Insect cytokine, growth-blocking peptide, is a primary regulator of melanin-synthesis enzymes in armyworm larval cuticle Yosuke Ninomiya 1 and Yoichi Hayakawa 2 1 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan 2 Department of Applied Biological Science, Saga University, Japan Animal color patterns have evolved under various selective pressures such as predator avoidance, sexual selection, and thermotolerance. The variety of color patterns reflects a complex interplay between natural selection and color phenotypes [1]. One of the most widespread pigments in the biological world is mel- anin, which consists of two classes: eumelanins, which are black or brown, and phaeomelanins, which are red, orange, or yellow [2]. In vertebrates, melanins are derived from the catecholamine precursors 3,4-dihyd- roxy-l-phenylalanine (Dopa) and dopamine, which are synthesized from tyrosine by two enzymes, tyrosinase and Dopa decarboxylase (DDC), that convert tyrosine to Dopa and dopamine, respectively [3,4]. In insects, although tyrosinase may play a role in melanin synthe- sis, tyrosine hydroxylase (TH) is important in provi- ding the dopamine precursor. Black and brown patterns are conserved in the cuticles of a broad range of insect species, which suggests their evolutionary importance as adaptive traits [4–6]. Keywords calcium ion; epidermal cell; growth-blocking peptide; insect; uric acid Correspondence Y. Hayakawa, Department of Applied Biological Science, Saga University, Honjo-1, Saga, 840-8502, Japan Fax ⁄ Tel: +81 952 28 8747 E-mail: hayakayo@cc.saga-u.ac.jp (Received 20 December 2006, revised 20 January 2007, accepted 1 February 2007) doi:10.1111/j.1742-4658.2007.05724.x The cuticles of most insect larvae have a variety of melanin patterns that function in the insects’ interactions with their biotic and abiotic environ- ments. Larvae of the armyworm Pseudaletia separata have black and white stripes running longitudinally along the body axis. This pattern is empha- sized after the last larval molt by an increase in the contrast between the lines. We have previously shown that 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxylase (DDC) is activated during the molt period by prefer- ential enhancement of its transcription in the epidermal cells beneath the black stripes. This study demonstrated that tyrosine hydroxylase (TH) expression is activated synchronously with DDC. Furthermore, enhance- ment of DDC and TH transcription is due to an increase in cyotoplasmic Ca 2+ , which is induced by the insect cytokine, growth-blocking peptide (GBP). Enhanced gene expression for both enzymes was induced by substi- tution of the calcium ionophore A23187, and completely blocked by EGTA. A GBP-induced increase in cytoplasmic Ca 2+ was seen in epider- mal cells under the black stripes but not those beneath the white stripes, suggesting that a difference in Ca 2+ concentration in stripe cells leads to the specific expression of DDC and TH genes. Based on the fact that epi- dermal cells beneath the white stripes contain abundant granules composed mainly of uric acid, which can form a complex with Ca 2+ and hence decrease its free concentration, we discuss the possibility that uric acid, as well as GBP, contributes to the difference in cytoplasmic Ca 2+ within the epidermal cells. Abbreviations DDC, Dopa decarboxylase; Dopa, 3,4-dihydroxy- L-phenylalanine; GBP, growth-blocking peptide; TH, tyrosine hydroxylase. 1768 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS Larvae of the armyworm Pseudaletia separata have a relatively simple color pattern composed largely of black and white stripes in the dorsal cuticle that run lon- gitudinally along the body axis [7]. It has been reported that dopamine melanin is the predominant black mel- anin in insect cuticles [3,8,9]. A previous study con- firmed this by demonstrating that DDC mRNA and protein are expressed specifically in the epidermal cells under the black stripes but not those under the white stripes [7]. Enhanced DDC expression increases the dop- amine concentration in the black-stripe dorsal cuticles. Furthermore, we have previously shown that an insect cytokine, growth-blocking peptide (GBP), enhances DDC expression in the integument [10,11]. We therefore inferred that local enhancement of DDC expression by GBP elevates the dopamine concentration in the epider- mal cells where dopamine melanin is actively synthes- ized to produce the black stripes in the