Holometabolous larvae metamorphose into adults by a stage termed the pupa.
More than 70 years ago, V. B. Wigglesworth discovered that the
entry into metamorphosis depends on juvenile hormone (JH) (3, 4).
In larvae, JH prevents the steroid molting hormones (ecdysteroids)
from initiating metamorphosis, so that after a molt another larval
stage follows (5, 6). Therefore, in most species, supply of JH makes
an insect reiterate its juvenile stage, whereas experimental removal
of JH causes it to metamorphose prematurely (1, 7–12). An
exception from this paradigm is the ‘‘higher’’ (cyclorrhaphous)
Diptera including the fruit fly, Drosophila melanogaster, where
exogenous JH cannot induce extra larval stages (13). In Drosophila,
ectopic JH blocks only adult differentiation of the abdominal
histoblasts, whereas the rest of the adult body that derives from
imaginal discs is insensitive to JH (14–17).
The mode of JH action remains an enigma, because neither its
receptor nor a signaling pathway is known (6, 18–20). In 1986,
Wilson and Fabian reported Drosophila mutants that survive
toxic doses of JH or its mimic methoprene (21). The Methoprenetolerant (Met) gene [also called Resistance to juvenile hormone,
Rst (1)JH] encodes a transcriptional regulator of the basic
helix–loop–helix (bHLH)- Per-Arnt-Sim (PAS) domain family
(22). Met has been shown to bind JH at physiological concentrations (23, 24) and therefore is a good candidate for the elusive
JH receptor (6, 25–27).
10488 –10493 兩 PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25
However, the critical evidence that Met is required for the
antimetamorphic JH function is missing, because Met-null mutants are fully viable (27–29). A likely reason for the lack of an
expected developmental phenotype is the weak effect of JH on
preadult Drosophila. Another explanation might be a potential
functional redundancy between Met and a paralogous Drosophila
gene germ-cell expressed (gce) (30, 31), whose protein product can
dimerize with Met in a JH-sensitive manner (26).
The beetle Tribolium castaneum shows the classical developmental response to JH, which can cause repetition of juvenile stages (ref.
32 and this work). Moreover, the recently available genome information indicates that the beetle possesses a single ortholog of
Drosophila Met and gce. Tribolium therefore offers an ideal opportunity to examine the role of Met in metamorphosis without the
complication of redundancy and the atypical JH response. Here, we
show that depletion of Met renders Tribolium resistant to JH, and
that in beetle larvae it causes premature pupal morphogenesis.
These results establish Met as an essential mediator of the antimetamorphic JH signal and support its putative receptor role.
Results
Met Is Conserved and Constitutively Expressed in Tribolium. Based on
the T. castaneum genome database and using rapid amplification
of cDNA ends, we have isolated a presumably full-length copy of
a transcript that encodes 516 amino acids. The deduced protein
is identical to a previously annotated Tribolium protein
(XM㛭961449), except that its N-terminal sequence is 69 amino
acids shorter than in the GenBank entry. Our ORF starts with
the Kozak translation start site consensus ACCAUGG and is
preceded by a stop codon. The protein contains the bHLH
domain, two PAS domains (A and B), and a PAS C-terminal
motif PAC (Fig. 1A) that are all conserved with D. melanogaster
Met (22), DmGce, and proteins from mosquitoes (31) and the
honey bee [see supporting information (SI)]. In its bHLH and
PAS-B (but not PAS-A and PAC) domains, the Tribolium
protein appears more similar to DmGce than DmMet. The
number and positions of Tribolium introns are better conserved
with those of Dmgce and the Aedes aegypti genes than with the
single intron in DmMet (Fig. 1 A and ref. 31). Similar to
mosquitoes and the honey bee, the Tribolium genome apparently
Author contributions: B.K. and M.J. designed research; B.K. and M.J. performed research;
B.K. and M.J. analyzed data; and B.K. and M.J. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: JH, juvenile hormone; Met, Methoprene-tolerant; bHLH, basic helix–loop–
helix domain; PAS, Per-Arnt-Sim domain.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. EF468474).
See Commentary on page 10297.
‡To
whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/
0703719104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703719104
contains only one counterpart of Dmgce and DmMet. Our
phylogenetic analysis confirms a previous notion that Dmgce and
DmMet are paralogs that arose by a lineage-specific duplication
in higher Diptera (31) and shows that the Tribolium gene is a
single ortholog of Drosophila gce and Met (see SI). Because of its
functional similarity to Met (see below) and in keeping with the
literature, we will refer to the Tribolium gene as TcMet.
