close

Вход

Забыли?

вход по аккаунту

?

Modulation of the ecdysteroid-induced cell death by juvenile hormone during pupal wing development of Lepidoptera.

код для вставкиСкачать
152
Lobbia et al.
Archives of Insect Biochemistry and Physiology 65:152–163 (2007)
Modulation of the Ecdysteroid-Induced Cell Death by
Juvenile Hormone During Pupal Wing Development
of Lepidoptera
Saori Lobbia, Ryo Futahashi, and Haruhiko Fujiwara*
Females of the tussock moth Orgyia recens have only vestigial wings, whereas the males have normal wings. We previously
found that ecdysteroid induces both apoptotic events and phagocytotic activation in sex-specific and region-specific manners.
To investigate whether different responses to ecdysteroid are controlled at the receptor level, we cloned ecdysteroid receptor
isoforms, EcR-A and EcR-B1, in O. recens. In both male and female wings, EcR-A signal was detected in the distal region of
the bordering lacuna (BL), whereas EcR-B1 signal was detected in the proximal region of the BL. The similar expression
patterns of both EcR isoforms suggested that molecules other than EcR should be involved in different ecdysteroid responses
between male and female of O. recens. We next tested juvenile hormone (JH) effects on pupal wing morphogenesis in O.
recens. Interestingly, both JH and 20E addition induced wing degeneration not only in females but also in males. In addition,
higher concentration of JH pre-treatment of the pupal wings of the silkworm, Bombyx mori, also caused wing degeneration
under ecdysteroid treatment. These results indicate that JH modulates the ecdysteroid action to induce the cell death on pupal
wings, generally in Lepidoptera. Arch. Insect Biochem. Physiol. 65:152–163, 2007. © 2007 Wiley-Liss, Inc.
KEYWORDS: pupal wing formation; Lepidoptera; ecdysteroid; juvenile hormone; ecdysone receptor
INTRODUCTION
The acquisition of wings suitable for sustained
directional flight undoubtedly played a crucial part
in the evolution of insects, and is a major reason
for their success. Yet in spite of obvious benefits,
numbers of species within most major insect orders have secondarily lost the flight ability (Whiting et al., 2003). There is no apparent phylogenetic
predisposition to such reduction in wings; it can
evolve in any family, provided each species originally evolved in each case through fortuitous adaptive mutations followed by ecological, biological,
or environmental adaptive maintenance. However,
very little is known about the developmental processes and the molecular mechanisms of “wingless formation.”
Females of the tussock moth Orgyia recens (Lepidoptera; Lymantriidae) have only vestigial wings,
whereas the males have normal wings (Yamada,
1982; Lobbia et al., 2003). During early pupal development, the female wings degenerated drastically compared with those of males. The larval wing
discs of both sexes develop normally; epithelial
evagination and tracheal migration into the lacunae space occur. The female and male pupal wings
also show the same morphology, and have a complete structure for “bordering lacuna (BL)” at pu-
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
Contract grant sponsor: Japan Society for the Promotion of Sciences (JSPS); Contract grant sponsor: Ministry of Education, Science and Culture of Japan.
Abbreviations used: JH = juvenile hormone; EcR = ecdysone receptor; 20E = 20-hydroxyecdysone; BL = bordering lacuna.
Saori Lobbia’s present address is Department of Molecular and Computational Biology, University of Southern California, 1050 Child’s Way, 201B, Los Angeles,
CA 90089-2910.
*Correspondence to: Haruhiko Fujiwara, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Bldg.
501, Kashiwa, Chiba 277-8562, Japan. E-mail: haruh@k.u-tokyo.ac.jp
© 2007 Wiley-Liss, Inc.
DOI: 10.1002/arch.20192
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
pation. This BL is known as a common wing structure in Lepidoptera, which runs parallel to the periphery of the pupal wing and represents the future
adult wing shape. The cells in the region distal to
BL degenerate and disappear during early pupal
development (Dohrmann and Nijhout, 1988,
Kodama et al., 1995), and the region proximal to
BL forms the adult wing. It has been demonstrated
that this peripheral degeneration is induced directly
by ecdysteroid (Fujiwara and Ogai, 2001). In the
wing development of O. recens, we previously
found that ecdysteroid induces both apoptotic
events and phagocytotic activation in sex-specific
and region-specific manners (Lobbia et al., 2003).
Various concentrations of 20-hydroxyecdysone
(20E) caused massive cell death in female and cell
death distal to the BL in male cultured wings. However, at any concentrations, male wings did not
show female-type degeneration and female wings
did not show normal cell death except in the region distal to BL (Lobbia et al., 2003).