cuticle. In this study, we confirmed this by demonstrating that GBP contributes to the enhancement of gene expression for two key enzymes of melanin synthesis, TH and DDC, and by further examining the mechan- ism of enhancement of expression for both enzymes in epidermal cells beneath the black stripes. We demon- strated that gene expression for both enzymes is enhanced by GBP-induced elevation of cytoplasmic Ca 2+ concentrations in the epidermal cells. Results TH activity and gene expression Prior studies have indicated that DCC expression in the cuticle of armyworm larvae (P. separata)is enhanced in the epidermal cells under the dorsal black stripes during the last larval molt [7]. Because TH is another key enzyme in the biosynthesis of Dopa and dopamine, precursors of Dopa and dopamine melanin, respectively, the predominant black melanin of the insect cuticle [3,9], we measured integument TH activ- ity during the last larval ecdysis. TH activity remained very low in the ventral cuticles, which are without black stripes, however, the activity in the dorsal cuti- cles increased sharply during the last ecdysis (Fig. 1). This pattern is also seen for DDC activity during the last ecdysis (Fig. 1, inset). To characterize the TH expression profile in the armyworm larvae, we cloned its cDNA using RT-PCR and RACE. The fact that two mRNA isoforms, one long and one short, are expressed in the epidermal and brain cells, respectively, is consistent with that reported in Drosophila TH (Fig. 2) [12,13]. The predicted sequence shares the highest similarity, 95%, with that of the butterfly Papilio xuthus. Similarities between P. separata TH and THs reported for other animals, including humans, are also > 70%, i.e. 79, 78, 72, 72, and 71% with those of Drosophila melanogaster, Apis melifera, Rattus norvegicus, Mus musculus, and Homo sapiens, respectively. Using anti-TH IgG and TH cDNA, western and northern blottings were carried out. Levels of both TH protein and mRNA were clearly increased in the integument of day 0 of the last instar larvae of the armyworm (Fig. 3). Furthermore, immunocytochemis- try and in situ hybridization showed that TH protein and mRNA are expressed in the epidermal cells directly under the black stripes (Fig. 4). Mechanism of TH and DDC gene expression in epidermal cells To find sequence motifs commonly present in the upstream regions of the TH and DDC genes, we per- formed BLAST searches of the Bombyx mori genome database to identify genes homologous to P. separata L5D2 TH activity (pmol/min/µg protein) 0 2 4 6 Dorsal Ventral vitca C D D n ( y t imom/lniµ / g p or n iet ) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 L5D2 L6D0 L6D1 a*) b*) 18 16 14 10 12 8 L6D0 L6D1 Fig. 1. TH activity in integuments of day 2 penultimate (L5D2), day 0 last instar (L6D0), and day 1 last instar (L6D1) larvae. (Inset) DDC activity in integuments from the same larval stages. a*, Signi- ficantly different from TH activity of L5D2 larval dorsal integument (P<0.005, Student’s t-test); b*, significantly different from TH activity of L6D0 larval ventral integument (P<0.005, Student’s t-test). Bar ¼ mean ± SD of five independent determinations. Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1769 TH and DDC. Analysis of ~ 5 kb of the 5¢-flanking region of both enzyme genes showed the presence of five common cis-elements responsible for gene expres- sion (Fig. 5). Three of these five cis-elements, AP-1, Ets, and CREB, which are found repeatedly in the 5¢-flanking region, have been reported to be regulated by Ca 2+ -dependent protein kinases [14–16]. Although we did not find any evidence that these cis-elements contribute to controlling gene expression in the insect epidermal cells, this information prompted us to test the possibility that the synchronous expression of both enzyme genes is regulated by Ca 2+ concentration. To test this possibility, isolated integuments were incubated with the calcium ionophore A23187, and the expression levels of TH and DDC genes were meas- ured. Both gene expression levels were enhanced at 6 h following incubation with A23187 in the dorsal integu- ment with black stripes, but not in the ventral integu- ment without black stripes (Fig. 6A). Because we have previously shown that the insect cytokine GBP strongly induces Ca 2+ influx into brain synaptosomes [20], we examined whether incubation of the dorsal integument with GBP enhanced the expression levels of both genes. Incubation