Insect
Molecular
Biology
Insect Molecular Biology (2009) 18(5), 571–581
doi: 10.1111/j.1365-2583.2009.00898.x
Vestigial and scalloped in the ladybird beetle: a
conserved function in wing development and a novel
function in pupal ecdysis
imb_898
571..582
T. Ohde, M. Masumoto, M. Morita-Miwa, H. Matsuura,
H. Yoshioka, T. Yaginuma and T. Niimi
Graduate School of Bioagricultural Sciences, Nagoya
University, Chikusa, Nagoya, Japan
Abstract
In Drosophila melanogaster, Vestigial (Vg) and Scalloped (Sd) form a transcription factor complex and play
a crucial role in wing development. To extend our
knowledge of insect wing formation, we isolated vg
and sd homologues from two ladybird beetle species,
Henosepilachna vigintioctopunctata and Harmonia
axyridis. Although the ladybird beetle vg homologues had only low homology with D. melanogaster
vg, ectopic expression of H. vigintioctopunctata vg
induced wing-like tissues in antennae and legs of D.
melanogaster. Subsequent larval RNA interference
(RNAi) analysis in H. vigintioctopunctata demonstrated
conserved functions of vg and sd in wing development,
and an unexpected novel function of sd in pupal
ecdysis. Furthermore, our results can be applied to the
production of a flightless ladybird beetle for biological
control purposes using larval RNAi.
Keywords: vestigial, scalloped, Henosepilachna vigintioctopunctata, wing development, pupal ecdysis.
Introduction
The insect wing is an evolutionary novelty. It has obviously
contributed to adaptive radiation in various ecological
niches. Thus, the insect wing is a key organ for helping to
understand the great success of insects on planet Earth.
Cumulative information about the molecular mechanism
of insect wing formation has been provided from studies of
Correspondence: Teruyuki Niimi, Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya
464-8601, Japan. Tel.: +81 52 789 5504; fax: +81 52 789 4036; e-mail:
[email protected].
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society
the fruit fly, Drosophila melanogaster, and it is known
that vestigial (vg) and scalloped (sd) play a central role in
D. melanogaster wing development. Both vg and sd are
co-expressed in the wing pouch region of the wing imaginal
disc, and mutants of these genes display a defect in wing
morphology (Daniels et al., 1985; Williams et al., 1991,
1993; Campbell et al., 1992). In vivo ectopic expression
analysis revealed that vg can induce ectopic wing-like
outgrowth on the compound eyes, legs and antennae, and
accordingly vg is regarded as a master gene of wing
development (Kim et al., 1996), like eyeless in eye development (Halder et al., 1995). Thus, vg is a potentially good
molecular marker for understanding insect wing formation.
Despite its importance, however, since the first cloning of
vg from D. melanogaster and Drosophila virilis in 1991
(Williams et al., 1991), isolation of vg homologues from
other insects using this sequence information has been
restricted to the mosquito Aedes aegypti (J. Williams and
S. Carroll, pers. comm.; Halder & Carroll, 2001) because
of the low sequence homology between Vgs of D. melanogaster and D. virilis (65%).
Sd is the only protein that belongs to the TEAD (TEF-1,
TEC-1, ABAA domain) protein family in D. melanogaster.
The TEAD transcription factors contain the TEA domain
as a conserved DNA-binding domain (Bürglin, 1991). The
Tead genes are often expressed in a wide range of tissues,
and they can mediate various functions by interacting with
tissue-specific co-factors. It has been demonstrated previously that Vg and Sd physically interact with each other both
in vivo and in vitro (Halder et al., 1998; Paumard-Rigal et al.,
1998; Simmonds et al., 1998).Although Vg does not contain
any characterized DNA-binding motif, Sd is able to localize
Vg in the nucleus and bind to DNA via the TEA domain
(Halder et al., 1998; Simmonds et al., 1998). In D. melanogaster wing development, Vg and Sd form a functional
transcription factor complex and promote cell proliferation
and cell survival of the wing disc (Delanoue et al., 2004).
In addition to the function of sd in wing development,
recent studies revealed that Sd can mediate multiple
functions cooperatively with other co-factors such as Yorkie
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T. Ohde et al.
(Yki) and Dmef2 (Drosophila homologue of Myocyte
enhancer factor-2). Yki is the downstream effector of the
Hippo signalling pathway, and functions in cell growth,
proliferation and apoptosis (Huang et al., 2005). It has
been shown that Sd and Yki work in concert to control organ
size (Goulev et al., 2008; Wu et al., 2008; Zhang et al.,
2008). The Mef2 protein family plays a key role in muscle
cell differentiation (Black & Olson, 1998). Together with
Dmef2, Sd has functions in cardiac and somatic muscle cell
differentiation in late embryonic development (Deng et al.,
2009). Similar to Sd, human TEAD family protein Transcription enhancer factor-1 (TEF-1) can interact with tissuespecific co-factors such as Vestigial-like 2 in the skeletal
muscle (Maeda et al., 2002) and Vestigial-like 4 in the heart
muscle (Chen et al., 2004).
It has also been reported that the dominant-negative
phenotypes, as a consequence of antagonizing Sd with
Vg constructs, suggested functions of Sd in the eye, leg
and optic lobe (Garg et al., 2007). Additionally, Sd restricts
the expression of a specific odour receptor gene to a
proper olfactory receptor neurone class in the maxillary
palp (Ray et al., 2008). However, co-factors which interact
with Sd in these tissues have not yet been identified.
