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: firstname.lastname@example.org © 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. 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