One of the key questions is what makes the differences of ecdysteroid responses on female and
male pupal wings in O. recens. One possibility is
that different 20E responses are regulated at the
receptor level. In lepidopteran species, ecdysteroid
receptor (EcR) has two isoforms that differ at Nterminal A/B trans-activation regions (Fujiwara et
al., 1995; Jindra et al., 1996; Kamimura et al.,
1997). To check this possibility, we first cloned two
isoforms of EcR, EcR-A, and EcR-B1, in O. recens
and performed in situ hybridization on pupal
wings. The other possibility is that juvenile hormone (JH) is involved in the difference of cellular
responses to ecdysteroid female and male wing development. JH is known as the key in determining
the nature of the response “status quo” to ecdysteroid (Riddiford, 1994). Besides insect metamorphosis regulation, JH is involved in longevity, caste
dividing in highly social insects, other polyphenism
and sexual maturation such as protein synthesis
in male accessory glands and female ovarian
growth (Yamamoto et al., 1988; Shemshedini et
al., 1990). The cells in larval wing discs of Lepidoptera proliferate while receiving ecdysteroid
pulses under JH. To examine JH effects on the puArchives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
153
pal wing development, we added JH to the medium of wing culture and observed the cellular responses and the wing morphogenesis.
We found that EcR-A specific signals were detected
only in the peripheral region, which corresponds to
the distal region of BL, whereas EcR-B1-specific signals were detected only in the proximal region of
BL generally for Lepidopteran pupal wings (data
for Orgyia was shown here; unpublished data for
other Lepidoptera). However, we here observed the
similar expression patterns of EcR isoform mRNA
for both male and female wings of the tussock
moth, which suggest that sex-specific ecdysone responses of O. recens are controlled by other molecules besides EcR. Interestingly, JH application
caused wing degeneration even in O. recens male
and the silkworm Bombyx mori under ecdysteroid
treatment. These results imply that JH is involved
in the sex-specific pupal wing development in O.
recens, and JH-dependent wing degeneration potentially exists widely in Lepidoptera.
MATERIALS AND METHODS
Insects
O. recens were collected at an apple orchard in
Gifu, Japan. The larvae were reared on an artificial
diet (Nihon Nosan Kogyo, Yokohama, Japan) developed for Bombyx mori. All developmental stages
of O. recens were maintained under a 16 h:8 h photoperiod (light:dark) at 26°C. Under these conditions, pupation occurs 2 days after the onset of
wandering. For JH experiments, silkworm, Bombyx
mori (C145 × N140, N4) were reared on an artificial diet (Nihon Nosan Kogyo, Yokohama, Japan)
under 12 h:12 h photoperiod (light:dark) at 26°C.
Cloning of Ecdysone Receptor Isoforms (OrEcR-A and
OrEcR-B1) From O. recens
Total RNA was extracted from the entire body
of female pupa at the P2 stage and reverse transcribed with random primer (N6) using FirstStrand cDNA Synthesis Kit (Amersham). Using the
cDNA as template, RT-PCR was performed with
154
Lobbia et al.
degenerate primer sets, which were designed based
on the EcR-A/-B1 sequences from B. mori (BmEcRA, Kamimura et al., 1997; BmEcR-B1, Kamimura
et al., 1996), M. sexta (MsEcR-A, Jindra et al., 1996;
MsEcR-B1, Fujiwara et al., 1995), and D. melanogaster (DmEcR-A, Koelle et al., 1991; DmEcR-B1,
Talbot et al., 1993). The primers for specific
isoforms are as follows: EcR-A (primer A), 5′GGACACGTNMGNGACTGGATG-3′; EcR-B1 (primer
B1), 5′-GCC-ATGGTNATGTCNCCGGA-3′; EcRA/B1
common downstream primer (primer C), 5′CATGTACATRTCCATYTCRCA-3′. PCR products
were isolated and sub-cloned into the TA cloning
vector (pGEM-T Easy vector, Promega) and sequenced by a 310 DNA sequencer (ABI).
times successively, and incubated in the hybridization solution (50% formamide, 5×SSC, 1% SDS,
50 µg/ml salmon sperm DNA and 50 µg/ml heparin in PBS) for 3 h at 44°C. Then, the wings were
hybridized with 500 ng probe RNA in 1 ml hybridization solution for 16 h at 46°C. After washing out the probe RNA with PBS, the samples were
treated with 5% skim milk in PBST. The 1:500 diluted anti-DIG-alkaline phosphatase conjugate
(Boehringer) in PBST was added and incubated for
16 h at 4°C. After washing by PBST eight times,
color reaction was performed with BCIP/NBT solution (Nakarai Tesque) for 20 min at room temperature. The samples were washed in PBST,
distilled water, and mounted into 50% glycerol.