Our current knowledge of insect wing development is
limited to D. melanogaster because of its well-established
gene functional analysis tools. To understand the molecular mechanisms of wing formation in diverse insect
species, the study of gene function in species other than
D. melanogaster is indispensable. Recent progress of
gene functional analysis systems enables us to study
gene function in nonmodel insects. One such system is
germ-line transformation using transposons. However,
gene functional analysis by germ-line transformation is still
limited in nonmodel insects. Alternatively, the established
ectopic gene expression system in D. melanogaster can
be utilized to analyse the function of exogenous genes
(Duffy, 2002; McGuire et al., 2004). However, an analysis
in D. melanogaster is insufficient to clarify the original
function of exogenous genes of other insects. The RNA
interference (RNAi) technique is able to circumvent these
difficulties. RNAi is a gene silencing mechanism using
double-stranded (ds) RNA, which triggers the sequencespecific degradation of mRNA and consequently disrupts
gene function (Fire et al., 1998). To date, RNAi is one of
the most important methods for gene functional analysis in
nonmodel insects. Larval RNAi is a potent gene functional
analysis tool (Tomoyasu & Denell, 2004). The technique is
performed by injecting dsRNA into the larval body cavity. It
has already been established in the coleopteran insects
Tribolium castaneum (Tomoyasu & Denell, 2004) and
Harmonia axyridis (Niimi et al., 2005), where it effectively
disrupted gene function during adult development.
In this study, we first cloned vg and sd homologues from
the 28-spotted ladybird beetle, Henosepilachna (previously
assigned to Epilachna) vigintioctopunctata and the
multicoloured Asian ladybird beetle, Harmonia axyridis.
As the H. vigintioctopunctata vg homologue (Hv-vg)
showed low homology with D. melanogaster vg, we next
performed ectopic expression analysis in D. melanogaster
to examine its function. Consequently, ectopic expression
of Hv-vg induced wing-like tissues in the antennae and legs
of D. melanogaster. To investigate in vivo functions of Hv-vg
and the H. vigintioctopunctata sd homologue (Hv-sd), we
performed larval RNAi analysis. As a result, it was revealed
that Hv-Vg and Hv-Sd play conserved roles in ladybird
beetle wing formation. Unexpectedly, a novel function of sd
in the pupal ecdysis was revealed from RNAi analysis.
Based on our studies, we developed the RNAi mediated
production of a flightless aphidophagous ladybird beetle as
a biological control agent.
Results and discussion
Cloning of vestigial and scalloped cDNAs from
ladybird beetles
To extend our understanding of wing development in
insects, we tried to clone the homologues of vg and
sd as the master genes for wing development. When we
designed degenerate primers for the cloning of vg from
H. vigintioctopunctata and H. axyridis, sequence information for vg homologues was limited to just D. melanogaster,
D. virilis and A. aegypti (J. Williams and S. Carroll, pers.
comm.) and the homology of Vg amongst them was surprisingly low, even between closely related species (65%
overall sequence homology between Vg from D. melanogaster and D. virilis) (Williams et al., 1991). Thus, several
primers were designed and possible combinations of
primer sets were tested for degenerate oligonucleotide
primed PCR (DOP-PCR). The initial isolation of a partial
Hv-vg cDNA was achieved by DOP-PCR of the first-strand
cDNA prepared from total RNA of wing discs of last larval
instars using the vg-1 and vg-4 primer sets (see Experimental procedures), whereas a partial Ha-vg cDNA was
prepared from total RNA of wing discs of last larval instars
using the vg-5 and vg-7 primer sets (see Experimental
procedures). These PCR products of H. vigintioctopunctata
and H. axyridis were 697 and 514 bp, respectively (data not
shown). For Hv-vg, full-length cDNA of 1183 bp was cloned
by 5′ and 3′ rapid amplification of cDNA ends (RACE) using
gene-specific primers as described in the Experimental
procedures. It encoded a polypeptide of 308 amino acids.
These results indicated the first cloning of vg in insects
outside dipteran insects.
In contrast to vg, designing a degenerate primer for sd
cloning was not difficult because of the high homology of
Sd amongst insects and mammals (Yasunami et al., 1995).
The size of the DOP-PCR products of H. vigintioctopunctata
and H. axyridis were 864 and 876 bp, respectively. For
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
Vestigial and scalloped in the ladybird beetle
Hv-sd, cloning of full-length cDNA was attempted by 5′ and
3′ RACEs using gene-specific primers as described in
the Experimental procedures. Only 3′ RACE product was
obtained and the size of the product containing the stop
codon was 391 bp.
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observed in the flies that ectopically expressed Dm-vg by
Dll-Gal4 (Fig. 3D). Wing-like structures induced by ectopic
Hv-vg expression revealed wing blade-specific characters,
such as the margin bristles and the morphology of hair,
as previously demonstrated (Fig. 3F, I; Kim et al., 1996;
Conservation of functional domains in Vg and Sd
(A)
To identify the important functional domains in Vg, we compared Vgs from insects and humans (Fig. 1). The entire
homology between Hv-Vg and D. melanogaster Vg (Dm-Vg)
was as low as 36%. In contrast to the low homology of Vg
between D. melanogaster and D. virilis, that of Vg between
H. vigintioctopunctata and H. axyridis was as high as 89%.
Comparison of Vg sequences of H. vigintioctopunctata,
H. axyridis, D. melanogaster and human homologues
such as Vestigial-like 2 (Vgl-2, also referred to as Vito-1)
(Maeda et al., 2002; Mielcarek et al., 2002) and Tondu
(Tdu, also referred to as Vestigial-like 1) (Vaudin et al., 1999;
Maeda et al., 2002) revealed highly conserved regions,
although the entire homology was quite low. The highly
conserved regions were restricted to small stretches of
amino acids in the N-terminus transactivation domain
(N-TA), Sd interaction domain (SID) (Simmonds et al., 1998),
SID franking domain (SFD), His-rich domain (HRD)
and C-terminus transactivation domain (C-TA). Although
N-terminus and C-terminus domains have been identified
as transcriptional activation domains (Vaudin et al., 1999;
MacKay et al., 2003), the functions of SFD and HRD are
not yet identified. All insect Vgs have these five conserved
regions. In human Vg homologues, however, Vgl-2 has four
conserved regions whereas Tdu has only SID.