of isolated tissues with 1 nm GBP elevated the expression levels of both genes, whereas the addition of 1 mm EGTA prevented GBP- dependent elevation of gene expression (Fig. 6B). When the concentration of added Ca 2+ was higher Met 219 376 Epidermis form of PsTH Stop 1884531 5’UTR 3’UTR 5’UTR 3’UTR 219 Met 376 1728 Stop Brain form of PsTH Fig. 2. Gene structures of P. separata TH expressed in integuments and brains. Epi- dermis (AB274834) and brain (AB274835) forms of P. separata TH genes. Note that the epidermal form has an additional coding sequence (156 bp portion shown by diago- nal lines) near the 5¢)end. L5D2 L6D0 L6D1 L5D2 L6D0 L6D1 116kDa A B 66kDa 42kDa Commassie blue Western(4CN) rRNA DV L5D2 L6D0 L6D1 DV D V Fig. 3. Western (A) and northern (B) blots of TH from P. separata penultimate and last instar larval integuments. (A) Coomassie Brilliant Blue-stained integument proteins (left) and immunoblots of TH with anti-TH IgG (right). (B) 32 P-labeled TH cDNA was hybridized to northern blot of total RNAs from dorsal (D) and ventral (V) integuments. Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa 1770 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS than the concentration of EGTA, expression of both enzyme genes was enhanced by GBP (Fig. 6B). Incuba- tion of the ventral integument with GBP did not ele- vate expression of either gene above the detectable level (data not shown). The data indicate that GBP- Anti PsTH IgG Rabbit IgG antisense probe sense probe A B Fig. 4. TH protein and mRNA expression in vertical sections of integument. (A) Immunohistochemical staining of TH protein with the P. separata anti-TH IgG. (B) In situ hybridization of TH mRNA probed with antisense or sense probes of TH RNAs. Tissue sec- tions were prepared from day 0 last instar larvae. Note that immu- noreactivity and hybridization signals are much stronger in the epidermal cells under the black stripe (as indicated by arrows) than those under the white stripe. -5.3 Bombyx mori DDC ORF -5.2 Bombyx mori TH ORF AP-1 Ets G motif CREB Grh Fig. 5. Cis-elements in the 5¢-flanking region of B. mori DDC and TH genes. TH DDC Actin B in vitro 6h incubation TH DDC Actin A 0 1 1 1 M m 1 A SB M n 1 1 M m m M M n aC lC PBG ATGE 2 PBG M n 1 M n PBG AT GE 20pmol BSA Dorsal Ventral Dorsal Ventral 20pmol GBP C in vivo 6h after injection TH DDC Actin in vitro 6h incubation Dorsal DorsalVentral Ventral A23187 0µM A23187 50µM Fig. 6. RT-PCR analysis of TH and DDC expression in the integu- ment of day 1 last instar larvae. (A) Clear bands of TH and DDC mRNAs expressed in the dorsal integument after coincubation with the calcium ionofore A23187. (B) GBP-induced expression of TH and DDC mRNAs was observed only when Ca 2+ is present in the integument incubation medium. (C) GBP-induced expression of TH and DDC mRNAs was observed only in the dorsal integument 6 h after injection of GBP. Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1771 dependent expression of the two enzyme genes in the dorsal integument requires cytoplasmic Ca 2+ . Specific expression of TH and DDC genes in the integument To produce black stripes, GBP has to activate expres- sion of TH and DDC genes in the epidermal cells directly under the black stripes. Therefore, we exam- ined whether GBP-dependent enhancement of gene expression occurs in the integument containing the black stripes. Injection of 20 pmol of GBP enhanced the expression of both enzymes in the dorsal integu- ment containing the black stripes only (Fig. 6C), indi- cating that GBP must increase Ca 2+ concentrations specifically in the epidermal cells under the black stripes. We monitored the Ca 2+ concentration in the epider- mal cells under both black and white stripes using a laser confocal microscope. Addition of GBP to the incubation medium containing isolated integuments elevated the cytoplasmic Ca 2+ concentration in the epidermal cells under the black stripes, but not in those under the white stripes (Fig. 7A). Furthermore, significant elevation of the cytoplasmic Ca 2+ concen- tration was not seen in white stripe cells even with addition of A23187, suggesting that calcium was not present as free ions. The data prompted us to examine how GBP main- tains stable cytoplasmic Ca 2+ concentrations in epi- dermal cells under the white stripes. We focused our attention on the abundant granules composed primar- ily of uric acid which have previously been found only in the epidermal cells under