In Situ Hybridization
Dissection and Culture of Wings
The plasmid clones carrying the EcR-A (225 nt)
and EcR-B1 (290 nt) -specific regions were purified
by a QIAGEN Plasmid Mini Kit (QIAGEN). After the
plasmid DNA was digested with SalI, a single-strand
RNA was synthesized with digoxigenin (DIG)-labeled
NTP (Boehringer) and T7 RNA polymerase (TaKaRa).
Both sense and anti-sense RNA probes were made
for EcR-A and EcR-B1, respectively.
The wings just after pupation were rinsed in
phosphate-buffered saline (PBS; 137 mM NaCl,
8.10 mM Na2 HPO 4, 2.68 mM KCl, 1.47 mM
KH 2PO 4, pH 7.4) and fixed with FEA (formalin:45% ethanol:acetic acid = 2:6:1) overnight and
washed in 70% ethanol three times. The samples
were immersed in PBST (0.1% Tween in PBS),
methanol: PBST (1:3), methanol: PBST (1:1),
methanol: PBST (3:1), and methanol three times
successively and stored at –20°C before use. The wings
were treated with hydrogen peroxide:methanol (1: 5)
solution for 5 h, rinsed with methanol three times
and PBS three times. After treatment with 2 µg/ml
proteinase K (Sigma, St. Louis, MO) at 37°C for
15 min, the samples were rinsed with PBS three
times and fixed in 10% formalin for 20 min. The
tissues were rinsed in PBS three times, PBST three
times, 0.1 M triethanolamine two times, and 0.25%
acetic anhydride/0.1 M triethanolamine, PBS three
P0 pupae (within 1 h after pupation) were surface-sterilized in 70% ethyl alcohol for 30 sec and
dissected on ice. Left forewings with the cuticle
were removed from the pupae by a scalpel as long
as they were not sclerotized and rinsed three times
in cold PBS. Each wing was incubated at 25°C on
the surface of 1.0 ml Grace’s medium (Gibco BRL)
including antibiotic-antimycotics (Gibco) in a 4well multi dish (Nunc). 20-hydroxyecdysone (20E)
(Sigma) were dissolved in 10% isopropanol, and
added to the medium to give the desired concentration prior to use. JH analog methoprene (Zoecon
Corp.) was dissolved in ethanol; fenoxycarb (Labor Dr. Ehrenstorfer-Schafers) and pyriproxyfen
(Labor Dr. Ehrenstorfer-Schafers) were dissolved in
acetone. JH analogs were kindly provided from Dr.
H. Kataoka of the University of Tokyo and added
to the medium to give the desired concentration
prior to use.
RESULTS
Region-Specific Expression Patterns of EcR Isoforms
in O. recens Pupal Wings
Using degenerated primers designed to amplify
respective EcR isoforms, the A/B and C regions in
the isoform cDNA were obtained by PCR (Fig.
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
155
Fig. 1. Identification of O. rences ecdysone receptoer
(EcR) sequences. A: Schematic structure of ecdysone receptor (EcR) (A/B, N-terminal transactivation; C, DNA
binding; D, hinge; E, ligand binding; F, C-terminal). The
primer sets used for cloning are shown by arrows. The
isoform-specific probes used for in situ hybridization are
diagrammatically represented by dotted lines. B: The
nucleotide and deduced amino acid sequences of the par-
tial isoform-specific region of the O. recens ecdysone receptor isoform. (a) OrEcR-A, (b) OrEcR-B1. C: Comparison
of amino acid sequences. (a) OrEcR-A (this study),
Manduca sexta EcR-A (Jindra et al., 1996) and Bombyx mori
EcR-A isoform (Kamimura et al., 1997). (b) OrEcR-B1 (this
study), MsEcR-B1 (Fujiwara et al., 1995) and BmEcR-B1
(Kamimura et al., 1997). *The conserved amino acid.
Dashes, gaps introduced to align sequences.
1A,B). The cloned regions were sequenced and
compared with the corresponding regions in two
isoforms of Manduca EcR (Jindra et al., 1996;
Fujiwara et al., 1995) and Bombyx EcR (Kamimura
et al., 1997) (Fig. 1C). The high conservation of
amino acid sequences among three insects indicates that these two sequences isolated here were
canonical EcR-A and -B1 in O. recens and named
OrEcRA and OrEcRB1, respectively (GenBank accession nos. AB086421-086422).
Using a specific probe for OrEcRA and OrEcRB1,
we next examined in situ hybridization for pupal
wings just after pupal ecdysis (P0). EcR-B1 specific
signals were detected only in the proximal region
of BL, both in male and female wings (Fig. 2A,C,H).
The wing samples hybridized with sense probes
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
156
Lobbia et al.