In contrast to Vg, the entire sequences of D.
melanogaster Sd (Dm-Sd) and its homologues from
H. vigintioctopunctata, H. axyridis and human TEF-1 (Xiao
et al., 1991) were well conserved. Characterized functional
domains of Sd, TEA domain (Bürglin, 1991) and Vestigial
interaction domain (VID) (Simmonds et al., 1998), also
show high homology (Fig. 2).
Hv-Vg
N-TA
SID SFD HRD C-TA
Ha-Vg
Dm-Vg
Aa-Vg
Hs-Vgl-2
Hs-Tdu
0
100
200
300
400
500
(B) N-TA
Hv-Vg
Dm-Vg
Aa-Vg
Hs-Vgl-2
M--SCSEVMYQAYYPYLYQRAG
MAVSCPEVMYGAYYPYLYGRAG
MAVSCPEVMYGAYYPYLYGRAG
M--SCLDVMYQVYGPPQPYFAA
(C) SID
Hv-Vg
Ha-Vg
Dm-Vg
Aa-Vg
Hs-Vgl-2
Hs-Tdu
RAQYVSANCVVFTHYQGDAASVVDEHFSRALD
RAQYVSANCVVFTHYQGDAASVVDEHFSKALD
QAQYLSASCVVFTNYSGDTASQVDEHFSRALN
RAQYVSATCVVFTHYSGDAASVVDEHFSRALN
EAEYINSRCVLFTYFQGDISSVVDEHFSRALS
IKTEWNSRCVLFTYFQGDISSVVDEHFSRALS
(D) SFD
Hv-Vg
Dm-Vg
Aa-Vg
Hs-Vgl-2
DSPMSARNFPPSFWNS
SSPMSNRNFPPSFWNS
YSSMSSRNFPPSFWNS
SFPMSQRSFPASFWNS
(E) HRD
Hv-Vg
Dm-Vg
Aa-Vg
Hs-Vgl-2
H-HRA-VHDYHHHNMAAQYGGL
HAHAAHAHAYHH-NM-AQYGSL
HAHAAHAHAYHH-NM-AQYGSL
HAHPHHAHP-HHPY--ALGGAL
Ectopic expression of Hv-vg in D. melanogaster
(F) C-TA
Hv-Vg
Dm-Vg
Aa-Vg
YSSYPTMSGLEA---QVQDSSKDLYWF
YSSYPTMAGLEAQVAQVQESSKDLYWF
YSSYPTMAGLEA---QVQESSKDLYWF
We next examined wing formation is a conserved function
in Hv-vg, despite its overall low homology with Dm-Vg,
using the D. melanogaster Gal4/Upstream Activation
Sequence (UAS) ectopic gene expression system (Brand
& Perrimon, 1993). It has been reported that ectopic
expression of vg, driven by Distal-less (Dll)-Gal4, induced
wing-like transformation of the distal regions of appendages where Dll is expressed (Weatherbee et al., 1998;
Baena-López & García-Bellido, 2003). We generated UASHv-vg insertion lines, and then crossed them with the
Dll-Gal4 lines. Subsequent morphological analysis was
performed for the progeny. Hv-vg expression by the DllGal4 driver induced ectopic wing-like structures in the
distal positions of the antennae and the legs (Fig. 3G), as
Figure 1. Comparison of conserved domains of Vestigial (Vg) amongst
Drosophila melanogaster Vg and Vg homologues of ladybird beetles,
mosquito and human. (A) Schematic structure of D. melanogaster Vg
(Dm-Vg) and its homologues from Henosepilachna vigintioctopunctata
(Hv-Vg), Harmonia axyridis (Ha-Vg), Aedes aegypti (Aa-Vg), human
Vgl-2 (Hs-Vgl-2) and human Tondu (Hs-Tdu). Conserved domains are
represented in coloured boxes: N-terminus transactivation domain (N-TA)
in red, Sd interaction domain (SID) in blue, SID franking domain (SFD) in
green, His-rich domain (HRD) in yellow, and C-terminus transactivation
domain (C-TA) in orange. The scale bar below indicates an amino acid
ruler. (B–F) Comparison of conserved domains of D. melanogaster Vg and
deduced amino acid sequences of ladybird beetles, mosquito and human
Vg homologues: (B) N-TA, (C) SID, (D) SFD, (E) HRD and (F) C-TA.
Conserved residues are shown by coloured letters, whereas
nonconserved residues are in grey. Residues that are conserved only
between D. melanogaster and A. aegypti are shown by purple letters.
Light blue letters in the SID alignment indicate residues conserved only
between human Vg homologues.