To find out whether TcMet is transcriptionally regulated in
diverse developmental stages, we analyzed its expression by
using RT-PCR (Fig. 1B). The transcript was detected in all stages
examined: embryos, fifth instar larvae, prepupae (i.e., immobile
pharate pupae that displayed a crooked posture), pupae, and
adults. Thus, at the level of mRNA TcMet seems to be continuously expressed throughout beetle development.
TcMet Silencing Confers Insensitivity to Ectopic JH. To establish
whether TcMet mediates JH response in the beetle as Met does
in Drosophila, we have tested for resistance against the hormone
and its mimic methoprene in TcMet(RNAi) Tribolium pupae. It
has been well documented that upon exposure to JH analogs,
pupae of a closely related beetle Tenebrio molitor, will produce
Fig. 2. TcMet knockdown provides resistance against a potent JH mimic
methoprene during pupal stage. (A) Topical application of methoprene
(0.3-mM solution) to a freshly ecdysed control pupa that had been injected
with egfp dsRNA before pupation induced formation of an abnormal second
pupal stage and death. The tip of the abdominal pupal cuticle was peeled off
(from the line indicated by the arrowhead) to reveal a second pupal cuticle
underneath it and a second set of pupal urogomphi (arrow). (B and C)
TcMet(RNAi) pupae after the same methoprene treatment developed into
apparently normal adults that either eclosed (B) or died unable to shed the
pupal cuticle (C). (Scale bar: 1 mm.)
a supernumerary (second) pupa instead of an adult (33–35).
When we treated early Tribolium pupae with methoprene or with
JH naturally occurring in beetles (JH-III), nearly all of the
animals died after molting to a second pupal stage, and none
emerged as adult (Table 1 and Fig. 2A). Geraniol and farnesol,
compounds that are structurally related to JH but lack JH
activity, had no such effect even at much higher doses (Table 1).
Strikingly, RNAi knockdown of TcMet was sufficient to neutralize the action of methoprene and JH-III on pupae (Fig. 2 and
Table 1). TcMet dsRNA was injected into early prepupae. This
late treatment caused no lethality while reducing TcMet mRNA
level at the beginning of the pupal stage (Fig. 1C). The newly
ecdysed TcMet(RNAi) pupae were given JH-III or methoprene
as previously. Only 2% of the JH-treated TcMet(RNAi) individuals arrested as second pupae, whereas 68% molted to normal
viable adults (Table 1). The remaining 30% died as pharate
adults just before imaginal eclosion, a phenotype that was likely
caused by the silencing of Met itself rather than by JH treatment,
because ecdysis defects were also frequent in TcMet(RNAi)
acetone controls (Table 1). Thus, depletion of TcMet was able
Table 1. TcMet RNAi confers resistance to JH-III and methoprene in Tribolium
dsRNA
egfp
TcMet
None
Treatment*
n
Second pupae
Uneclosed adults
Normal adults
Methoprene (0.3 mM)
Methoprene (0.03 mM)
JH-III (0.3 mM)
Acetone
Methoprene (0.3 mM)
Methoprene (0.03 mM)
JH-III (0.3 mM)
Acetone
Geraniol (57 mM)
Geraniol (0.6 mM)
Farnesol (40 mM)
Farnesol (0.4 mM)
20
8
41
33
26
6
47
11
35
15
31
15
20
8
38
—
4
1
1
—
—
—
—
—
—
—
3
—
3
—
14
6
—
—
—
—
—
—
—
33
19
5
32
5
35
15
31
15
*Early seventh- or eighth-instar prepupae were injected with dsRNA and allowed to pupate, then, within 0 –12
h after ecdysis, briefly dipped in acetone or acetone-diluted compounds.
Konopova and Jindra
PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10489
DEVELOPMENTAL
BIOLOGY
Fig. 1. Structure and expression of Tribolium Met. (A) Schematic representation of Tribolium Met as compared with orthologous proteins from A.
aegypti (Aa) and D. melanogaster (Dm). Numbers indicate amino acid identities within the conserved domains. Black arrowheads mark intron positions
in all four respective genes. See also SI for detailed alignment and phylogenetic analysis. (B) Total RNA prepared from staged embryos or from 4 to 11
larvae, prepupae (i.e., pharate pupae), pupae, and adults was treated with
DNase and assayed for TcMet expression by using RT-PCR. Contamination with
genomic DNA was excluded by PCR on samples processed without reverse
transcriptase (not shown). In addition, the TcMet primers flanked a 4.4-kb
intron. Numbers denote age (in days) of the indicated stages. F, female; M,
male. (C) Early prepupae were injected with either TcMet or egfp dsRNA and
allowed to pupate. Reduced TcMet expression is shown in two such individual
pupae (a, b) aged 0 –12 h after ecdysis. A similar transcript depletion was seen
after dsRNA injection in larvae (not shown). Expression of rp49 served for
control.