Fig. 2. Different expression patterns of EcR isoforms A and B1 in
pupal wings of O. recens. Using EcR
isoform specific probes (Fig. 1),
whole mount in situ hybridization
for pupal wing (P0 or P1) of O.
rencens was performed. A,B: EcR-B1
(A) and EcR-A (B) expressions in
male P0 forewings. Scale bar = 1
mm. C,D: EcRB1 (C) and EcR-A (D)
expression in female P0 forewings.
E,F: Negative control with using
OrEcR-B1 sense probe for female P0
wing (E) and using OrEcR-A sense
probe for male P0 wing (F). G: EcRA expression in female P1 forewings. H–J: Magnified view of C, D,
and G, respectively. Arrowheads indicate the position for bordering lacuna (BL). In J, degenetrating female
wings (red dotted line indicates the
margin) still have the BL (black dotted line). K: Schematic model of
EcR expression in pupal wings of O.
recens. Grey regions indicate localization of the intact cells. Blue regions indicate the expression of EcR
mRNAs. In female wings, successive
degradation occurs after PC.
showed no signals (negative controls) (Fig. 2E,F).
In contrast, EcR-A specific signals were detected
only in the peripheral region of the male wing (Fig.
2B), which corresponds to the distal region of BL.
In the female wing, weak EcR-A signals were detected in the distal region of the BL (Fig, 2D,I). In
the female wing at day 1 after pupation (P1) when
the wing tissue begins to degenerate, however, weak
EcR-A signals were also detected in the distal region of BL (Fig. 2G,J). It is noteworthy that the
female wings of P1 had the intact BL structure (dotted black line in Fig. 2J) when the wing margin
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
(dotted red line in Fig. 2J) has shrunken. As a
whole, the above results suggest the region-specific
expression patterns of EcR-A and EcR-B1 are very
similar between pupal wings of male and female
O. recens, although we observed a different expression pattern of EcR-A mRNA specifically in the female wing at the P1 stage (Fig. 2K).
Effects of Juvenile Hormone to Pupal Cultured Wings in
Males and Females of O. recens
To know whether JH is involved in the sexual
dimorphism formation in wings, we first investigated the effects of JH on pupal wings of O. recens
Fig. 3. The effects of JH on cultured pupal wings of O.
recens. Wings of male and female of O. recens were dissected from pupae just after pupation and cultured with
1 µg/ml-20E and with various doses (0.1, 1, and 5 µg/
ml) of JH analogs (m: methoprene, p: pyriproxyfen, and
f: fenoxycarb). A: The schematic diagrams summarizing
the results of JH effects on male (left) and female (right)
wings. The numbers of sample tested are shown in parenArchives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
157
males. The wings were dissected from the newly
molted pupae (at least 1 h after pupation) and cultured in Grace’s medium with or without 20E/JH
analogs at various concentrations. We used three
kinds of JH analogs: methoprene, pyriproxyfen,
and fenoxycarb (Fig. 3).
Interestingly, pupal wings of O. recens male
showed the female-type degeneration when cultured for 2 days with both 1 µg/ml of 20E and of
1 µg/ml of methoprene (denoted as m, in Fig. 3A).
A photo of a typical example that exhibits the “female-type degeneration” is shown in Figure 3B
(designated “D” for this phenotype). Under 1 µg/
ml of 20E, any concentration of JH (methoprene,
theses. There are three different tissue responses: D (black
column), whole wing degeneration as shown in Orygia
females; BL (grey column), cell death in the distal region
to BL, as shown in Orgyia males; N (white column), no
morphological change. B: The representative pictures of
three morphological changes (D, BL, and N) are shown,
respectively.
158
Lobbia et al.
0.1 to 5 µg/ml) could induce wing degeneration
(Fig. 3A, Table 1). Methoprene only or acetone
(solvent) could not induce the degeneration; no
cell death occurred in any region of wings (data
not shown). With a lower concentration of 20E
(0.5 and 0.1 µg/ml), 0.1 to 5 µg/µl of methoprene
did not induce the female-type degeneration but
induce cell death in the region distal to BL (Table
1 and a typical photograph [BL] in Fig. 3B).
Pyriproxyfen (denoted as p) and fenoxycarb (denoted as f) also induced the degeneration of O.
recens male pupal wings when cultured with 1 µg/
ml-20E (Fig. 3A, Table 1).