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
574
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
T. Ohde et al.
MKNITSSSTCSTGLLQLQNNLSCSELEVAEKTEQQAVGPGTIPSPWTPVNAGPPGALGSA
TEA domain
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
SPDIEQSFQEALAIYPPCGRRKIILSD
SPDIEQSFQEALAIYPPCGRRKIILSD
DTNGSMVDSKNLDVGDMSDDEKDLSSADAEGVWSPDIEQSFQEALSIYPPCGRRKIILSD
MERMSDSADKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSD
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
EGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKLREIQAKLKVQ---------EGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKLREIQAKLKVQ---------EGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKLREIQAKIKVQ---------EGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKSRDFHSKLKDQTAKDKALQHM
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
---------------------------------FWQPGLQ---PGTSQDVKPFTQAAYP---------------------------------FWQPGLQ---PGTSQDVKPFTQAAYP---------------------------------FWQPGLQ---PSTSQDFYDYSIKPFPQ
AAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYP-
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
-TKPATAVFSGDVGALQEPPP--PVWEGRAIATHKLN-CEFQHYGVSNRE--SISTSSFV
-TKPATAVSSGEVGALQEPPP--PVWEGRAIATHKLRLVEFSAFMESRNREEAYQRHLFV
PPYPAGKTSTAVSGDETGIPPSQLPWEGRAIATHKFRLLEFTAFMEIQRD-EIYHRHLFV
-IQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFV
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
HIGGP-LIFHRPLLEPVDVRQIYDKFPEKKGGLKELYDKGPQNAFFLVKFWADLNSNFQD
HIGGPALSYSDPLLEAVDVRQIYDKFPEKKGGLKELYDKGPQNAFFLVKFWADLNSNFQD
QLGGK-PSFSDPLLETVDIRQIFDKFPEKSGGLKDLYEKGPQNAFYLVKCWADLNTDLTT
HIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQD
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
--EAGAFYGVTSSYESNENMIITCSTKVCSFGKQVVEKVETEYARFENGRFMYRIHRSPM
--EAGAFYGVTSSYESNENMIITCSTKVCSFGKQVVEKVETEYARFENGRFVYRIHRSPM
GSETGDFYGVTSQYESNENVVLVCSTIVCSFGKQVVEKVESEYSRLENNRYVYRIQRSPM
--DAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPM
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
CEYMINFIHKLKHLPEKYMMNSVLENFTILQVVSNRDTQETLLCTAYVFEVSTSEHGAQH
CEYMINFIHKLKHLPEK
CEYMINFIQKLKNLPERYMMNSVLENFTILQVMRARETQETLLCIAYVFEVAAQNSGTTH
CEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQH
Hv-Sd
Ha-Sd
Dm-Sd
Hs-TEF-1
HIYRLIKD
VID
HIYRLIKE
HIYRLVKD
Weatherbee et al., 1998; Baena-López & García-Bellido,
2003). These observations suggest that Hv-vg is able to
function to form the wing blade at least in D. melanogaster,
despite its low entire sequence homology with Dm-vg.
It has been reported that the human Vg homologue Tdu
shows homology with Vg only in SID as an identified functional domain and is unable to form wing-like outgrowths by
ectopic expression using Gal4 driver despite Tdu physically
interacting with Sd (Vaudin et al., 1999). In contrast, our data
show that Hv-vg, with all identified functional domains, was
able to induce an ectopic wing in D. melanogaster. From this
result, it is likely that a transactivation domain of Vg is
required to form an ectopic wing-like structure.
Functional analysis of Hv-vg and Hv-sd by larval RNAi
in ladybird beetles
To investigate functions of Hv-vg and Hv-sd in H.
vigintioctopunctata, we performed a larval RNAi experi-
Figure 2. Multiple alignment of Drosophila
melanogaster Scalloped (Dm-Sd) and its homologues
from Henosepilachna vigintioctopunctata (Hv-Sd),
Harmonia axyridis (Ha-Sd), and human (Hs-TEF-1).
Functional domains are boxed in colour: TEA domain
in pink and Vg interacting domain (VID) in green.
Conserved residues of functional domains are shown
by coloured letters, whereas conserved residues
outside of functional domains are shown by black
letters. Nonconserved residues are shown by grey
letters.
ment to knock-down the gene function. To explore a stage
in which Hv-vg and Hv-sd play important roles, we injected
dsRNAs of Hv-vg and Hv-sd into larvae of different instars.
Hv-vg RNAi adults exhibited defects in wing formation
(Fig. 4A–D) similar to the loss-of-function mutant of vg
in D. melanogaster adults (Williams et al., 1991). The most
severe adult phenotype – an almost complete loss of wing
– was induced when dsRNA was injected into third instar
larvae (Fig. 4B,C), whereas only a weak phenotype – slight
deformation of wing – was observed when dsRNA was
injected into first instar larvae (Fig. 4A). In D. melanogaster,
it was demonstrated that the domain of vg-expressing wing
disc cells expands dramatically by Wingless signalling
during the mid- and late third larval instar (Zecca & Struhl,
2007). Similarly, the dramatic wing disc cell proliferations
during the last larval instar stage are also observed in the
silkworm, Bombyx mori (Kurushima & Ohtaki, 1975), the
greater wax moth, Galleria mellonella (Meyer et al., 1980),
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
Vestigial and scalloped in the ladybird beetle
Figure 3. Ectopic expressions of Drosophila melanogaster vestigial (Dmvg) and Henosepilachna vigintioctopunctata vg (Hv-vg) in D. melanogaster
using a Distal-less-Gal4 (Dll-Gal4) driver. The scanning electron
microscope images of wild-type adult (A) and pharate adults that ectopically
express Upstream Activation Sequence (UAS)-Dm-vg (D) or UAS-Hv-vg
(G) under the control of Dll-Gal4 driver are shown. Wings of pharate adults
dissected from the pupal cases are not extended because they could not
accomplish ecdysis because of the ectopic wing-like structures. Ectopic
wing-like structures were formed in all of the Dll-Gal4 > UAS-Hv-vg pharate
adults (n = 34). (B) Wing and (C) T2 legs of wild-type flies. (E, H) Wings and
(F, I) ectopic wing-like structures in T2 legs of pharate adults that express
UAS-Dm-vg or UAS-Hv-vg driven by Dll-Gal4. Highly magnified images of
the red-boxed regions are shown in the insets. Arrows in insets indicate the
margin bristles. Scale bars = 300 mm in (A), (D), (G), 50 mm in insets and
100 mm in the other panels.