C
B
SEE COMMENTARY
A
A
B
C
D
E
F
Fig. 3. Silencing of TcMet in Tribolium larvae induces premature metamorphosis. (A) Summary of the fate of animals injected with dsRNA during third to fifth
instars (L3–L5). Numbers in gray box indicate how many larval instars (since hatching) occurred either before the animals died or before they reached adulthood.
Whereas 7 and 8 represent the normal count of larval instars, the numbers 5 and 6 imply precocious metamorphic development, manifest as prothetelic larvae
(blue; for phenotypes, see Fig. 5), prepupae (pink; for phenotypes, see Fig. 4), or pupae (yellow), all of which were lethal. Examples of premature pupation (yellow)
after TcMet dsRNA injection in the third and fourth instars are depicted in C, D, and F, respectively. Larvae injected with egfp dsRNA pupated in the seventh (E)
or eighth instar (B). Some of the TcMet(RNAi) premature pupae did not ecdyse properly (C) and had larval exuvia attached along the ventral side (arrows).
Arrowheads indicate formation of compound eyes. (Scale bar: 1 mm.)
to rescue 98% of pupae (n ⫽ 47) from the JH-induced reiteration
of pupal development. Similar results were obtained with methoprene (Fig. 2 and Table 1). The increased incidence of second
pupae in methoprene-treated TcMet(RNAi) animals was likely
due to the fact that methoprene is metabolically nondegradable
and therefore more potent than JH-III.
Our data clearly demonstrate that TcMet knockdown renders
Tribolium pupae insensitive to natural JH as well as to its analog
methoprene. This finding suggests a developmental role for
TcMet in JH signaling.
Loss of TcMet Causes Premature Pupation. We next asked whether
TcMet mediates the antimetamorphic function of JH, which is
manifested as extranumerary larval stages instead of pupation in
T. castaneum (see SI and ref. 32). Conversely, experimental
depletion of JH in many insects including beetles (11, 36) induces
premature pupation, and exactly this phenotype would be expected upon TcMet knockdown if JH prevented precocious
metamorphosis by TcMet. Under our breeding conditions Tribolium larvae normally pass through seven or (less frequently)
eight instars before they pupate.
Injection of TcMet dsRNA into larvae at the beginning of the
third and fourth instars caused precocious metamorphosis (Fig.
3). Most notably, RNAi triggered during the third instar induced
pupation of four of 52 animals at the end of the fifth instar (Fig.
3 A, C, and D). Similarly, silencing of TcMet from the beginning
of the fourth instar caused 22% of the larvae to pupate early,
usually at the end of their sixth instar (Fig. 3 A and F). Some of
the minute pupae resulting from these experiments could not
completely ecdyse from their larval cuticles and soon died (Fig.
3C), whereas others survived for several days, forming superficially perfect yet eventually lethal pharate adults that were
10490 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703719104
unable to eclose (Fig. 3F). When TcMet dsRNA was diluted
2-fold before injection, we occasionally obtained miniature adult
beetles from sixth instar pupae (not shown). By contrast to TcMet
RNAi, no signs of precocious metamorphosis were seen in
controls that had been injected with egfp dsRNA as third to fifth
instar larvae (Fig. 3 A, B, and E). On average, 91% of these
controls developed to adults normally (Fig. 3A), except that
relative to noninjected animals, their pupation was often delayed
until the eighth instar, presumably in response to the injury.
Because the same injury occurred also in the injected TcMet(RNAi) larvae, their pupation might have been early not by
one or two but rather by two or three molts. In addition to early
metamorphic phenotypes (see also below), TcMet(RNAi) animals at various stages also suffered ecdysis defects (Figs. 2C, 3C,
and 4A). Even larvae that were injected early in the final
(seventh) instar and that pupated at the right stage died, being
unable to shed the larval or pupal cuticles (not shown). Disrupted ecdysis might thus be a second effect of TcMet deficiency.
On average, 46% of larvae that had been injected with TcMet
dsRNA in their third and fourth instars, and nearly all of those
injected in the fifth instar, were unable to shed the larval cuticle
and died as undersized prepupae (pharate pupae) during the
next, i.e., the fifth (Figs. 3A and 4) or sixth instar (Fig. 3A).
Before death, these animals displayed a wandering-like behavior
that is typical of the final larval instar (37), and premature
compound eye development became visible underneath their
apolysed larval cuticle (Fig. 4A). To provide further evidence
that these TcMet(RNAi) animals were true pharate pupae, two
of those that had arrested at the fifth instar were removed from
the larval cuticle and subjected to scanning electron microscopy.