The female pupal wings of O. recens degenerated in a whole wing region when cultured with
0.1 to 1 µg/ml of 20E (Lobbia et al., 2003). The
addition of 0.1 to 5 µg/ml of methoprene to the
culture medium did not suppress this female-type
ecdysone-triggered degeneration (black bar (D) in
Fig. 3A; Table 1). The lower dose (0.1 µg/ml) of
pyriproxyfen (p) did not inhibit the female-type
ecdysone-elicited cell death in O. recens female
wings ((D) in Fig. 3A, Table 1), and the lower dose
TABLE 1. The Dose-Effectiveness of Ecdysteroid and Juvenile Hormone
Analogs on Cell Death in Orgyia recens Wing*
O. recens male
JH (µ/ml)
20E (µ/ml)
D
M5
1
0.5
0.1
1
0.5
0.1
1
0.5
0.1
3
M1
M 0.1
P5
P1
P 0.1
F5
F1
F 0.1
BL
O. recens female
N
3
3
1
2
2
3
2
2
3
2
2
3
2
2
2
2
1
1
2
2
3
2
7
5
1
0.1
1
1
0.1
3
2
2
2
1
0.1
1
1
0.1
2
D
N
2
2
2
2
2
2
2
1
1
1
BL
1
1
2
*M, methoprene; P, pyriproxyfen; F, fenoxycarb; D, degeneration; BL, cell death of
distal to bordering lacuna; N, no cell death. The numbers of samples showing
each response are indicated.
(0.1 µg/ml) of fenoxycarb (f) also did not inhibit
the female-type ecdysone-elicited cell death with
0.1 µg/ml of 20E (Table 1), while higher concentration of these two JH analogs inhibited cell death
(Fig. 3A,B, Table 1). The typical photograph for
this response (N) is shown in Figure 3B. Interestingly, cell death only in the region distal to BL,
which is a male-type wing degeneration, did not
occur at any combination of 20E and JH analogs
for female wings (Fig. 3A, Table 1).
Effects of Juvenile Hormone to Cultured Pupal Wings
of B. mori
To know the effect of JH on pupal wings on
Lepidoptera other than O. recens, we next used the
silkworm, Bombyx mori, which is a standard lepidopteran insect to show the peripheral wing degeneration in males and females (Fujiwara and
Ogai, 2001). It was an unexpected result that JH
treatment before 20E addition in the wing culture
also induced the whole wing degeneration for the
pupal wings of B. mori (Fig. 4A, Table 2). Wings
from B. mori just after pupal ecdysis were pre-incubated with 5 µg/ml of methoprene for 6 h, and
then cultured with 1 µg/ml of 20E and 5 µg/ml of
methoprene. This treatment induced whole wing
degeneration such as wings of O. recens female or
20E/JH-treated O. recens male, even in B. mori wings
(Fig. 4A,B, Table 2). However, when cultured with
1 µg/ml of 20E and 5 µg/ml of methoprene (without pre-incubation of methoprene), B. mori wings
lost cells only in regions distal to BL (Fig. 4A-(a),
Table 2). Even if pre-treated with JH analogs, lower
concentrations of methoprene (0.1 and 1 µg/ml)
did not induce the female-type degeneration in the
silkworm wings (Fig. 4A-(b), Table 2). When incubated with higher concentrations of pyriproxyfen
(1 and 5 µg/ml), pre-treatment was not necessary
to cause the whole wing degeneration (Fig. 4A,
Table 1). Pre-incubation with 0.1 and 1 µg/ml of
fenoxycarb and incubation with 1 µg/ml of 20E
induced the whole wing degeneration (Fig. 4A,
Table 2), although a higher concentration of
fenoxycarb rather inhibited cell death even in a region distal to the BL (Fig. 4A, Table 2).
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
159
Fig. 4. The effects of JH on cultured pupal wings of B.
mori. A: The dose-effectiveness of juvenile hormone analogs on cell death in Bombyx wing. The diagrams show
the effects of JH treatment (a) and additional JH pre-treatment (b) on B. mori pupal wings when wings just after
pupal ecdysis were cultured with 1 µg/ml-20E and JH analogs (m: methoprene, p: pyriproxyfen, and f: fenoxycarb).
There are three different tissue responses: D (black col-
umn), whole wing degeneration as shown in Orygia females; BL (grey column), cell death in the distal region
to BL, as shown in Orgyia males; N (white column), no
morphological change. The numbers of sample tested are
shown in parentheses. B: The representative pictures of
three morphological changes (D, BL, and N) of the silkworm wings are shown, respectively.
DISCUSSION
cause the cells proximal to BL to die in normal
development, resulting in fully developed wings;
JH addition to the culture medium could switch
the cell fate in the region proximal to BL, leading
to reduced wing experimentally. Recently, it has
been shown that JH inhibits the growth of imaginal discs during larval stages in the tobacco hornworm, Manduca sexta (Truman et al., 2006). Our
results suggest that JH not only prevents wing disc
from growing but also degenerates developed
wing tissues.