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the intertidal chironomid, Clunio marinus (Neumann &
Spindler, 1991) and H. vigintioctopunctata (T. Niimi,
unpubl. data). Therefore, one possible interpretation of our
data is that, like D. melanogaster, Hv-vg plays a role in wing
disc cell proliferation; thus, the most intensive effect on
wing formation was caused by RNAi treatment at just
before the last instar (third instar). In contrast, dsRNA
injected into first instar larvae may become ineffective
during larval development, and consequently the effect of
RNAi on wing formation was reduced in such adults.
Likewise for Hv-vg RNAi, wing size reduction in adults was
observed when Hv-sd dsRNA was injected into first instar
larvae (Fig. 4E); however, an unexpected phenotype of
prepupal arrest just before ecdysis to pupa was observed
when dsRNAs were injected into second, third and fourth
instar larvae (Fig. 4F–H). We removed the larval cuticle of
the individuals that showed the prepupal arrest phenotype in
order to observe the stage at which pupal formation was
stopped. Apart from small-sized wings, normal pupal characters such as coloration and hair patterning were observed
(Fig. 4I compared to N). Within several days of removing
the larval cuticle, the individual died and never reached
the adult stage. Importantly, from this observation it seems
that pupal formation was accomplished but ecdysis behaviour was prevented specifically by Hv-sd dsRNA injection. However, only when Hv-sd dsRNA was injected into
first instar larvae, the ecdysial arrest phenotype was not
observed. This result suggests that the effect of RNAi was
sufficiently maintained to prevent wing formation at the
larval stage, but was gradually weakened during larval and
pupal development. It is likely that the RNAi effect was so
weak at pupal ecdysis stage that it was unable to prevent
ecdysis. This novel function of sd supports the idea that
TEAD protein is involved in a variety of biological processes.
Recently, the role of sd in organ size regulation has been
suggested from studies of D. melanogaster (Goulev et al.,
2008; Wu et al., 2008; Zhang et al., 2008).We note that any
significant size defects in organs other than the wing were
not induced by Hv-sd dsRNA injection. In this RNAi experiment, the same amount of Enhanced Green Fluorescent
Protein (EGFP) dsRNAs was injected into larvae of each
stage as a negative control, and never induced the phenotypes which were observed in Hv-vg or Hv-sd RNAi (Fig. 4J–
N). Therefore, phenotypes observed in Hv-vg and Hv-sd
RNAi reflect gene-specific functions.
Analyses of Hv-vg and Hv-sd expression by
semiquantitative reverse transcriptase PCR
To understand more about the ecdysial arrest phenotype
observed in the Hv-sd RNAi experiment, we examined the
expression profiles of Hv-sd and Hv-vg in each tissue of the
prepupal stage by semiquantitative reverse transcriptase
PCR (RT-PCR). cDNAs, synthesized from total RNAs
extracted from tissues of H. vigintioctopunctata prepupa,
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
576
T. Ohde et al.
Figure 4. RNA interference (RNAi) phenotypic analyses of Henosepilachna vigintioctopunctata vestigial (Hv-vg) and Hv scalloped (Hv-sd). DsRNAs of
(A–D) Hv-vg, (E–I) Hv-sd and (J–N) Enhanced Green Fluorescent Protein (EGFP) were injected into H. vigintioctopunctata larvae. (A) Elytra were
deformed when Hv-vg dsRNA was injected into first instar H. vigintioctopunctata larvae. (B–D) Reductions in wing size occurred when Hv-vg dsRNA was
injected into early or late third and fourth instar larvae. (E) A similar wing defect in adults was induced when Hv-sd dsRNA was injected into first instar
larva. (F–H) Arrests at ecdysis occurred and larvae were unable to moult to pupae when Hv-sd dsRNA was injected into second, third and fourth instar
larvae. (J–M) EGFP dsRNA was injected as a negative control and revealed no obvious effect. (I) After removing the larval cuticle, wing size reduction
and pupal characters were seen in Hv-sd RNAi larvae arrested at ecdysis (compared to N: EGFP dsRNA injected pupa). Note that after removing the
larval cuticle, the individual was dead within several days. The Roman number at the bottom left of the panel shows injected instar of the larva. For
Hv-vg RNAi, wing-specific adult phenotypes were observed in 60% of cases (n = 5) for first instar larval injection, and 100% of cases (n = 5) for the other
injections. For Hv-sd RNAi, wing-specific adult phenotypes were observed in 81% of cases (n = 21) for first instar larval injection, and pupal ecdysial
arrest phenotypes were observed in 75 (n = 8), 78 (n = 23) and 55% (n = 22) of cases for second, third and fourth instar larval injections, respectively.