the white stripes A a) b) d) f) c) a) b) d) c) e) B Fig. 7. Ca 2+ influx imaging of GBP (A) and calcium ionophore (B) in the dorsal integu- ment of day 1 last instar larvae. (A) a, c, e, Laser transmission images show the center of the dorsal integument; b, d, f, Ca 2+ indi- cator Fluo-3 fluorescence (green) of the same integument was overlaid with the laser transmission image. Note that GBP strongly induced the Ca 2+ influx only in the black stripe integument (d), and the influx was largely abolished by removal of extra- cellular Ca 2+ by EGTA (f). (B) a, c, Laser transmission images show the center of the dorsal integument; b, d, Fluo-3 fluorescence (green) of the same integument was over- laid with the laser transmission image. Note that calcium ionophore A23187 solubilized in dimethylsulfoxide strongly induced the Ca 2+ influx only in the black stripe integument (d). Control integument was treated with Grace’s medium containing 0.1% dimethyl- sulfoxide (the same concentration as d) (a, b), B and W boxes indicate the black and white stripe regions, respectively. Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa 1772 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS and ventral epidermal cells (Fig. 8, insert) [7]. The effect of uric acid on soluble calcium ion concentra- tions was examined by coincubating CaCl 2 with uric acid. We found that incubation with > 70 mgÆmL )1 uric acid significantly decreased the free Ca 2+ concen- tration (Fig. 8). The decrease in free Ca 2+ occurred in a time-dependent manner: free Ca 2+ in the incuba- tion medium had disappeared completely after coincu- bation for 3 h (Fig. 8). Based on these observations, we presume that the abundant uric acid granules con- tributed to the change in cytoplasmic calcium from the soluble to insoluble solid phase. However, we need more data to substantiate this because we do not have any direct evidence that a high level of uric acid is also present in the cytosol of white stripe epi- dermal cells. Discussion Prior studies indicated that, during the last larval ecdy- sis, DDC is preferentially expressed only in the epider- mal cells under the black stripes of armyworm last instar larvae [7]. High expression of DDC produces dopamine, which is a precursor for dopamine melanin, the predominant black pigment of the insect cuticle; the blackish color is thereby enhanced in the black stripes. Although the DDC gene is not expressed in detectable levels in epidermal cells under the white stripes, uric acid accumulates and forms abundant white granules. We therefore proposed that the differ- ence in DDC activity and the presence of white uric acid granules produce the black and white stripes seen in the cuticles of armyworm larvae. However, the mechanism by which epidermal cells actively express the DDC gene directly under the black stripes remains unknown. In this study, we focused on another key enzyme, TH, which is involved in the dopamine syn- thesis pathway, and characterized its gene structure and expression pattern. TH expression in epidermal cells is basically the same as that of DDC: TH is act- ively expressed during larval ecdysis only in the epider- mal cells under the black stripes. Furthermore, we found that the 5¢-flanking regions of B. mori TH and DDC genes contain five common cis-elements (AP-1, Ets, CREB, G-motif and Grh), three (AP-1, Ets and CREB) of which have been reported to be regulated by Ca 2+ -dependent protein kinases [14–16]. The importance of these cis-elements, especially in terms of TH expression, was demonstrated, mutation of either the AP-1 or CREB site abolished expression in adult transgenic mice [17]. Furthermore, it has been reported that, in Drosophila transgenic embryos, the Ddc pro- moter-green fluorescent protein reporter gene with point mutations in the single CREB and the three AP-1-like consensus sites showed a marked reduction in wound-induced activation compared with wild-type reporter controls [18]. This information prompted us to examine the relationship between cytoplasmic Ca 2+ concentration and the expression levels of both enzymes. A calcium ionophore (A23187) elevated expression of both enzyme genes, suggesting that Ca 2+ is a regulatory factor leading to optimal activation of the expression of both genes. The next logical step was to find the principal agent causing cytoplasmic Ca 2+ elevation in the epidermal cells. We considered GBP to be the most promising candidate based on two