From the above results, we present a hypothetical model for ecdysteroid-induced pupal cell
death modulated by JH in Lepidoptera (Fig. 5B).
The prepupal wing epithelial cells proximal to BL
of females of Orgyia might be determined to die
under the prepupal JH influence and die during
pupal stage due to increase of 20E titer, which
results in female-specific wing degeneration. In
O. recens male and B. mori, prepupal JH does not
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
160
Lobbia et al.
TABLE 2. The Dose-Effectiveness of Ecdysteroid and Juvenile Hormone
Analogs on Cell Death in Bombyx mori Wing*
No pre-incubation
JH (µ/ml)
20E (µ/ml)
M 10
M5
1
1
0.1
1
0.1
1
0.1
M1
M 0.1
P 10
P5
P1
P 0.1
1
1
1
1
F 10
F5
F1
F 0.1
1
1
1
1
D
BL
N
3
7
3
7
7
5
5
1
2
1
D
BL
N
3
6
7
5
3
3
7
5
1
5
1
2
7
5
Pre-incubation with JHAs
1
7
5
3
2
*M, methoprene; P, pyriproxyfen; F, fenoxycarb; D, degeneration; BL, cell death of
distal to bordering lacuna; N, no cell death. The numbers of samples showing
each response are indicated.
JH may alter the cell fates, either cell death or
cell proliferation, in response to the stimulus of
20E. In O. recens female wings, methoprene did
not suppress the ecdysteroid-elicited cell death
(neither the female-type whole wing degeneration
nor cell death at distal to BL) (Fig. 3), suggesting
that methoprene has no effects on the pre-determined female wings. The observation that a higher
concentration of methoprene is required for changing the fate of the silkworm wings (Fig. 4A-(b)) suggests that methoprene has milder effects than other
JH analogs. In contrast, pyriproxyfen and fenoxycarb seem more effective in reducing ecdysteroid action, because a higher concentration of pyriproxyfen
and fenoxycarb completely blocked all ecdysteroidelicited cell death events, whole cell degeneration,
and peripheral degeneration in female wings (Fig.
3A). It is noteworthy that any hormonal treatment
on O. recens female wings could not induce the
Fig. 5. Thematic model of 20Einduced cell death modulated by
JH in lepidopteran pupal wings. A:
Male (left) and female (right) adult
O. recens. Arrowhead indicates vestigial female wing. Scale bars = 1
mm. B: Working model of the
ecdysteroid-induced cell death modulated by JH in pupal wings. In
normal specimen, 20E induces
apoptosis only in the distal to BL
in male wing of Orgyia and other
Lepidoptera (top), whereas apoptosis
is also induced in cells inside of
the BL in female wings of Orgyia
(center). In Orgyia males and the
silkworm, the fate of cells inside
the BL can be changed to female
Orgyia-type ecdysteroid-induced
cell death by JH treatment (bottom).
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
male-type peripheral degeneration (BL) (Fig. 3A),
implying that the fate of female wings just after
pupal ecdysis has been determined already and
could not be changed. In O. recens male, however,
JH can alter their fate even after the pupal ecdysis,
from peripheral degeneration to whole wing degeneration. JH also induced O. recens female-type
degeneration in the silkworm wings when pupal
wings were pre-incubated with JH analogs and then
cultured with 20E and JH analogs, although pyrproxyfen could induce such a degeneration without preincubation (Fig. 4). The difference of JH response
between O. recens male and B. mori may be dependent on the dose and type of JH analogs. These
results suggest that JH works potentially as a
“modulator” for ecdysteroid-elicited-wing degeneration in Lepidoptera.
Although the EcR expression patterns did not
differ between the male and female of O. recens
wings (Fig. 2), it supports a working hypothesis of
programmed cell death, which occurs in the peripheral region of lepidopteran pupal wings. The
pupal peak of 20E is the normal developmental
trigger for programmed cell death of wing periphery in Lepidoptera. 20E acts directly on the wing
epithelial cells distal to the BL to trigger the cellautonomous, region-specific pattern of apoptosis
(Fujiwara and Ogai, 2001). The present result suggests that isoform-specific expression of EcR is involved in the region-specific pattern of apoptosis
in pupal wings. In female wing, EcR-A signal was
weaker than male wing (Fig. 2B,D), which corresponds with the fact that the peripheral region of
the female wing remains longer than male wing
(Lobbia et al., 2003). Several histological and genetical analysis in ecdysone-triggered cell death
during lepidopteran postembryonic development
have been reported in Galleria mellonella anterior
silk glands (Jindra and Riddiford, 1996), B. mori
anterior silk glands (Tsuzuki et al., 2001), in M.
sexta prothoracic glands (Dai and Gilbert, 1999),
M. sexta motoneurons (Hoffman and Weeks, 2001;
Kinch et al., 2003), though it is not confirmed yet
which transcriptional regulatory factors such as EcR
or caspases (executor enzymes) are associated with
lepidopteran tissue degradation. Cherbas et al.