One of the most severe phenotypes induced by dsRNA injections is shown for each panel. Arrowheads indicate deformed wings. Arrows indicate almost
complete loss of wings. Red and blue colours indicate forewings and hindwings, respectively. Scale bars = 2 mm.
were used as templates for the PCR reaction. Hv-sd
mRNAs were detected in brain, forewing disc, hindwing
disc, whole gut (including fore, mid and hindgut), Malpighian
tubules, testis and ovary (Fig. 5A). Hv-vg mRNAs were
detected in brain, forewing disc, hindwing disc and testis
(Fig. 5B). With respect to arthropod ecdysis, it is well known
that hormones have a crucial role in changing the internal
physiological state and regulating moulting behaviour
(Truman, 2005). For example, eclosion hormone (EH) is a
neuropeptide that is located in one or two pairs of neurones
in the insect brain, called ventromedial (VM) cells, and the
involvement of EH in pupal ecdysis has been suggested
from a study of the tobacco horn worm, Manduca sexta
(Truman & Copenhaver, 1989; Hesterlee & Morton, 2000).
From the expression profile of Hv-sd mRNA, it seems
possible that Hv-Sd directly or indirectly regulates the
expression of genes encoding hormones such as EH in
the brain, and also functions in pupal ecdysis. In D. melanogaster, it has been previously reported that sd was localized
to a subset of cells in the brain during the third instar, but its
function is still unknown (Campbell et al., 1992). Although
the functions of sd in whole gut, Malpighian tubules, testis
and ovary are also unknown, it seems that these tissues
are not related to the ecdysial arrest phenotype. However,
it is interesting that Hv-sd represents such a broad expression profile, even in tissues where Hv-vg mRNAs were not
detected. It is known that TEAD proteins, such as Sd and
its human homologue TEF-1, interact with tissue-specific
co-factors, such as Vg in the wing, which provide transactivation activity (Halder et al., 1998; Simmonds et al.,
1998; Vaudin et al., 1999; Maeda et al., 2002; Chen et al.,
2004). Our result suggests that Hv-Sd may interact
with as-yet-unidentified partners in tissues other than that
of the wing.
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
Vestigial and scalloped in the ladybird beetle
577
Figure 5. Reverse transcriptase PCR (RT-PCR)
analysis showing tissue expression profile of
Henosepilachna vigintioctopunctata vestigial (Hv-vg)
and Hv scalloped (Hv-sd) in H. vigintioctopunctata
prepupa. (A) The PCR products for Hv-sd mRNA
were detected in the brain, forewing and hindwing
discs, whole gut, Malpighian tubules, testis and ovary.
(B) Hv-vg mRNA was detected in the brain, forewing
and hindwing discs and testis. (C) Hv-rp49 was used
as an internal control. The amounts of Hv-rp49
mRNA in each tissue were at almost constant levels.
Abbreviations: -RT, the negative control in which
cDNA synthesis reaction was performed without
reverse transcriptase; CNS, central nervous system
excluding the brain.
In this RT-PCR experiment, both Hv-vg and Hv-sd
mRNAs were detected in the brain. In the larval RNAi
analysis, however, Hv-vg dsRNAnever induced the ecdysial
arrest phenotype. Therefore, it seems that Hv-sd may play
a role in pupal ecdysis independently of Hv-vg. In order to
understand the relationship between Hv-sd and hormones
underlying pupal ecdysis regulation, future studies aimed
to determine whether Hv-sd mRNA is localized in hormone
secreting cells, such as the VM cell, and to explore the
functional partner of Hv-Sd in the brain, are required.
Production of a flightless ladybird beetle for biological
control using RNAi method
H. vigintioctopunctata feeds on potato leaves and
damages field crops and accordingly it is regarded as a
destructive insect in terms of pest management. In contrast, the multicoloured Asian ladybird beetle, H. axyridis
is a predator of numerous aphid species, and its use as a
biological control agent has already been established (eg
commercially available in North America and Japan). However, because adult beetles tend to fly away from field crops
immediately, sustainable use in biological control has been
restricted (Trouve et al., 1997). To resolve this problem and
make this biological control more efficient, a wingless, and
therefore flightless, strain unable to disperse from crops
was developed (Marples et al., 1993; Tourniaire et al.,
1999) and effectively used for biological control (Koch,
2003). As shown in Fig. 4, wing size reduction was induced
in H. vigintioctopunctata adult by Hv-vg or Hv-sd RNAi.
We considered that the RNAi method could be applied
as a tool for the production of a wingless H. axyridis as a
biological control agent against aphids, and thus performed
larval RNAi experiments with Ha-vg and Ha-sd. Ha-vg and
Ha-sd dsRNAs were injected into third and first instar
larvae, respectively, to induce severe wing defect phenotypes [as per the results of the H. vigintioctopunctata RNAi
experiment (Fig. 4)]. As a consequence, H. axyridis adults
almost completely lacking wings were obtained using
Ha-vg or Ha-sd RNAi (Fig. 6). The dsRNA injection did not
have any obvious effect on feeding behaviour, and the
voracious trait of H. axyridis remained unchanged. From
these observations, it should be possible to use the RNAi
method for flightless beetle production. As this RNAi
method depends not on genetic transformation, but on
posttranscriptional regulation, we consider that it would
be easily accepted by the public from environmental and
safety points of view. It may also be possible to obtain the
flightless beetle by knock-down of other wing formation
genes via the same method. In contrast to the conventional
methods, we can improve the insect as a biological control
agent by additional gene knock-down using multiple
gene-targeted RNAi. A variety of genes involved in organ
formation and behavioural trait could be targeted to make
this biopesticide more effective and useful. We should be
able to design a ‘tailor-made insect’ for ideal biological
control using this method.