As expected, both of them showed typical pupal hallmarks such
as the presence of wings, gin traps, and pupal genital papillae
Konopova and Jindra
B
Fig. 4. Most TcMet(RNAi) larvae are unable to ecdyse and arrest as premature prepupae (see Fig. 3A, pink). (A) Arrested fifth instar TcMet(RNAi) prepupa and a control (egfp dsRNA) eighth instar prepupa are shown; both had
been injected with dsRNAs as early-third instar larvae. Note the smaller body
size and advanced eye pigmentation (arrow) in TcMet(RNAi) prepupa. (B) The
premature TcMet(RNAi) prepupa from A after it had been removed from the
larval cuticle bears typical pupal features including wings (asterisks), gin traps
(arrowheads and Inset), and pupal (female) genital papillae (arrow). (Scale
bars: A, 1 mm; B, 500 ␮m.)
(Fig. 4B). Thus, up to 67% of the third-to-fifth instar larvae
injected with TcMet dsRNA form premature pupae, although
most of them fail to ecdyse.
Prothetely Marks Precocious Metamorphosis in TcMet(RNAi) Animals.
Approximately 44% and 21%, respectively, of the larvae injected
in third and fourth instars arrested after two or three molts (Fig.
3A). During the instar in which they arrested, the larvae did not
feed, sometimes had reduced pigmentation, and finally died
within 1 or 2 days after ecdysis, occasionally with pieces of old
(previous instar) cuticle attached to their legs or mouthparts.
Despite their overall larval appearance, many of these animals
displayed a mix of pupal characters (prothetely) (Fig. 5). Some
had larva-like body only with prematurely advanced development of antennae and eyes (Fig. 5 K and L) or wings of various
sizes (Fig. 5M). Other pupal characters in these larvae included
gin traps, elongated urogomphi, or pupal genital papillae (compare Fig. 5 N and O with D and I and with E and J, respectively).
We also noticed elongated legs with double claws and reduced
number of sensillae (not shown). Taken together, our observations reveal that silencing of TcMet in young Tribolium larvae
induces precocious pupal morphogenesis.
Discussion
How JH controls metamorphosis at the genetic and molecular
level is poorly understood, mainly because neither a JH receptor
nor downstream components of JH signaling have been clearly
identified. Genetic studies on JH action have been hampered by
the fact that the highly evolved Drosophila metamorphosis is only
marginally sensitive to JH (16, 17). Thus, although Drosophila
has been an excellent system for disclosing important genes such
Konopova and Jindra
Methods
Tribolium Breeding and Staging. Wild-type T. castaneum (strain
San Bernardino) beetles were maintained on flour–yeast diet at
32°C in constant darkness. Eggs were collected by sifting, and
larvae were kept on food in Petri dishes until the desired stage.
Because second to final instar larvae cannot be distinguished by
obvious morphological markers, staging into instars was based
on the number of undergone ecdyses. Under constant condiPNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10491
SEE COMMENTARY
as Met, it has not revealed its role in the fundamental function
of JH: maintaining the status quo in juvenile insects.
In this study, we show that Tribolium Met mediates JH
response and is required in the beetle larvae for the proper
timing of entry to the metamorphic pupal program. Silencing of
TcMet produced two major lines of evidence. First, by making
pupae resistant to high doses of JH-III and methoprene, it
established that the single Tribolium ortholog of the Met/gce
genes has a similar function to Met. The function of gce is
presently unknown, and future studies in Drosophila should yield
interesting comparisons. Second, deficiency of TcMet in young
larvae induced premature pupation and other heterochronic
phenotypes that mark precocious metamorphosis. The presence
of prothetelic larvae likely reflected temporal differences of
diverse tissues in the acquisition of the pupal fate and/or their
uneven sensitivity to TcMet RNAi. Both variables might bear
some influence, because pupal characters in these larvae did not
appear in an exact sequence, and individual animals displayed
different sets of phenotypes. Generally, however, unlike the
appendages, eyes, or wings, the thoracic and abdominal cuticle
often retained its larval character, suggesting that, as in other
insects (38, 39), Tribolium epidermis becomes pupally committed later than these organs.
Premature metamorphosis can be elicited in many insects by
removal of the JH-producing corpora allata gland. However,
when such ablation is carried out in very young larvae, one or two
additional ecdyses are usually necessary before signs of metamorphosis appear (2, 7, 10). Depletion of JH via continuous
overexpression of JH esterase also fails to cause premature
pupation in silkworm larvae that are younger than third instar
(12), suggesting that insect tissues require certain time of
postembryonic development before acquiring the competence to
metamorphose in response to ecdysteroid in the absence of JH.