previous observations: GBP acted directly on the epidermal cells to induce expres- sion of the DDC gene [11,19,20], and GBP enhanced Ca 2+ influx into brain synaptosomes in a concentra- 0 1 60 180 Time (min) Relative fluorescence intensity 2µm hWit ts eriep a l Bc epir ts k 2.0 1.8 1.6 1.4 1.2 1 Fig. 8. Affect of uric acid on free Ca 2+ concentration in the incuba- tion medium. CaCl 2 (250 nM) was incubated with 70 mgÆ mL )1 uric acid for the indicated periods. Relative fluorescence intensity was calculated as the difference between each fluorescent value and that of the deionized water. Each bar represents the mean of two independent determinations. (Inset) Transmission electron micro- graphs of epidermal cells under the black and white stripes. Note that only white stripe cells contain a large number of white gran- ules composed mainly of uric acid. Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1773 tion-dependent manner [21]. As expected, GBP activa- ted TH and DDC gene expression only in the presence of free Ca 2+ (Fig. 6B). Because we used a type of EGTA that is not able to enter the cytosol [22], we interpreted the results as suggesting that GBP stimu- lates the epidermal cells to trigger Ca 2+ entry via cer- tain Ca 2+ channels. Although we do not have substantial data on GBP receptors and Ca 2+ channels in the epidermal cells, it is reasonable to speculate that activation of a GBP receptor opens a certain type of Ca 2+ channel. Voltage-independent calcium channels might contribute to GBP-induced Ca 2+ entry into the epidermal cells [23]; furthermore, it is also possible that the recently identified Ca 2+ release-activated Ca 2+ (CRAC) channels play a role in this system [24,25]. Monitoring cytoplasmic Ca 2+ concentration in the cuticles revealed a significant difference, GBP increased the Ca 2+ concentration in epidermal cells under the black stripes, but not those under the white stripes, suggesting that the GBP receptor population is higher in the epidermal cells under the black stripes than those under the white stripes. Preliminary experiments of competitive receptor-binding assays using isolated cuticles and 125 I-labeled GBP were carried out, but we did not find any significant difference in the ligand- binding capacities of black and white cuticles (data not shown), indicating that the populations of GBP recep- tors in the epidermal cells under both stripes are not significantly different. Thus, we proposed another mechanism by which DDC and TH genes are preferen- tially expressed in the epidermal cells under the black stripes. It has been reported that uric acid is involved in urinary stone formation; the presence of uric acid increases the rate of stone growth from calcium salts such as calcium oxalate and calcium phosphate in the human kidney [26–28]. If a similar reaction proceeds in the armyworm epidermal cells under the white stripes, cytoplasmic free calcium ions could be decreased. As partial confirmation of this, using laser confocal micr- oscopy, Ca 2+ -induced fluorescence of Fluo-3 was not seen in the cells under the white stripes of integument that had been treated with GBP or calcium ionophore (Fig. 7). We also showed that coincubation of CaCl 2 with uric acid decreased the free Ca 2+ concentration in the incubation medium (Fig. 8). However, we were unable to measure the cytoplasmic uric acid concentra- tion in the epidermal cells because it is too difficult to prepare a sample containing only the uric acid solubi- lized in the cytoplasm. Therefore, the role of uric acid in controlling Ca 2+ concentration in white stripe epi- dermal cells should be carefully substantiated in the future. We also cannot exclude the possibility that the white stripe epidermal cells possess other Ca 2+ -buffer- ing systems. It will be interesting to determine the mechanisms by which white stripe cells efficiently take up large amounts of uric acid from the hemocoel. In mammals, including humans, several types of urate transporter, such as a voltage-sensitive urate transporter and a ura- te ⁄ anion exchanger, have been identified [29–31]. Although the molecular mechanisms underlying urate transport in insects are largely unknown, it is reason- able to expect the presence of an active urate transpor- ter in the plasma membrane of the epidermal cells under the white stripes of the armyworm larvae. In summary, two key enzymes in the dopamine syn- thesis pathway, TH and DDC, are preferentially