Experimental procedures
Insects
The offspring of H. vigintioctopunctata adults collected from a
potato field at Nagoya University were used in all experiments.
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
578
T. Ohde et al.
Figure 6. Production of a flightless ladybird beetle
(Harmonia axyridis) for biological control. To induce
severe phenotypes, (A) double-stranded RNA
(dsRNA) of H. axyridis vestigial (Ha-vg) was injected
into third instar larvae whereas (B) dsRNA of H.
axyridis scalloped (Ha-sd) was injected into first instar
larva. Wing-specific adult phenotypes were observed
in 79 (n = 14) and 50% (n = 6) of cases for Ha-vg
and Ha-sd RNA interference, respectively. Adults
displaying one of the most severe phenotypes are
shown. (C) An adult which was injected with
Enhanced Green Fluroescent Protein (EGFP) dsRNA
in third instar larva as a negative control. Scale
bars = 1 mm.
They were maintained at 25 °C under constant illumination. They
were reared on potato leaves from field and laboratory culture.
Experimental larvae were derived from a few batches of eggs and
were staged after every ecdysis.
Laboratory stocks of H. axyridis were derived from field collections in Aichi. They were reared as described by Niimi et al. (2005).
D. melanogaster was reared on a standard medium at 25 °C.
The embryos used as recipients for DNA injection to generate
transgenic lines were yellow white (y ac w1118) flies. The Dll-Gal4
and UAS-vg lines were described by Calleja et al. (1996) and Kim
et al. (1996), respectively.
Cloning
Total RNA was extracted from wing discs of H. vigintioctopunctata
and H. axyridis with TRIzol (Gibco BRL, Paisley, UK) according to
the manufacturer’s instructions. The first-strand cDNA was synthesized with SMART PCR cDNA Amplification Kit (Clontech,
Mountain View, CA, USA) using 1 mg of total RNA. Hv-vg and
Ha-vg cDNA fragments were amplified using vg-1 and vg-4,
and vg-5 and vg-7 primer sets, respectively. Hv-sd and Ha-sd
cDNA fragments were amplified using sd-1 and sd-4 primer sets. Hv-rp49 cDNA fragments were amplified using rp49-1
and rp49-2 primer sets. The following degenerate primers
were designed corresponding to highly conserved amino acid
sequences found in Vg amongst D. melanogaster Vg (S72379),
D. virilis Vg (GJ20351) and A. aegypti Vg (J. Williams and S.
Carroll, pers. comm.); in Sd amongst D. melanogaster Sd
(M83787), human TEF-1 (M63896) and mouse TEF-1 (L13853);
and in Rp49 amongst D. melanogaster Rp49 (U92431), mouse
Rp49 (M23453), rat Rp49 (X06483) and yeast Rp49 (Y13134).
Degenerate primers for vg
vg-1: 5′-GTIWSITGYCCIGARGTIATGTA-3′
vg-4: 5′-RTAYTGIGCCATRTTRTGRTGRTA-3′
vg-5: 5′-ATGTAYSRIGCITAYTAYCCITAYYTITA-3′
vg-7: 5′-SWRTTCCARAAISWIGGIGGRAARTT-3′
Degenerate primers for sd
sd-1: 5′-GAYGCIGARGGIGTITGG-3′
sd-4: 5′-TTYTCIARIACISWRTTCATCATRTA-3′
Degenerate primers for rp49
rp49–1: 5′-ACIAARMAITTYATIMGICA -3′
rp49–2: 5′-TGIGCIATYTCISCRCARTA-3′
(W = A + T, R = A + G, Y = T + C, S = C + G, M = C + A,
I = inosine)
PCRs were performed using 2.5 ml of the 10-fold diluted firststrand cDNA, a pair of primers from the list above and AmpliTaq
Gold (Perkin Elmer, Boston, MA, USA).
To obtain a full-length cDNA, 5′ RACE and 3′ RACE were
performed with the following gene-specific primers and the
SMART PCR cDNA Amplification Kit according to the manufacturer’s instructions.
Gene-specific primer for 5′ RACE
Hv-vg-01: 5′-GTCACCATAGCAACGCAGCAGACCTCTA-3′
Hv-vg-02: 5′-ATAGTCAGCACTACAGCGGTGCCAGTGG-3′
Hv-sd-01: 5′-CGCCAGCGCTTCCTGGAAACTCTG-3′
Hv-sd-02: 5′-ATTTTTCGTCGTCCGCAAGGTGG-3′
Gene-specific primer for 3′ RACE
Hv-vg-03: 5′-GTGAGTGCGCCTCCTGTAGCCTCTGTAC-3′
Hv-vg-04: 5′-GGTCCAGTCCTGGGTACCGGATGGGCCG-3′
Hv-sd-03: 5′-GTTCTTTCGGAAAGCAGGTGGTTG-3′
Hv-sd-04: 5′-GAAGCCCCATGTGTGAGTACATG-3′
5′ RACE and 3′ RACE were performed using 2.5 ml of the
10-fold-diluted first-strand cDNA, 10 ¥ universal primer mix,
Hv-vg-01 or Hv-sd-01 for 5′ RACE, Hv-vg-03 or Hv-sd-03 for 3′
RACE, and Advantage 2 Polymerase Mix (Clontech). The nested
PCR for 5′ RACE and 3′ RACE were performed using 0.2 ml of the
primary PCR product, nested universal primer, Hv-vg-02 or
Hv-sd-02 for 5′ RACE, Hv-vg-04 or Hv-sd-04 for 3′ RACE, and
Advantage 2 Polymerase Mix.