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
161
(2003) showed the EcR-A during larval stage is required for normal development of wing margin in
Drosophila. In Diptera, the wing discs have no BL
structure and there is no programmed cell death
that occurs during pupal development but the
mechanism of shaping wings involved in EcR
isoforms may be common among Pterygota.
Our results showed no remarkable differences
in the region-specific expression patterns of EcR
isoforms in the male and female wings of O. recens,
at least immediately after pupal ecdysis. Although
the different expression patterns of EcR isoforms
could not be detected at some stages before pupation (data not shown), we could not exclude the
possibility that EcR isoforms differently express for
a short period at very specific timing. We have observed weak signals of EcR-A isoform in the proximal region to BL of female wings of Orgyia at the
P0 (Fig. 2D) and P1 stages (Fig. 2G). These EcR-A
signals may be related to the whole wing degeneration of the female Orgyia, but further studies
are needed to confirm this idea. The EcR molecule
functions as a heterodimer with ultraspiracle protein (USP), and its function is sometimes modulated by another partner, such as hormone receptor
3 (DHR3) (Lam et al., 1999), Seven-up (Svp) (Kerber
et al., 1998), or Broad Complex (BRC) (Zhou et al.,
1998). Interestingly, USP has been reported to be
bound with JH in Drosophila melanogaster (Jones
and Sharp, 1997; Jones et al., 2001). BRC is upregulated by JH at the pupal stage both in D.
melanogaster and M. sexta (Zhou and Riddiford,
2002). Therefore, another possible explanation for
the effects of ecdysteroid on wing development of
female O. recens involves an alteration of the EcR
partner in the EcR-USP heterodimer.
JH plays important roles in not only insect
ecdysis and metamorphosis (Riddiford, 1994) but
also sexual maturation (Yamamoto et al., 1988;
Shemshedini et al., 1990). JH has been considered
not as a cause of sexual differentiation but as a
trigger. In pterygote insects, possession of wings
by individuals of a wing-dimorphic species is
known to carry a fitness cost, which appears in females to be reduced fecundity and a delay in the
start of oviposition of the winged morph; the ap-
162
Lobbia et al.
pearance of brachyptery (short wing) and its ovarian growth are considered to be controlled by JH
in Orthoptera (crickets and grasshoppers) (Roff,
1986; Ayoade et al., 1999). This is the first report
that JH is participating in the lepidopteran wing
dimorphism, the development and specificity of
O. recens female wing. By comparing the wing development of females and males, or other macropterous (full wing) lepidopteran insects, we hope
to gain further insight into how adaptive morphology is hormonally controlled.
ACKNOWLEDGMENTS
We thank Mr. K. Yamada, Mr. S. Niitsu, and Mr.
J. Nagase for collecting the original O. recens larvae. This work was supported by a Grant-in-Aid
from the Research for the Future Program of the
Japan Society for the Promotion of Sciences (JSPS)
and grants from the Ministry and Education, Science and Culture of Japan.
LITERATURE CITED
Ayoade O, Morooka S. Tojo S. 1999. Enhancement of short
wing formation and ovarian growth in the genetically defined macropterous strain of the brown planthopper,
Nilaparvata lugens. J Insect Physiol 45:93–100.
Hoffman KL, Weeks JC. 2001. Role of caspases and mitochondria in the steroid-induced programmed cell death of a
motoneuron during metamorphosis. Dev Biol 229:517–
536.
Jindra M, Riddiford LM. 1996. Expression of ecdysteroid-regulated transcripts in the silk gland of the wax moth, Galleria mellonella. Dev Genes Evol 206:305–314.
Jindra M, Malone F, Hiruma K, Riddiford LM. 1996. Developmental profiles and ecdysteroid regulation of the
mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta.
Dev Biol 180:258–272.
Jones G, Sharp PA. 1997. Ultraspiracle: an invertebrate nuclear
receptor for juvenile hormones. Proc Natl Acad Sci USA
94:13499–13503.
Jones G, Wozniak M, Chu Y, Dhar S, Jones D. 2001. Juvenile
hormone III-dependent conformational changes of the
nuclear receptor ultraspiracle. Insect Biochem Mol Biol
32:33–49.
Kamimura M, Tomita S, Fujiwara H. 1996. Molecular cloning of an ecdysone receptor (B1 isoform) homologue from
the silkworm, Bombyx mori, and its mRNA expression during wing disc development. Comp Biochem Physiol
113:341–347.