These findings help explain why in our experiments metamorphic changes typically followed two or three molts after injection
of TcMet dsRNA. Another reason for this delay may be a time
lag before sufficient depletion of the protein.
How might TcMet prevent metamorphosis in response to JH?
Drosophila Met has been linked with JH in several ways. Met
mutants tolerate harmful effects of JH (21, 28), and their tissues
display reduced affinity to JH (23). The recombinant Met
protein has been shown to bind JH and to stimulate transcription
in response to JH-III (24). Finally, Met can form homodimers
and heterodimers with Gce in the absence of JH (26). Met thus
presents the best known candidate for the JH receptor role.
bHLH-PAS domain proteins are particularly interesting signaling molecules, because many of them act as sensors of external
stimuli such as hypoxia, xenobiotics or hormones (reviewed in
ref. 40). At this point, we have no biochemical evidence for JH
binding and JH-dependent transactivation by TcMet; however,
our results encourage such studies.
The work presented here discloses the long-elusive role of Met
in the JH-controlled process of entry into metamorphosis. This
evidence supports the hypothesis that Met might serve as a JH
receptor or its essential constituent. Further genetic studies in
Tribolium should address interactions of TcMet with other
signaling pathways and ultimately improve our understanding of
insect metamorphosis and its hormonal regulation.
DEVELOPMENTAL
BIOLOGY
A
A
F
K
B
G
L
C
D
E
H
M
I
N
J
O
Fig. 5. TcMet RNAi causes larval prothetely. TcMet dsRNA was injected at the beginning of the third or fourth larval instar. Shown are examples of heterochronic
pupal characters in several different TcMet(RNAi) individuals. When compared with wild-type larvae (A–E) and pupae (F–J), TcMet(RNAi) larvae (K–O) display
pupa-like antennae (K, an) and eyes (L). Wings (arrows in H and M) and gin traps (gt) appear in normal pupae and TcMet(RNAi) animals (compare H with M and
I with N) but are absent in larvae (see their presumptive positions marked with asterisks in C and D). Urogomphi at the distal abdomen of TcMet(RNAi) animals
are elongated like those in pupae (arrows, compare O with E and J). Larval pygopods (E, py) disappear, whereas genital papillae on the ninth abdominal segment
develop (arrowheads in J and O, females shown). The larva in A is of the second instar, and larvae in B–E are of the seventh instar. Anterior is to the left in all
images; I is a dorsal and N a ventral view. (Scale bars: B, C, G, H, L, and M, 500 ␮m; D–F and I–K, 200 ␮m; O, 100 ␮m; and A and N, 50 ␮m.)
tions, larvae developed at a similar rate, and successive ecdyses
thus took place at roughly constant time points since hatching.
Therefore, ecdysis into the desired instar was assessed from the
timetable of ecdyses that had been prepared as follows: eggs
from a 6-h egg collection were placed individually into wells of
a cell culture plate and checked daily to mark the time of
hatching and of each ecdysis.
Isolation of Tribolium Met cDNA. Conserved region of TcMet spanning the bHLH through PAC domains was identified in the
Tribolium genome database (www.bioinformatics.ksu.edu/
BeetleBase) by using TBLASTN search with the Drosophila Met
protein sequence. A cDNA of 2,710 bp was obtained by using
RACE (Invitrogen) from embryonic and larval RNA with two
gene-specific primers for 5⬘RACE (5⬘-TGGCACTTCTTGCGACCATC-3⬘ and nested 5⬘-TGTCCCTTCGCATCTTTTCC-3⬘) and
two for 3⬘RACE (5⬘-ACCACGACCGCCTGCGTATG-3⬘ and
nested 5⬘-GAAGCTTCAAGAGAGGAATATG-3⬘). The fulllength transcript was compared with the genome database to
identify intron positions.
10492 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703719104
mRNA Expression Analysis. Total RNA was isolated from decho-
rionated eggs, whole larvae, pupae, or adults by using TRIzol
(Invitrogen, Carlsbad, CA). After treatment with DNase
(Roche, Mannheim, Germany), 2 ␮g of the RNA was used for
first-strand cDNA synthesis with oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen). One microliter of
5-fold-diluted cDNA was used for PCR run for 28 cycles with a
pair of primers designed for TcMet (5⬘-GAAGCTTCAAGAGAGGAATATG-3⬘ and 5⬘-TTTCAACAGTTCCCTGGTCG-3⬘) and for 24 cycles with a pair of primers for a Tribolium
ribosomal protein gene rp49 (5⬘-T TATGGCA A ACTCA A ACGCA AC-3⬘ and 5⬘-GGTAGCATGTGCT TCGTTTTG-3⬘). That contamination with genomic DNA did not
occur was verified by PCR on samples processed without reverse
transcriptase. In addition, the primers for TcMet flanked an
intron of 4.4 kb.