expressed in the epidermal cells under the black stripes on the armyworm larva cuticle. Expression of both enzyme genes is enhanced by the insect cytokine, GBP, via an increase of cytoplasmic Ca 2+ concentrations. Our preliminary data showed that the epidermal cells under the white stripes contain as many GBP receptors just like the black stripe cells (data not shown). How- ever, GBP-dependent enhancement of expression of the two enzyme genes does not occur in the white stripe cells. Although we do not have sufficient data to explain this mechanism, one possible mechanisms is that extremely high concentrations of uric acid decrease the cytoplasmic Ca 2+ concentration, thereby preventing expression of both enzyme genes in the cells under the white stripes. As a consequence, melanin synthesis proceeds only in the epidermal cells under the black stripes, which produce the unique stripe pat- tern in the cuticle of armyworm larvae. Experimental procedures Animals Pseudaletia separata larvae were reared on an artificial diet at 25 ± 1 °C in a photoperiod of 16:8 light ⁄ dark [10]. Pen- ultimate instar larvae undergoing ecdysis between 4 and 4.5 h after starting the light period were designated as day 0 last instar larvae. Chemicals l-(3,5-H 3 )Tyrosine was purchased from Amersham Bio- science (Uppsala, Sweden). l-Tyrosine and A23187 (calcium ionophore) were obtained from Nacalai Tesque Co. (Kyoto, Japan) and Fluo-3 from Dojindo Laboratories (Kumamoto, Japan), respectively. Grace’s insect cell culture medium was purchased from Gibco-BRL (Rockville, MD). All other chemicals were reagent grade. Regulation of melanin synthesis in insect cuticle Y. Ninomiya and Y. Hayakawa 1774 FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS TH assay Dissected tissue was homogenized in 100 lL of ice-cold 50 mm Hepes-KOH buffer (pH 7.0) containing 0.2 m sucrose and 0.1% phenylthiourea by sonication (10 pulses at 50 W). The homogenate was assayed directly for TH activity using a slightly modified version of the method des- cribed by Vie et al. [32]. The reaction mixture (total vol- ume: 200 lL) consisted of 50 mm Hepes-KOH buffer (pH 7.0), 1 mm dithiothreitol, 0.3 mm (6R)-5,6,7,8-tetra- hydrobiopterin dihydrochloride, 10 lm ferrous ammonium sulfate, 10 000 U catalase, 50 lml-tyrosine, 12.5 mCiÆ- mmol )1 l-(3,5-H 3 )tyrosine and the enzyme preparation. The mixture, without (6R)-5,6,7,8-tetrahydrobiopterin dihy- drochloride, was equilibrated at 37 °C for 5 min and after adding (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride, the reaction was performed for 30 min. After adding 600 lL of ice-cold 0.5 m trichloroacetic acid to stop the reaction, the mixture was centrifuged at 20 000 g for 10 min at 4 °C. The supernatant was then transferred into a microtest tube containing 120 mg of Norit A. The tube was mixed occasionally for 30 min at 25 °C, and then cen- trifuged at 20 000 g for 10 min at 4 °C. One hundred microliters of the supernatant were transferred to a vial with 1 mL of scintillation cocktail, and the radioactivity was counted in a liquid scintillation counter (Aloka LSC- 5100, Tokyo, Japan). Cloning and sequence analysis of TH cDNA Total RNA was isolated from integuments of day 0 last instar larvae using TRIzol reagent (Gibco-BRL) according to the manufacturer’s instructions. Five micrograms of total RNA was reverse transcribed with oligo(dT) primer using ReverTra Ace (TOYOBO, Osaka, Japan). Degenerated oligonucleotide primers were designed on the basis of sequences of D. melanogaster and H. sapiens :5¢-TTYGCN CARTTYWSNCARGA-3¢ and 5¢-TGRTCRTGRTANGG YTGNAC-3¢. PCR cycling conditions were 35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min. PCR products were isolated and subcloned into the TA clo- ning vector (pGEM-T Easy vector, Promega, Madison, WI) and sequenced by a 310 DNA sequencer (ABI, Wellesley, MA, USA). Full-length cDNA was isolated using the RACE technique with a RACE system kit (Gibco-BRL). Computer-assisted sequence analyses were performed by genetyx-mac v. 10.0 (Software Development Co., Tokyo, Japan). RT-PCR Two micrograms of total epidermal RNA was reverse transcribed with oligo(dT) primer using ReverTra Ace (TOYOBO). The cDNA was amplified with TH-specific primer pair (5¢-CAGCTGCCCAGAAGAACCGCGAGA TG-3¢, +11 to +36; and 5¢-GAACTCCACGGTGAACC AGT-3¢, +1286 to +1305 bp), DDC-specific primer pair (5¢-ATGGAGGCCGGAGATTTCAAAG-3¢, +1 to +22 bp; and 5¢-ACGGGCTTTAAGTATTTCATCAGGC-3¢, +1405 to +1428 bp) and actin primer pair (5¢-TTCGAGCAG GAGATGGCCACC-3¢ and 5¢-GAGATCCACATCTGYTG GAAGGT-3¢). PCR was conducted under the following conditions: 25 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C 2 min. Northern hybridization Twenty micrograms of total RNA was separated on a 1% formaldehyde–agarose gel and transferred onto a Hybond N + nylon membrane. Hybridization was performed at 42 °C for 16 h in 50% formaldehyde containing 5· SSPE and 0.5% SDS. The cDNA (nucleotides 11–1305) labeled with [ 32 P]dCTP was used as a probe. The membrane was washed with 2· NaCl ⁄ Cit containing 0.1% SDS at 42 °C, according to the protocols of Sambrook et al. [33]. Autora- diogram was analyzed using a BAS-1500 imaging analyzer (Fuji Film, Tokyo, Japan). Production of polyclonal antibody The cDNA fragment containing the ORF of P. separata TH was cloned into pET32a (Novagen, San Diego, CA, USA) and expressed as a recombinant protein in Escheri- chia coli, BL21(DE3). Production of the protein containing 6 histidine-tag residues was induced by 0.4 mm isopropyl thio-b-d-galactoside for 3 h at 37 °C. The recombinant pro- tein was purified by a Chelating Sepharose Fast Flow col- umn (Amersham Pharmacia Biotech, Piscataway, NJ) charged with nickel. The purified protein was emulsified by Titer Max Gold (CytRx Corporation, Los Angeles, CA, USA) and injected into a rabbit to generate an anti-TH IgG. Anti-TH IgG was precipitated by adding ammonium sulfate to 40% saturation and further purified by an affinity column of protein G–Sepharose (Amersham Bioscience). Immunoblotting and immunocytochemical analyses Integuments dissected from larvae were homogenized in 80 mm Tris ⁄ HCl buffer (pH 8.8) containing 1% SDS and 2.5% 2-mercaptoethanol, and centrifuged at 20 000 g for 10 min at 4 °C. The supernatant was boiled for 5 min and applied to a SDS ⁄ PAGE gel. Proteins separated by SDS ⁄ PAGE were electrically transferred to a poly(vinylid- ene difluoride) membrane filter, blocked and probed with the primary antibody, anti-TH IgG. After washing thor- oughly with 0.05% Tween 20 in Tris-buffered saline (10 mm, 150 mm NaCl, pH 7.5), antigens were detected Y. Ninomiya and Y. Hayakawa Regulation of melanin synthesis in insect cuticle FEBS Journal 274 (2007) 1768–1777 ª 2007 The Authors Journal compilation ª 2007 FEBS 1775 using peroxidase-conjugated secondary antibody and a 4-chloro-1-naphtol Immun-Blot Colorimetric Assay kit (Bio-Rad Laboratories, Hercules, CA) [34]. Immunohistochemistry examination of integument sec- tions was performed essentially as described by Somogyi & Takagi [35], except that isolated tissues were fixed with 4% paraformaldehyde in NaCl ⁄ P i (pH 7.4) for 2 h on ice. Anti- gens were detected using HRP-conjugated anti-rabbit IgG. In situ hybridization The DIG-labeled TH RNA probe was prepared using the Roche Biochemicals kit (Roche Molecular Biochemicals, Indianapolis, IN). Hybridization and washing were carried out as described previously [36]. Dissection and culture of integument A whole abdominal integument (day 1 last instar larva) of the test armyworm larva was dissected between the first and second segments. Care was taken to remove all the adhering fat body tissue from the integument. The dissected integument was separated into dorsal and ventral parts. After washing with NaCl ⁄ P i , the tissues were lightly blotted with filter paper, weighed and immediately used for experi- ments. Pieces of dorsal larval integument were cultured in Grace’s medium with or without 1 nm GBP at 25 °C. As a control, 1 n m BSA was added to the medium. To remove extracellular free Ca 2+ , Grace’s medium containing 1 mm EGTA was used. A23187 was dissolved in dimethylsulfox- ide and added to the medium. In vivo experiment Total RNAs were extracted from the dorsal and ventral integuments of day 1 last instar larvae 6 h after injection of 20 pmol of GBP. Control larvae were injected with 20 pmol of BSA. RT-PCR was done as described above. Confocal calcium imaging and electron microscopy A dissected dorsal integument (day 1 last instar larva) was washed with Ca 2+ -free Carlson solution (120 mm NaCl, 2.7 mm KCl, 0.5 mm MgCl 2 , 1.7 mm NaH 2 PO 4 , 1.4 mm NaHCO 3 , 2.2 mm glucose) and loaded with 10 lm Fluo-3 (Dojindo) at 25 °C for 30 min. After loading, the tissue was washed twice with Ca 2+ -free Carlson solution, lightly blot- ted with filter paper, and placed on a glass slide. 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