Cherbas L, Hu X, Zhimulev I, Belyaeva E, Cherbas, P. 2003. EcR
isoforms in Drosophila: testing tissue-specific requirements by
targeted blockade and rescue. Development 130:271–284.
Kamimura M, Tomita S, Kiuchi M, Fujiwara H. 1997. Tissuespecific and stage-specific expression of two silkworm
ecdysone receptor isoforms ecdysteroid-dependent transcription in cultured anterior silk glands. Eur J Biochem
248:786–793.
Dai J, Gilbert LI. 1999. An in vitro analysis of ecdysteroidelicited cell death in the prothoracic gland of Manduca
sexta. Cell Tissue Res 297:319–327.
Kerber B, Fellert S, Hoch M. 1998. Seven-up, the Drosophila
homolog of the COUP-TF orphan receptors, controls cell
proliferation in the insect kidney. Genes Dev 12:1781–1786.
Dohrman CF, Nijhout NF. 1988. Development of the wing
margin in Precis coenia (Lepidoptera: Nymphalidae). J Res
Lepi 27:151–159.
Kinch G., Hoffman KL, Rodrigues EM, Zee MC, Weeks JC.
2003. Steroid-triggered programmed cell death of a motoneuron is autophagic and involves structural changes in
mitochondria. J Comp Neurol 457:384–403.
Fujiwara H, Jindra M, Newitt R, Palli SR, Hiruma K, Riddiford
LM. 1995. Cloning of an ecdysone receptor homolog from
Manduca sexta and the developmental profile of its mRNA
in wings. Insect Biochem Mol Biol 25:845–856.
Fujiwara H, Ogai S. 2001. Ecdysteroid-induced programmed
cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori. Dev Genes Evol
211:118–123.
Kodama R, Yoshida A, Mitsui T. 1995. Programmed cell death
at the periphery of the pupal wing of the butterfly, Pieris
rapae. Roux’s Arch Dev Biol 204:418–526.
Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P,
Hogness DS. 1991. The Drosophila EcR gene encodes an
ecdysone receptor, a new member of the steroid receptor
superfamily. Cell 67:59–77.
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Modulation of Pupal Wing Fate by JH
163
Lam G., Hall BL, Bender M, Thummel CS, 1999. DHR3 is
required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis. Dev Biol 212:204–216.
Truman JW, Hiruma K, Allee JP, MacWhinnie SGB, Champlin
DT, Riddiford LM. 2006. Juvenile hormone is required to
couple imaginal disc formation with nutrition in insects.
Science 312:1385–1388.
Lobbia S, Niitsu S, Fujiwara, H. 2003. Female-specific wing
degeneration caused by ecdysteroid in the Tussock Moth,
Orgyia recens: Hormonal and developmental regulation of
sexual dimorphism. J Insect Sci 3:1–7.
Tsuzuki S, Iwami M, Sakurai S. 2001. Ecdysteroid-inducible
genes in the programmed cell death during insect metamorphosis. Insect Biochem Mol Biol 31:321–331.
Riddiford LM. 1994. Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Adv Insect Physiol 24:213–274.
Roff DA. 1986. The evolution of wing dimorphism in insects.
Evolution 4: 1009-1020.
Shemshedini L, Lanoue M, Wilson TG. 1990. Evidence for a
juvenile hormone receptor involved in protein synthesis
in Drosophila melanogaster. J Biol Chem 265:1913–1918.
Talbot WS, Swyryd EA, Hogness DS. 1993. Drosophila tissues
with different metamorphic responses to ecdysone express
different ecdysone receptor isoforms. Cell 73:1323–1337.
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Whiting MF, Bradler S, Maxwell T. 2003. Loss and recovery
of wings in stick insects. Nature 421:264–267.
Yamamoto K, Chadarevian A, Pellegrini M. 1988. Juvenile
hormone action mediated in male accessory glands of
Drosophila by calcium and kinase C. Science 239:916–919.
Zhou B, Riddiford LM. 2002. Broad specifies pupal development and mediates the ‘status quo’ action of juvenile hormone on the pupal-adult transformation in Drosophila and
Manduca. Development 129:2259–2269.
Zhou B, Hiruma K, Shinoda T, Riddiford LM. 1998. Juvenile
hormone prevents ecdysteroid-induced expression of
Broad Complex RNAs in the epidermis of the tobacco
hornworm, Manduca sexta. Dev Biol 203:233–244.
Документ
Категория
Без категории
Просмотров
3
Размер файла
391 Кб
Теги
development, lepidoptera, death, induced, wing, pupal, juvenile, modulation, ecdysteroids, hormone, cells
1/--страниц
Пожаловаться на содержимое документа