RNAi. TcMet and egfp dsRNAs, at lengths 564 and 720 bp,
respectively, were prepared as described (41) by using the T3 and
T7 MEGAscript kit (Ambion, Austin, TX). Larvae and pupae
Konopova and Jindra
Hormonal Treatments. Early-seventh or -eighth instar prepupae
were injected with TcMet or egfp dsRNA and allowed to pupate.
Within 12 h after ecdysis, the pupae were briefly dipped into
acetone (control) or acetone-diluted methoprene (VUOS, Pardubice, Czech Republic), juvenile hormone III, geraniol, or
farnesol (the latter three from Sigma, St. Louis, MO).
postfixed with 1% osmium tetroxide, dehydrated in ethanol,
critical point dried, gold coated, and observed under a JEOL
(Tokyo, Japan) 6300 scanning electron microscope.
We thank G. Bucher for advice on beetle rearing and RNAi; Y.
Tomoyasu for the phylogenetic analysis and comments on the manuscript; T. Shippy, S. Brown, P. Svacha, K. Slama, and F. Sehnal for
valuable input; and D. Kodrik for advice on the use of methoprene. This
work was supported by Grant IAA5007305 and Project Z5007907 from
the Czech Academy of Sciences and Grant LC07032 from the Czech
Ministry of Education. B.K. was supported by Student Grant 23/2005/
P-BF from the University of South Bohemia.
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Scanning-Electron Microscopy. Samples were fixed in 80% ethanol,
SEE COMMENTARY
just after ecdysis were anesthetized with CO2 and injected into
abdomens with dsRNA at concentration 5 ␮g/␮l until inflation
was visible (42). Insects were then kept individually in 24-well
microtiter plates supplied with food and were checked daily to
count all ecdyses until a developmental arrest occurred.
Konopova and Jindra
PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10493
Supporting Results
Supporting Fig. 6
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
1
1
1
1
1
--------MDSGDCETEEDSAPEACVSPEGSLLPSNSREMRNRAEKMRRDKLNSYIGELA
------------MKVKFAKLFCIRLALTSAYCKIRNGREARNRAEKNRRDKLNGSIQELS
-----------------------------MQTAVQNGREARNRAEKNRRDKLNGSIQELS
------------------------------------------MAEKQRRDNLNTNISAMA
MAAPETGNTGSTGSAGSTGSGSGSGSGSGSSSDPANGREARNLAEKQRRDKLNASIQELA
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
53
49
32
19
61
TLVPMVARSAKRMDKTSILRLAATHLRIYQTLLSGKNHPHIQLPKHVDQ----------GMVPHVAESPRRVDKTAVLRFSAHALRLKYVFDTEQEQTKQEPSENAAG----------TMVPHVAESPRRVDKTAVLRFAAHALRLKHAFGNSLMQQRP------------------ALVPTVAESPRKMDKISILRLAANFLRIHYTVGRGVSDFLPRELGDLDL----------TMVPHAAESSRRLDKTAVLRFATHGLRLQYVFGKSASRRRKKTGLKGTGMSASPVGDLPN
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
102
98
73
68
121
--YLLEQLVCEQLGGFLLILTPNGKIVFVSHTVEHLLGHLQTDLMGQSIFNITSPDDHDR
QKTEVHDALFRMLNGFLLTVTCRGQIVLVSASVEHFLGHCQTDLYGQNLFNLIHPDDHNL
---QITDTLMDMLDSFFLTLTCHGHILLISASIEQHLGHCQSDLYGQSIMQITHPEDQNM
--EQYIGDNLIKNGSFFIVVTTTGKIVYVSRQVQEHLGHTQADLLGHSLYAFIHPKDEEE
PSLHLTDTLMQLLDCCFLTLTCSGQIVLVSTSVEQLLGHCQSDLYGQNLLQITHPDDQDL
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
160
158
130
126
181
LRMYIN---TESVLDGDWK----------------------------------------LKQQLVPNNLVNLFDSAVSAPSTSRTSSGTETSAE---------------------EQQR
LKQQLIPTELENLFDAHGDSDAEGEP------------------------------RQRS
LARNLNPDEMQGVVSSLPQITDGTND-------------------------------NSN