Sequencing and sequence analysis
The PCR product was subcloned into the EcoRV site of the
pBluescript KS + vector (Stratagene, La Jolla, CA, USA). The
nucleotide sequences of the PCR products and the flanking
regions around the restriction enzyme-digested fragments, which
were inserted into the vectors during cloning procedures, were
confirmed using the dideoxy chain-termination method by an
automatic DNA sequencer (CEQ 2000XL; Beckman Coulter,
Fullerton, CA, USA). Sequence analysis was carried out using a
DNASIS system (Hitachi Software Engineering, Tokyo, Japan).
Deduced amino acid sequences were aligned with CLUSTALW
and then adjusted manually to determine amino acid sequence
identities.
Sequence accession numbers
DDBJ/EMBL/GenBank accession numbers for the sequences
reported in this article are AB480199 for Hv-vg, AB480200 for
© 2009 The Authors
Journal compilation © 2009 The Royal Entomological Society, 18, 571–581
Vestigial and scalloped in the ladybird beetle
Hv-sd, AB480201 for Hv-rp49, AB480202 for Ha-vg and
AB480203 for Ha-sd.
Construction of vectors
To create EcoRI restriction sites at both ends of the Hv-vg open
reading frame, a PCR was performed using the first-strand cDNA
described above as a template, a primer set comprising the REHvvg-01 primer (5′-CCGGAATTCATGTGTAATATGAGTTGTTC-3′)
and the RE-Hvvg-02 primer (5′-CCGGAATTCAGATCTTCAGAACCAGTAGAGATCCT-3′), and a high-fidelity DNA polymerase
(Pyrobest; Takara, Kyoto, Japan). This PCR product was inserted into
the EcoRV site of the pBluescript KS + vector, and nucleotide
sequences were confirmed. The plasmid was then digested with
EcoRI and inserted into the EcoRI site in the pUAST vector (Brand
& Perrimon, 1993).
579
Hv-sd-05: 5′-AGTCCAGATATTGAACAGAG-3′
Hv-sd-06: 5′-TCCGTGCCAATACTTGAATA-3′
Hv-rp49-01: 5′-CAGACCGATATGGAAAATTG-3′
Hv-rp49-02: 5′-TCCTGTTTTGCATCATCAAG-3′
Acknowledgements
We express our gratitude to Dr J. Williams and Dr S. B.
Carroll for communication of results prior to publication, to
Dr M. Kobayashi and Dr M. Ikeda for helpful discussions,
to Dr W. J. Gehring and Dr T. Kadowaki for providing
vectors and fly strains, and to Ms M. Yoshioka and Dr K.
Kawakita for providing potato leaves. This study was
supported by a Grant-in-Aid from the Japan Society for
the Promotion of Science.
Generation of transgenic flies
Thirteen transgenic flies carrying the UAS-Hv-vg construct were
generated and analysed as described by Rubin & Spradling
(1982).
Scanning electron microscope (SEM) analysis
Adult or pharate adult flies were fixed in 70% EtOH, dehydrated in
a graded ethyl alcohol series and then transferred to acetone.
The flies were dried in a critical-point dryer, coated with platinum,
and observed under a S-3000N electron microscope (Hitachi,
Tokyo, Japan).
Larval RNAi
Preparation and injection of dsRNA into H. vigintioctopunctata
and H. axyridis larvae were performed according to Niimi et al.
(2005). Plasmids containing Hv-vg (the 697 bp of RT-PCR product),
Ha-vg (the 514 bp of RT-PCR product), Hv-sd (either 864 bp of
RT-PCR product or 303 bp of 3′ RACE product), Ha-sd (the 876 bp
of RT-PCR product) and EGFP (Kuwayama et al., 2006) were used
as templates for dsRNA syntheses. For the Hv-sd RNAi experiment, we used two different dsRNAs corresponding to two nonoverlapping regions of the Hv-sd cDNA, and similar results were
obtained from these two experiments. Approximately 0.1, 0.2, 0.4
and 0.6 mg of dsRNA were injected into first, second, third and
fourth (last) instar larvae, respectively. EGFP dsRNA was injected
as a negative control.
Semiquantitative RT-PCR
H. vigintioctopunctata prepupa was dissected in ice-cold
phosphate-buffered saline (137 mM NaCl, 2.68 mM KCl,
10.14 mM Na2HPO4, pH 7.2) and frozen in liquid nitrogen soon
after dissection. RNA extractions from each of the dissected
tissues and the subsequent first-strand cDNA syntheses (using
60 ng total RNA for each tissue) were performed as described
above. A reaction without reverse transcriptase was performed
with cDNA synthesis and was used as a negative control for the
RT-PCR experiment. The PCR cycle numbers were 38 cycles for
Hv-vg and Hv-rp49 and 50 cycles for Hv-sd. The following primers
were used. Hv-rp49 was used as an internal control.
Gene-specific primer for RT-PCR
Hv-vg-T02: 5′-AGAGTCTTGCACTTGAGCTT-3′
Hv-vg-T03: 5′-ATCAGTGGTCGACGAACATT-3′
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