LRQQLIPRDIETLFYQHQHHQQQGHNPQQHSTSTSASASGSDLEEEEMETEEHRLGRQQG
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
176
197
160
155
241
----------------------------------KCFNIRLKRAGPRTESA-VYEPVRIM
KSQDEED-----------------EIDRKLRQDRRKFTLRIARAGPRSEPT-AYELVTID
KAEED-------------------AIDRKLREDRRSFRVRLARAGPRSEPT-AYEVVKID
SSEDSTS----------------SKNGKTFQNQRRTFELRMLHRTSSRREHTQYEWFEFS
EADDDEDHPYNRRTPSPRRMAHLATIDDRLRMDWRCFTVRLARASTRAEATRHYERVKID
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
201
239
200
199
301
GVHRPGFDNDCNKNTST-----------------------------------------SK
GFFRRADAAPRGERPSG-PSGLQLLRRARG-------------------RDDGIT---LQ
GCFRRSDEAPRGVRSNHFSSNLQLIRRTRG-------------------RDDVIP---LH
GILRLADACKTSTSNVNRSRHR------------------------------------EI
GCFRRSDSSLTGGAAANYPIVSQLIRRSRNNNMLAAAAAVAAEAATVPPQHDAIAQAALH
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
220
276
238
223
361
EIALNNDVLLFFVKVFR---PEPLCE--------RLFEASREEYVTRHLIDGRIIGCDQR
SINGNDIVLVAVARVQK---VPTICD--------RLIEACRYEYKTRHLIDGRIVQCDHR
TISGNDIILTGCARIIR---PPKIAS--------RLIDANTLEYKTRHLIDGRIIDCDQR
TSTSNDIVFVGVAQLLMK-QPLTKIS---------IIDANKNEYITRHLVDGRIIYCDHR
GISGNDIVLVAMARVLREERPPEETEGTVGLTIYRQPEPYQLEYHTRHLIDGSIIDCDQR
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
269
325
287
273
421
ISFIAGYMTEEVSGLSAFKFMHREDVRWVMIALRQMYDRGESKGSSCYRLLSRNGQFIYL
ISVVAGYLTTEVSGLSPFTFMHKDDVRWVIVALRQMYDYSQNYGESCYRLMTRTGDFVYL
IGIVAGYMTDEVRNLSPFTFMHNDDVRWVIVALRQMYDCNSSYGESTYRLFTRNGNIIYL
VSVVAGYLSEEVSGMSAFGFMHKDDRIWAMVALRQMYDRAETCGSSCYRLTSKTGEPIYL
IGLVAGYMKDEVRNLSPFCFMHLDDVRWVIVALRQMYDCNSDYGESCYRLLSRNGRFIYL
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
329
385
347
333
481
RTFGFLEIDDQG-TVESFVCVNTLVSEQEGLQLINEMKKRYSALINSQSCPITSSG---KTRGYLEVDDSSKKVQSFVCINTLVSDEEGRRLVREMKHKFSVIVEADELP--------QSKGYLEIDKETNKVHSFVCVNTLLGEEEGKRRVQEMKKKFSVIINT-QIP--------RTHGYLEVDKDTQIAVSLVCINTLVSEEEGIQLMQQMKKRFSATISETMRAIIQNGDDAS
HTKGFLEVDRGSNKVHSFLCVNTLLDEEAGRQKVQEMKEKFSTIIKA-EMPTQ-------
1
Supporting Fig. 6 (Continued)
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
384
436
397
393
533
-------STDSSSQSVEDPQQVEAAIVHLIANLPSPG------------------------------DESDEPAVENPTQIEKAVLNLLTNLHSEDDEPSERALPSNTSQETDGSEGSQ
-------QSTIDVPASEHPALLEKAVLRLIQNLQKSGENGGH-----DDGDEDDDAQDGD
IDLGSDSQNPNSKSNMEDPAQLEDAITYLVSDLSSPLPEDCLSPPTQNEQYAKAAMISQH
-------SSSPDLPASQAPQQLERIVLYLIENLQKS------------------------
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
414
488
445
453
562
-----------------------------SDQRSTPSPRVYGN----------------LAIIAPSSKAVKSAIVKSMNVIAIASKKMSNGSNSPLRDSTGASPRSKDHSGSPSAIITM
DDEEDDDDDQDD-----------------GARSMSEFGDPYGSHHGRSHHG---SSALSS
LPPAEAQARRLG--------IKKIDRYLMVQAKATNNQKSESKTNNNKNNSNERQENTRS
----VDSAETVG-----------------GQGMESLMDDGYSSP----------ANTLTL
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
428
548
485
505
591
-------------------------------------VNENQDCSTPTENSPTKPYYKLK
QRPSVLQKAHSPALESRVPPLHGTDHDFIQPFSPAGSTSSSSSSSGSVFSPASHQRNSRS
HG---HGNAKTPPLALVPP--------------EASSVKSAITKSISVVNVTAAKHLRGI
SRKTVHEVTKQTRNSTQDE--------------QNSSRINNMSNVESCVNTQRKPGISQL
EE---LAPSPTPALALVPP--------------APSSVKSSISKSVSVVNVTAARKFQQE
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
451
608
528
551
634
TANNKRPPSTELGTNIYTSSKRQRTSPQLSPMSSLPPYPNRTQITEVTQQTEP------PFGGSIPPTSPPAIAATPSTSVSRIVDNTASGHSRSISVLKRTHSNSCLDSSDGSSIGHT
HASTAVKSPSSLGSCTCSDS--HSPCDFCQGAPTTDLQAVGSNLKRGSTAHVE------T
ELHQNVDILNPNTTVKKPVATVENNPCNVKIERNMDVYTPETMIEYFEKNAID-----NN
HQKQRERDREQLKERTNSTQGVIRQLSSCLSEAETASCILSPASSLSASEAPD------T
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
504
668
580
606
688
-------------------------------------SIYQHDQLLRNKV---------ADFVVQKRPKINTILDLDTCRTRLSNPATDLITMLPQAFHAVDQSLLKVDDDTAKLRAQV
EEKLSKRRF----------------IPSTEIEHVLHTSLDQIGRNLTQQLNVARNLREQS
SDLKSHPYP----------------LKRIYSDEDIKTGCTKKRQYNNVPYASSDTSDEQN
PDPHSNTSP----------------PPS------LHTRPSVLHRTLTSTLR---------
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
-----------------------------------------------------------728 SGYENLQGKSLQQLDSIVEASHEQRNVLQSIKQELTQFQSNNGSNDQDGAGGRGPIVVND
624 QRYELPHAN--QRFDEIMQEHQKQSELYVNIKSEYEVQLQHKASTRKSSDSDRN-----650 ISYIDCKPFVDEEFSELSTDFNNINPQSLKIAESPNSSLSEVITDYQQVDSSIPLIAPNP
------------------------------------------------------------
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
-----------------------------------------------------------788 LLPGGGAIVDDLDPPSLMMEDFPSDSSNLMADLDNNGSLLEGDSPLEFFSSINTPLISPT
676 ---------QEQPPPPLQEDDQD------------------------------------710 ELEAEYGDLQDNSLLSPGLEANPGTELSTVTLHLQTK----------------------------------------------------------------------------------
Tribolium
Aedes
Dm_Gce
Apis
Dm_Met
------------------------------------848 TATASVITSSRSVGGTGTGGASGTAGSFYQGADLNPS
-------------------------------------------------------------------------------------------------------------
Supporting Fig. 6. Alignment of Tribolium castaneum Met with related proteins from Aedes
aegypti (EAT47132), Apis mellifera (XP_395005) and two Drosophila melanogaster proteins, Met
(AAC14350) and Gce (NP_511160). The Tribolium protein can be accessed under EF468474 or
XM_961449 (amino acid residues 70-585). Conserved domains are indicated with lines above the
sequence: bHLH (red), PAS-A (green), PAS-B (blue) and PAC (yellow).
2
Supporting Fig. 7. Phylogenetic analysis. The indicated proteins were aligned by using the
ClustalW algorithm implemented in the MEGA 3.1 software (Kumar S, Tamura K, Nei M. 2004.
Briefings in Bioinformatics 5:150-163), and the combined conserved domains (outlined in
Supporting Fig. 6) were used for construction of phylogenetic trees. (A) Neighbor-joining plot
generated by MEGA 3.1 with bootstraping using 5000 replicates. (B) A plot generated with the
Treepuzzle maximum likelihood algorithm (http://bioweb.pasteur.fr/seqanal/interfaces/Puzzle.html)
(Schmidt HA, Strimmer K, Vingron M, von Haeseler A. 2002. Bioinformatics 18:502-504) using
standard settings. Both trees reveal essentially the same information. The Drosophila melanogaster
bHLH-PAS protein Spineless (Ss) (NP_476748) served as an outgroup in both types of analysis.
3
Supporting Fig. 8. Anti-metamorphic effect of methoprene on Tribolium. Topical application of
methoprene at the beginning of the eighth (final) larval instar produced supernumerary larvae. A
giant larva that has reached its eleventh instar (i.e., it completed 10 postembryonic ecdyses) is
shown on top in comparison with a normal sized eighth instar larva treated only with acetone.
4